Dr. Rajadas is currently working on the molecular mechanism of neurodegenerative disorders caused by aggregated tau and abeta proteinsproteins that are synergically involved in Alzheimers disease development. He uses various biophysical approaches such as AFM, fluorescence, and NMR to understand the structural details of neurotoxic oligomeric forms of these two proteins.
For the last 7 years, our lab has also been involved in transforming biophysical ideas into biomaterial and drug delivery technologies. These technologies include microencapsulation of drugs, vascular grafts, bio-implants, development of small molecule and protein-based drugs, regeneration of nerve and cardiovascular tissues, and wound healing applications
Stanford, CA
Stanford, CA
Stanford, CA
Stanford, CA
Stanford, CA
View details for Web of Science ID 000463117000011
View details for Web of Science ID 000463117000043
4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intra-vital 2-photon microscopy, we found that 4-MUG (a non-fluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.
View details for PubMedID 30914479
View details for DOI 10.1111/bph.14528
View details for Web of Science ID 000455517100003
Improved methods are needed to reliably assess CFTR function in vivo in light of recent therapeutic developments targeting the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Saliva from patients with cystic fibrosis (CF) and healthy controls (HC) was studied using colorimetry and non-resonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients under-going treatment with CFTR modulators including ivacaftor, lumacaftor and tezacaftor, showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, non-invasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations, compared to colorimetry. Moreover, samples from patients can be collected remotely, as they are stable and can be shipped in dry state. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual pa-tients as well as development of new ones.
View details for DOI 10.1021/acs.analchem.9b01800
View details for PubMedID 31117414
Combined pulmonary fibrosis and emphysema (CPFE) is a syndrome that predominantly affects male smokers or ex-smokers and it has a mortality rate of 55% and a median survival of 5 years. Pulmonary hypertension (PH) is a highly fatal complication of CPFE. Despite this dismal prognosis, no curative therapies exist for patients with CPFE outside of lung transplantation and no therapies are recommended to treat PH. This highlights the need to develop novel treatment approaches for CPFE. Studies from our group have demonstrated that adenosine and its receptor, ADORA2B, are elevated in chronic lung diseases. Activation of ADORA2B leads to elevated levels of hyaluronan synthases and increased hyaluronan, a glycosaminoglycan which contributes to chronic lung injury. We hypothesize that ADORA2B and hyaluronan contribute to CPFE. Using isolated CPFE lung tissue, we characterized expression levels of ADORA2B and hyaluronan synthases (HAS). Next using a unique model of experimental lung injury that replicates features of CPFE, namely airspace enlargement, PH and fibrotic deposition, we investigated whether 4MU, a hyaluronan synthase inhibitor, was able to inhibit features of CPFE. Increased protein levels of ADORA2B and HAS3 were detected in CPFE and in our experimental model of CPFE. Treatment with 4MU was able to attenuate PH and fibrosis but not airspace enlargement. This was accompanied by a reduction of HAS3-positive macrophages. We have generated pre-clinical data demonstrating the capacity of 4MU, a FDA-approved drug, to attenuate features of CPFE in an experimental model of chronic lung injury.
View details for PubMedID 31036697
Microtubules (MTs) are intracellular polymers that provide structure to the cell, serve as railways for intracellular transport, and regulate many cellular activities, including cell migration. The dynamicity and function of the MT cytoskeleton are determined in large part by its regulatory proteins, including the recently discovered MT severing enzyme Fidgetin-like 2 (FL2). Downregulation of FL2 expression with small interfering RNA (siRNA) results in a more than twofold increase in cell migration rate in vitro as well as translates into improved wound-healing outcomes in in vivo mouse models. Here we utilized a commercially available surfactant polymer dressing (SPD) as a vehicle to deliver FL2 siRNA. To this end we incorporated collagen microparticles containing FL2 siRNA into SPD (SPD-FL2-siRNA) for direct application to the injury site. Topical application of SPD-FL2 siRNA to murine models of full-thickness excision wounds and full-thickness burn wounds resulted in significant improvements in the rate and quality of wound healing, as measured clinically and histologically, compared with controls. Wound healing occurred more rapidly and with high fidelity, resulting in properly organized collagen substructure. Taken together, these findings indicate that the incorporation of FL2 siRNA into existing treatment options is a promising avenue to improve wound outcomes.
View details for PubMedID 30911440
View details for DOI 10.1523/JNEUROSCI.0825-17.2018
View details for Web of Science ID 000452854200011
The immunopathological states of the brain induced by bacterial lipoproteins have been well-characterized by employing biochemical and histological assays. However, these studies have limitations in determining functional states of damaged brains involving aberrant synaptic activity and network, which makes it difficult to diagnose brain disorders during bacterial infection. To address this, we investigated the effect of Pam3CSK4 (PAM), a synthetic bacterial lipopeptide, on synaptic dysfunction of female mice brains and cultured neurons in parallel. Our functional brain imaging using PET with [18F]-FDG and [18F]-FMZ revealed the brain dysfunction induced by PAM is closely aligned to disruption of neurotransmitter-related neuronal activity and functional correlation in the region of the limbic system rather than to decrease of metabolic activity of neurons in the injection area. This finding was verified by in vivo tissue experiments that analyzed synaptic and dendritic alterations in the regions where PET imaging showed abnormal neuronal activity and network. Recording of synaptic activity also revealed that PAM reorganized synaptic distribution and decreased synaptic plasticity in hippocampus. Further study using in vitro neuron cultures demonstrated that PAM decreased the number of presynapses and the frequency of mEPSC, which suggests PAM disrupts neuronal function by damaging presynapses exclusively. We also showed PAM caused aggregation of synapses around dendrites, which may have caused no significant change in expression level of synaptic proteins while synaptic number and function was impaired by PAM. New findings of this study could provide a useful guide for diagnosis and treatment of brain disorders specific to bacterial infection.SIGNIFICANCE STATEMENTIt is challenging to diagnose brain disorders caused by bacterial infection because neural damage induced by bacterial products involves non-specific neurological symptoms, which is rarely detected by laboratory tests with low spatiotemporal resolution. To better understand brain pathology, it is essential to detect functional abnormalities of brain over time. To this end, we investigated characteristic patterns of altered neuronal integrity and functional correlation between various regions in mice brains injected with bacterial lipopeptides by using PET with a goal to apply new findings to diagnosis of brain disorder specific to bacterial infection. In addition, we analyzed altered synaptic density and function using both in vivo and in vitro experimental models to understand how bacterial lipopeptides impair brain function and network.
View details for PubMedID 30381406
BACKGROUND AND PURPOSE: Previous studies have shown that Creosote bush-derived NDGA exerts beneficial actions on the key components of MetS including dyslipidemia, insulin resistance and hypertension in several relevant rodent models. Here we synthesized and screened a total of 6 antihyperlipidemic analogs of NDGA and tested their efficacy against hepatic lipid metabolism in a high-fructose diet (HFrD) fed dyslipidemic rat model.EXPERIMENTAL APPROACH: HFrD fed Sprague-Dawley (SD) treated with NDGA or one of the 6 analogs were used here. Serum samples were analyzed for blood metabolites, whereas liver samples were quantified for changes in various mRNA levels by real-time RT-PCR.KEY RESULTS: Oral gavage of HFrD-fed rats for 4 days with NDGA analogs 1 and 2 (100 mg/kg, once daily) suppressed the hepatic triglyceride content, whereas treatment of rats with NDGA analogs 2, 3 and 4, like NDGA, decreased the plasma triglyceride levels by 70-75%. qRT-PCR measurements demonstrated that among the NDGA analogs 1, 2, 4 and 5, analog 4 was most effective in inhibiting the mRNA levels of some key enzymes and transcription factors involved in lipogenesis. All four analogs almost equally inhibited the key genes involved in triglyceride synthesis and fatty acid elongation. Unlike NDGA, none of the analogs affected the genes of hepatic fatty acid oxidation or transport CONCLUSIONS AND IMPLICATIONS: Our data suggest that NDGA analogs 1, 2, 4 and 5, particularly analog 4, exert their anti-hyperlipidemic actions by negatively targeting genes of key enzymes and transcription factors involved in lipogenesis, triglyceride synthesis and fatty acid elongation. These analogs have therapeutic implications.
View details for PubMedID 30374952
View details for DOI 10.1089/wound.2018.0827
View details for Web of Science ID 000448369800001
Chronic wounds are a significant medical and economic problem worldwide. Individuals over the age of 65 are particularly vulnerable to pressure ulcers and impaired wound healing. With this demographic growing rapidly there is a need for effective treatments. We have previously shown that defective hypoxia signaling through destabilization of the master hypoxia-inducible factor 1alpha (HIF-1alpha) underlies impairments in both aging and diabetic wound healing. To stabilize HIF-1alpha, we developed a transdermal delivery system of the FDA-approved small molecule deferoxamine (DFO) and found that transdermal DFO could both prevent and treat ulcers in diabetic mice. Here, we show that transdermal DFO can similarly prevent pressure ulcers and normalize aged wound healing. Enhanced wound healing by DFO is brought about by stabilization of HIF-1alpha and improvements in neovascularization. Transdermal DFO can be rapidly translated into the clinic and may represent a new approach to prevent and treat pressure ulcers in aged patients. This article is protected by copyright. All rights reserved.
View details for PubMedID 30152571
Cytokine therapies have emerged during the past decade as promising noninvasive treatments for heart disease. In general, current drug treatments are directed towards symptom control and prevention of disease progression; however, many agents also produce cause side effects that alter quality of life. Cytokine based therapies have the potential to reduce post-infarct heart failure and chronic ischemia by stimulating the proliferation and differentiation of endothelial cells and bone marrow hematopoietic stem cells and mobilizing these cells toward ischemic tissue. In turn, these mobilized cell populations contribute to myocardial regeneration. In contrast, over-expression of several cytokines has been linked to a variety of heart diseases; thus, therapies targeting and monitoring these cytokines are of great interest. Here we summarize results from clinical studies on cytokines as therapeutic agents or therapeutic targets in the treatment for heart disease as well as cytokines involved in the evolution of heart disease.
View details for PubMedID 30170892
View details for Web of Science ID 000451464000057
The increasing population of patients with heart disease and the limited availability of organs for transplantation have encouraged multiple strategies to fabricate healthy implantable cardiac tissues. One of the main challenges in cardiac tissue engineering is to direct cell behaviors to form functional three-dimensional (3D) biomimetic constructs. This article provides a brief review on various cell sources used in cardiac tissue engineering and highlights the effect of scaffold-based signals such as topographical and biochemical cues and stiffness. Then, conventional and novel micro-engineered bioreactors for the development of functional cardiac tissues will be explained. Bioreactor-based signals including mechanical and electrical cues to control cardiac cell behavior will also be elaborated in detail. Finally, the application of computational fluid dynamics to design suitable bioreactors will be discussed. This review presents the current state-of-the-art, emerging directions and future trends that critically appraise the concepts involved in various approaches to direct cells for building functional hearts and heart parts.
View details for DOI 10.1039/c7bm01176h
View details for Web of Science ID 000447710700001
View details for PubMedID 29767196
A novel benzimidazole compound ZLN005 was previously identified as a transcriptional activator of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in certain metabolic tissues. Upregulation of PGC-1α by ZLN005 has been shown to have beneficial effect in a diabetic mouse model and in a coronary artery disease model in vitro. ZLN005 could also have therapeutic potential in neurodegenerative diseases involving down-regulation of PGC-1α. Given the phenotypic efficacy of ZLN005 in several animal models of human disease, its metabolic profile was investigated to guide the development of novel therapeutics using ZLN005 as the lead compound.ZLN005 was incubated with both rat and human liver microsomes and S9 fractions to identify in vitro metabolites. Urine from rats dosed with ZLN005 was used to identify in vivo metabolites. Extracted metabolites were analyzed by LC-MS/MS using a hybrid linear ion trap triple quadrupole mass spectrometer under full scan, enhanced product ion scan, neutral loss scan and precursor scan modes. Metabolites in plasma and brain of ZLN005-treated rats were also profiled using multiple reaction monitoring.Identified in vitro transformations of ZLN005 include mono- and dihydroxylation, further oxidation to carboxylic acids, and mono-O-glucuronide and sulfate conjugation to hydroxy ZLN005 as well as glutathione conjugation. Identified in vivo metabolites are mainly glucuronide and sulfate conjugates of dihydroxyl, carboxyl, and hydroxy acid of the parent compound. The parent compound as well as several major phase I metabolites were found in rat plasma and brain.Using both in vitro and in vivo methods, we elucidated the metabolic pathway of ZLN005. Phase I metabolites with hydroxylation and carboxylation, as well as phase II metabolites with glucuronide, sulfate and glutathione conjugation, were identified.
View details for PubMedID 29334584
Vascular dysfunctions, hypometabolism, and insulin resistance are high and early risk factors for Alzheimer's disease (AD), a leading neurological disease associated with memory decline and cognitive dysfunctions. Early defects in glucose transporters and glycolysis occur during the course of AD progression. Hypometabolism begins well before the onset of early AD symptoms; this timing implicates the vulnerability of hypometabolic brain regions to beta-secretase 1 (BACE-1) upregulation, oxidative stress, inflammation, synaptic failure, and cell death. Despite the fact that ketone bodies, astrocyte-neuron lactate shuttle, pentose phosphate pathway (PPP), and glycogenolysis compensate to provide energy to the starving AD brain, a considerable energy crisis still persists and increases during disease progression. Studies that track brain energy metabolism in humans, animal models of AD, and in vitro studies reveal striking upregulation of beta-amyloid precursor protein (β-APP) and carboxy-terminal fragments (CTFs). Currently, the precise role of CTFs is unclear, but evidence supports increased endosomal-lysosomal trafficking of β-APP and CTFs through autophagy through a vague mechanism. While intracellular accumulation of Aβ is attributed as both the cause and consequence of a defective endolysosomal-autophagic system, much remains to be explored about the other β-APP cleavage products. Many recent works report altered amino acid catabolism and expression of several urea cycle enzymes in AD brains, but the precise cause for this dysregulation is not fully explained. In this paper, we try to connect the role of CTFs in the energy translation process in AD brain based on recent findings.
View details for PubMedID 30420907
View details for PubMedCentralID PMC6215552
Lyme disease accounts for >90% of all vector-borne disease cases in the United States and affect ~300,000 persons annually in North America. Though traditional tetracycline antibiotic therapy is generally prescribed for Lyme disease, still 10%-20% of patients treated with current antibiotic therapy still show lingering symptoms.In order to identify new drugs, we have evaluated four cephalosporins as a therapeutic alternative to commonly used antibiotics for the treatment of Lyme disease by using microdilution techniques like minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). We have determined the MIC and MBC of four drugs for three Borrelia burgdorferi s.s strains namely CA8, JLB31 and NP40. The binding studies were performed using in silico analysis.The MIC order of the four drugs tested is cefoxitin (1.25 µM/mL) > cefamandole (2.5 µM/mL), > cefuroxime (5 µM/mL) > cefapirin (10 µM/mL). Among the drugs that are tested in this study using in vivo C3H/HeN mouse model, cefoxitin effectively kills B. burgdorferi. The in silico analysis revealed that all four cephalosporins studied binds effectively to B. burgdorferi proteins, SecA subunit penicillin-binding protein (PBP) and Outer surface protein E (OspE).Based on the data obtained, cefoxitin has shown high efficacy killing B. burgdorferi at concentration of 1.25 µM/mL. In addition to it, cefoxitin cleared B. burgdorferi infection in C3H/HeN mice model at 20 mg/kg.
View details for PubMedID 30254421
View details for DOI 10.2147/DDDT.S164966
View details for Web of Science ID 000444298700004
View details for Web of Science ID 000430308600139
View details for Web of Science ID 000430308600036
View details for Web of Science ID 000430308600039
Formation of scars following wounding or trauma represents a significant healthcare burden costing the economy billions of dollars every year. Activation of focal adhesion kinase (FAK) has been shown to play a pivotal role in transducing mechanical signals to elicit fibrotic responses and scar formation during wound repair. We have previously shown that inhibition of FAK using local injections of a small molecule FAK inhibitor (FAKI) can attenuate scar development in a hypertrophic scar model. Clinical translation of FAKI therapy has been challenging, however, due to the lack of an effective drug delivery system for extensive burn injuries, blast injuries, and large excisional injuries. To address this issue, we have developed a pullulan collagen-based hydrogel to deliver FAKI to excisional and burn wounds in mice. Specifically, two distinct drug-laden hydrogels were developed for rapid or sustained release of FAKI for treatment of burn wounds and excisional wounds, respectively. Controlled delivery of FAKI via pullulan collagen hydrogels accelerated wound healing, reduced collagen deposition and activation of scar forming myofibroblasts in both wound healing models. Our study highlights a biomaterial-based drug delivery approach for wound and scar management that has significant translational implications.
View details for PubMedID 29775632
To determine the effects of NDGA on metabolic and molecular changes in response to feeding mice typical American fast food or Western diet, mice were fed with ALIOS diet and subjected to metabolic analysis. Male C57BL/6J mice were randomly assigned to: ALIOS, ALIOS + NDGA, or control diet and maintained on the specific diet for 8 weeks. Mice fed ALIOS diet, showed increased body, liver and epididymal fat pad weight, plasma ALT and AST levels (a measure of liver injury), and liver triglyceride (TG) content. Co-administration of NDGA normalized body and epididymal fat pad weight, ALT and AST levels, and liver TG. NDGA treatment also improved insulin sensitivity but not glucose intolerance in ALIOS diet fed mice. In ALIOS diet fed mice, NDGA supplementation induced PPARα (the master regulator of fatty acid oxidation) and mRNA levels of Cpt1c and Cpt2, key genes involved in fatty acid oxidation as compared to ALIOS diet. NDGA significantly reduced liver ER stress response CHOP protein, as compared to chow or ALIOS diet and also ameliorated ALIOS diet-induced elevation of apoptosis signaling protein, CASP3. Likewise, NDGA downregulated the ALIOS-diet induced mRNA levels of Pparg, Fasn, and Dgat2. NDGA treatment of ALIOS fed mice upregulated the hepatic expression of antioxidant enzymes, GPX4 and PRDX3 proteins. In conclusion, we provide evidence that NDGA improves metabolic dysregulation by simultaneously modulating PPARα transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways.
View details for PubMedID 29472517
Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen-dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.
View details for PubMedID 29721363
Invasive pulmonary disease due to the moldAspergillus fumigatuscan be life-threatening in lung transplant recipients, but the risk factors remain poorly understood. To study this process, we used a tracheal allograft mouse model that recapitulates large airway changes observed in patients undergoing lung transplantation. We report that microhemorrhage-related iron content may be a major determinant ofA. fumigatusinvasion and, consequently, its virulence. Invasive growth was increased during progressive alloimmune-mediated graft rejection associated with high concentrations of ferric iron in the graft. The role of iron inA. fumigatusinvasive growth was further confirmed by showing that this invasive phenotype was increased in tracheal transplants from donor mice lacking the hemochromatosis gene (Hfe -/- ). The invasive phenotype was also increased in mouse syngrafts treated with topical iron solution and in allograft recipients receiving deferoxamine, a chelator that increases iron bioavailability to the mold. The invasive growth of the iron-intolerantA. fumigatusdouble-knockout mutant (ΔsreA/ΔcccA) was lower than that of the wild-type mold. Alloimmune-mediated microvascular damage and iron overload did not appear to impair the host's immune response. In human lung transplant recipients, positive staining for iron in lung transplant tissue was more commonly seen in endobronchial biopsy sections from transplanted airways than in biopsies from the patients' own airways. Collectively, these data identify iron as a major determinant ofA. fumigatusinvasive growth and a potential target to treat or preventA. fumigatusinfections in lung transplant patients.
View details for PubMedID 29467298
Despite preliminary confidence on biosafety of polymer coated iron oxide nanoparticles (SPIONs), toxicity concerns have hampered their clinical translation. SPIONs toxicity is known to be due to catalytic activity of their surface and release of toxic Fe ions originating from the core biodegradation, leading to the generation of reactive oxygen species (ROS). Here, we hypothesized that a double-layer polymeric corona comprising of dextran as an interior, and polyethylene glycol (PEG) as an exterior layer better shields the core SPIONs. We found that ROS generation was cell specific and depended on SPIONs concentration, although it was reduced by sufficient PEG immobilization or 100 µM deferoxamine. 24 h following injection, PEGylated samples showed reduction of biodistribution in liver, heterogenous biodistribution profile in spleen, and no influence on NPs blood retention. Sufficient surface masking or administration of deferoxamine could be beneficial strategies in designing and clinical translation of future biomedical SPIONs.
View details for PubMedID 29523826
A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.
View details for DOI 10.1016/j.nano.2016.10.008
View details for Web of Science ID 000401089100037
View details for DOI 10.1038/ja.2016.131
View details for PubMedID 27826144
A major challenge in CT screening for lung cancer is limited specificity when distinguishing between malignant and non-malignant pulmonary nodules (PN). Malignant nodules have different mechanical properties and tissue characteristics ('stiffness') from non-malignant nodules. This study seeks to improve CT specificity by demonstrating in rats that measurements of volumetric ratios in PNs with varying composition can be determined by respiratory-gated dynamic CT imaging and that these ratios correlate with direct physical measurements of PN stiffness.Respiratory-gated MicroCT images acquired at extreme tidal volumes of 9 rats with PNs from talc, matrigel and A549 human lung carcinoma were analyzed and their volumetric ratios (δ) derived. PN stiffness was determined by measuring the Young's modulus using atomic force microscopy (AFM) for each nodule excised immediately after MicroCT imaging.There was significant correlation (p=0.0002) between PN volumetric ratios determined by respiratory-gated CT imaging and the physical stiffness of the PNs determined from AFM measurements.We demonstrated proof of concept that PN volume changes measured non-invasively correlate with direct physical measurements of stiffness. These results may translate clinically into a means of improving the specificity of CT screening for lung cancer and/or improving individual prognostic assessments based on lung tumor stiffness.
View details for DOI 10.1016/j.radonc.2016.11.019
View details for PubMedID 27989402
View details for PubMedCentralID PMC5319913
Autism Spectrum Disorders (ASDs) are a group of complex neurodevelopmental conditions characterized by abnormal patterns of attention, and impaired social and communication skills. ASDs are also associated with a number of functional challenges and potentially harmful deficits, including restricted and repetitive behaviors, anxiety, irritability, seizures, and self-harm. Although the exact causes of ASDs are currently unknown, it is suggested that genetic, epigenetic and environmental factors play critical roles. Recent findings support evidence for synaptic defects and impairments in brain information processing that are linked to social and perceptual skills. Owing to the clinical heterogeneity and lack of precise diagnostic tools, current therapeutic approaches aimed at managing ASD-associated conditions are not definitive. In this review, we seek to provide a contemporary account of the key pathological events pertaining to autism: the theory of oxidative stress and inflammatory causes; ideas of immune dysfunction; the probable biomarkers that can be used for diagnostics - and the use of pharmaceuticals and stem cells as possible candidates for the treatment of ASDs.
View details for DOI 10.1016/j.wneu.2016.09.100
View details for Web of Science ID 000397028300082
Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.
View details for DOI 10.1126/scitranslmed.aag2809
View details for PubMedID 28077677
Anastomosis with tissue adhesives is an alternative method for conventional anastomosis. However, this method has several technical challenges. It requires the use of suture to prevent leakage into lumen and precise application onto all surfaces of the anastomosis site. To solve these problems, poloxamer 407 (P 407) was previously used as a stent. In this study, we made heparinized P 407 (h-P 407) as a new formula. We aimed to successfully use h-P 407 as a stent in sutureless anastomosis in a rat abdominal aorta model.Sixty Sprague-Dawley rats were used. In the first group, end-to-end anastomoses were performed with suture; in the second and third groups, sutureless anastomoses were performed with 2-octyl cyanoacrylate. As an intraluminal stent, P 407 was used in the second group, and h-P 407 was used in the third group. Anastomosis time was measured. Lumen width, intimal hyperplasia, and foreign body reaction were assessed histologically. Velocity flow rates and vessel diameters were measured radiologically. Burst strength was measured, and the results were statistically evaluated.Sutureless anastomosis was more rapid than conventional anastomosis. Lumen width was narrower in the suture group. İnflammation and foreign body reaction were more severe in the suture group. There was no radiologic and biomechanical difference between the groups. We found that intimal hyperplasia was less in h-P 407 than in P 407.h-P407 can be successfully used as an intraluminal stent for sutureless microvascular anastomosis with tissue adhesives.
View details for PubMedID 27939908
View details for DOI 10.1039/C6TB03247H
View details for DOI 10.1016/B978-0-323-52725-5.00013-7
View details for Web of Science ID 000439410000015
View details for DOI 10.1016/B978-0-08-100963-5.00008-2
Group III Pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood.Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis.In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA dependent manner.Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.
View details for PubMedID 28688167
Treatment of type 2 diabetes mellitus continues to pose an important clinical challenge, with most existing therapies lacking demonstrable ability to improve cardiovascular outcomes. The atheroprotective peptide apelin (APLN) enhances glucose utilization and improves insulin sensitivity. However, the mechanism of these effects remains poorly defined. We demonstrate that the expression of APLNR (APJ/AGTRL1), the only known receptor for apelin, is predominantly restricted to the endothelial cells (ECs) of multiple adult metabolic organs, including skeletal muscle and adipose tissue. Conditional endothelial-specific deletion of Aplnr (Aplnr(ECKO) ) resulted in markedly impaired glucose utilization and abrogation of apelin-induced glucose lowering. Furthermore, we identified inactivation of Forkhead box protein O1 (FOXO1) and inhibition of endothelial expression of fatty acid (FA) binding protein 4 (FABP4) as key downstream signaling targets of apelin/APLNR signaling. Both the Apln(-/-) and Aplnr(ECKO) mice demonstrated increased endothelial FABP4 expression and excess tissue FA accumulation, whereas concurrent endothelial Foxo1 deletion or pharmacologic FABP4 inhibition rescued the excess FA accumulation phenotype of the Apln(-/-) mice. The impaired glucose utilization in the Aplnr(ECKO) mice was associated with excess FA accumulation in the skeletal muscle. Treatment of these mice with an FABP4 inhibitor abrogated these metabolic phenotypes. These findings provide mechanistic insights that could greatly expand the therapeutic repertoire for type 2 diabetes and related metabolic disorders.
View details for PubMedID 28904225
The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.
View details for PubMedID 28978794
The beta-1 adrenergic receptor (ADRB1) is a promising therapeutic target intrinsically involved in the cognitive deficits and pathological features associated with Alzheimer's disease (AD). Evidence indicates that ADRB1 plays an important role in regulating neuroinflammatory processes, and activation of ADRB1 may produce neuroprotective effects in neuroinflammatory diseases. Novel small molecule modulators of ADRB1, engineered to be highly brain permeable and functionally selective for the G protein with partial agonistic activity, could have tremendous value both as pharmacological tools and potential lead molecules for further preclinical development. The present study describes our ongoing efforts toward the discovery of functionally selective partial agonists of ADRB1 that have potential therapeutic value for AD and neuroinflammatory disorders, which has led to the identification of the molecule STD-101-D1. As a functionally selective agonist of ADRB1, STD-101-D1 produces partial agonistic activity on G protein signaling with an EC50 value in the low nanomolar range, but engages very little beta-arrestin recruitment compared to the unbiased agonist isoproterenol. STD-101-D1 also inhibits the tumor necrosis factor α (TNFα) response induced by lipopolysaccharide (LPS) both in vitro and in vivo, and shows high brain penetration. Other than the therapeutic role, this newly identified, functionally selective, partial agonist of ADRB1 is an invaluable research tool to study mechanisms of G protein-coupled receptor signal transduction.
View details for PubMedID 28746336
Neurotrophin (NT) receptors are coupled to numerous signaling networks that play critical roles in neuronal survival and plasticity. Several non-peptide small molecule ligands have recently been reported that bind to and activate specific tropomyosin-receptor-kinase (Trk) NT receptors, stimulate their downstream signaling, and cause biologic effects similar to, though not completely overlapping, those of the native NT ligands. Here, in silico screening, coupled with low-throughput neuronal survival screening, identified a compound, LM22B-10, that, unlike prior small molecule Trk ligands, binds to and activates TrkB as well as TrkC. LM22B-10 increased cell survival and strongly accelerated neurite outgrowth, superseding the effects of brain-derived neurotrophic factor (BDNF), NT-3 or the two combined. Additionally, unlike the NTs, LM22B-10 supported substantial early neurite outgrowth in the presence of inhibiting glycoproteins. Examination of the mechanisms of these actions suggested contributions of the activation of both Trks and differential interactions with p75(NTR), as well as a requirement for involvement of the Trk extracellular domain. In aged mice, LM22B-10 activated hippocampal and striatal TrkB and TrkC, and their downstream signaling, and increased hippocampal dendritic spine density. Thus, LM22B-10 may constitute a new tool for the study of TrkB and TrkC signaling and their interactions with p75(NTR), and provides groundwork for the development of ligands that stimulate unique combinations of Trk receptors and activity patterns for application to selected neuronal populations and deficits present in various disease states.
View details for DOI 10.1016/j.neuropharm.2016.06.015
View details for PubMedID 27334657
A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.
View details for DOI 10.1016/j.nano.2016.10.008
View details for PubMedID 27793788
Autism Spectrum Disorders (ASDs) are a group of complex neurodevelopmental conditions characterized by abnormal patterns of attention, and impaired social and communication skills. ASDs are also associated with a number of functional challenges and potentially harmful deficits, including restricted and repetitive behaviors, anxiety, irritability, seizures, and self-harm. Although the exact causes of ASDs are currently unknown, it is suggested that genetic, epigenetic and environmental factors play critical roles. Recent findings support evidence for synaptic defects and impairments in brain information processing that are linked to social and perceptual skills. Owing to the clinical heterogeneity and lack of precise diagnostic tools, current therapeutic approaches aimed at managing ASD-associated conditions are not definitive. In this review, we seek to provide a contemporary account of the key pathological events pertaining to autism: the theory of oxidative stress and inflammatory causes; ideas of immune dysfunction; the probable biomarkers that can be used for diagnostics - and the use of pharmaceuticals and stem cells as possible candidates for the treatment of ASDs.
View details for DOI 10.1016/j.wneu.2016.09.100
View details for PubMedID 27725300
Alzheimer's disease (AD) is a neurodegenerative disease characterized by genotypic and phenotypic heterogeneity. Critical components of the two AD pathological pathways, Aβ-amyloidosis and Tauopathy, have been considered as therapeutic targets. Among them, much effort is focused on aberrant Tau phosphorylation and targeting Tau-phosphorylating kinases. Methylene blue (MB), a phenothiazine dye that crosses the blood-brain barrier, has been shown to hit multiple molecular targets involved in AD and have beneficial effects in clinical studies. Here we present evidence that microtubule affinity-regulating kinase (MARK4) is a novel target of MB. MB partially rescued the synaptic toxicity in Drosophila larva overexpressing PAR1 (MARK analog). In 293T culture, MB decreased MARK4-mediated Tau phosphorylation in a dose dependent manner. Further studies revealed a two-fold mechanism by MB including down-regulation of MARK4 protein level through ubiquitin-proteasome pathway and inhibition of MARK4 kinase activity in vitro. This study highlights the importance of MARK4 as a viable target for Tauopathy and provides fresh insight into the complex mechanism used by MB to treat AD.
View details for DOI 10.1038/srep34784
View details for PubMedID 27708431
View details for DOI 10.1016/j.surfcoat.2016.01.017
View details for Web of Science ID 000381169300018
Iron plays an especially important role in human physiological functions and pathological impairments. The superior properties of carbon nanotubes (CNTs) and their modification with bismuth and magnetic nanoparticles as developed in this work have led to an extraordinary and novel material to facilitate ultrasensitive detection in the nanomolar range. Here, we present the development of an electrochemical sensor for detection of ferrous (Fe(2+)) and ferric (Fe(3+)) iron by means of CNTs modified with bismuth and magnetic nanoparticles for higher sensitivity of detection. The sensor fabrication includes microfabrication methodologies, soft lithography, and electrodeposition. Cyclic voltammetry and differential pulse voltammetry are used for the electroanalytical studies and detection of the ions in samples. The sensor has a dynamic range of detection from 0.01 nm to 10 mm. The performance of the sensor with modified CNTs was explored for sensitivity and specificity. CNTs, modified with bismuth and magnetic nanoparticles by means of electrodeposition, enhanced the detection limit significantly down to 0.01 nm.
View details for DOI 10.1002/cnma.201600174
View details for Web of Science ID 000383766800009
View details for PubMedCentralID PMC5110256
Prion diseases are characterized by a conformational change in prion protein from its native state into beta-sheet rich aggregates that are neurotoxic. The central domain that contain a highly conserved hydrophobic region of the protein play an important role in the toxicity. The conformation of the proteins is largely influenced by various solvent environments. Here we report results of study of hydrophobic prion fragment peptide PrP(113-127) under different pH and osmolytes solution conditions. The secondary structure and the folding of PrP(113-127) was determined using circular dichroism and fluorescence spectroscopic methods. The results indicate that PrP(113-127) adopts a random coil conformation in aqueous buffer at neutral pH and that converted into beta sheet on aging. Even though the initial random coil conformation was similar in different pH conditions, the acidic as well as basic pH conditions delays the conformational transition to beta sheet. FRET results indicate that the distance between N and C-terminal regions increased on aging due to unfolding by self-assembly of the peptide into an organized beta sheet structure. Presence of osmolytes, prevented or decelerated the aggregation process of PrP(113-127) peptide.
View details for DOI 10.1016/j.npep.2016.02.004
View details for Web of Science ID 000378193100002
View details for PubMedID 26919915
Protein misfolding and aggregation are responsible for a large number of diseases called protein conformational diseases or disorders that include Alzheimer׳s disease, Huntington׳s diseases, Prion related encephalopathies and type-II diabetes (http://dx.doi.org/10.1038/35041139) (Kopito and Ron, 2000) [1]. A variety of studies have shown that some small organic molecules, known as osmolytes have the ability to stabilize native conformation of proteins and prevent misfolding and aggregation (http://www.la-press.com/article.php?article_id=447) (Zhao et al., 2008) [2]. It has been shown that certain short segment or fragment of respective proteins can also form amyloids, and the segments also promote the aggregation in the full-length protein (http://dx.doi.org/10.2174/0929867023369187) (Gazit, 2002) [3]. This article presents circular dichroism spectroscopic data on conformational analysis and effect of osmolytes on Aβ peptide fragments, different lengths of polyglutamine peptide and the amyloidogenic segment of islet amyloid polypeptide.
View details for DOI 10.1016/j.dib.2016.04.070
View details for PubMedID 27222868
View details for PubMedCentralID PMC4872718
Huntington's disease (HD) is recognized as a currently incurable, inherited neurodegenerative disorder caused by the accumulation of misfolded polyglutamine (polyQ) peptide aggregates in neuronal cells. Yet, the mechanism by which newly formed polyQ chains interact and assemble into toxic oligomeric structures remains a critical, unresolved issue. In order to shed further light on the matter, our group elected to investigate the folding of polyQ peptides - examining glutamine repeat lengths ranging from 3 to 44 residues. To characterize these aggregates we employed a diverse array of technologies, including: nuclear magnetic resonance; circular dichroism; Fourier transform infrared spectroscopy; fluorescence resonance energy transfer (FRET), and atomic force microscopy. The data we obtained suggest that an increase in the number of glutamine repeats above 14 residues results in disordered loop structures, with different repeat lengths demonstrating unique folding characteristics. This differential folding manifests in the formation of distinct nano-sized fibrils, and on this basis, we postulate the idea of 14 polyQ repeats representing a critical loop length for neurotoxicity - a property that we hope may prove amenable to future therapeutic intervention. Furthermore, FRET measurements on aged assemblages indicate an increase in the end-to-end distance of the peptide with time, most probably due to the intermixing of individual peptide strands within the nanofibril. Further insight into this apparent time-dependent reorganization of aggregated polyQ peptides may influence future disease modeling of polyQ-related proteinopathies, in addition to directing novel clinical innovations.
View details for DOI 10.1016/j.npep.2016.01.011
View details for Web of Science ID 000378193100010
View details for PubMedID 26874369
Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.
View details for DOI 10.1089/ten.tea.2015.0277
View details for Web of Science ID 000369987900012
View details for PubMedID 26871860
View details for PubMedCentralID PMC4779321
The rapid ascent of nanotechnology and regenerative therapeutics as applied to medicine and surgery has seen an exponential rise in the scale of research generated in this field. This is evidenced not only by the sheer volume of papers dedicated to nanotechnology but also in a large number of new journals dedicated to nanotechnology and regenerative therapeutics specifically to medicine and surgery. Aspects of nanotechnology that have already brought benefits to these areas include advanced drug delivery platforms, molecular imaging and materials engineering for surgical implants. Particular areas of interest include nerve regeneration, burns and wound care, artificial skin with nanoelectronic sensors and head and neck surgery. This study presents a review of nanotechnology and regenerative therapeutics, with focus on its applications and implications in plastic surgery.
View details for PubMedID 26422652
View details for PubMedCentralID PMC4703458
Iron plays an especially important role in human physiological functions and pathological impairments. The superior properties of carbon nanotubes (CNTs) and their modification with bismuth and magnetic nanoparticles as developed in this work have led to an extraordinary and novel material to facilitate ultrasensitive detection in the nanomolar range. Here, we present the development of an electrochemical sensor for detection of ferrous (Fe(2+)) and ferric (Fe(3+)) iron by means of CNTs modified with bismuth and magnetic nanoparticles for higher sensitivity of detection. The sensor fabrication includes microfabrication methodologies, soft lithography, and electrodeposition. Cyclic voltammetry and differential pulse voltammetry are used for the electroanalytical studies and detection of the ions in samples. The sensor has a dynamic range of detection from 0.01 nm to 10 mm. The performance of the sensor with modified CNTs was explored for sensitivity and specificity. CNTs, modified with bismuth and magnetic nanoparticles by means of electrodeposition, enhanced the detection limit significantly down to 0.01 nm.
View details for PubMedID 27857882
View details for DOI 10.1016/j.surfcoat.2016.01.017
Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%-20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as lead molecules for further advanced studies.
View details for DOI 10.2147/DDDT.S101486
View details for Web of Science ID 000373575000001
View details for PubMedID 27103785
View details for PubMedCentralID PMC4827596
View details for DOI 10.1016/j.bjps.2015.08.028
View details for Web of Science ID 000367236800006
View details for DOI 10.1002/smll.201402579
View details for Web of Science ID 000367916600001
Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications.
View details for DOI 10.1007/s10439-015-1353-0
View details for PubMedID 26066056
Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.
View details for DOI 10.1016/j.chom.2015.10.013
View details for Web of Science ID 000365113100008
View details for PubMedID 26567508
View details for PubMedCentralID PMC4653043
View details for DOI 10.1016/j.msec.2015.05.045
View details for Web of Science ID 000356744600027
Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility. In this study, we aim to identify potential FOXM1 small molecule inhibitors to develop a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC.We used in silico screening of compounds against a solved structure of FOXM1 and subsequently to derive a list of possible compounds that could inhibit FOXM1. Three compounds were tested for in vitro cytotoxicity and FOXM1 expression level was confirmed by RT-PCR and Western blot in EOC cell lines.The FOXM1 structure obtained from 3G73 represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1/DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, N-phenylphenanthren-9-amine (related to hit #225) successfully showed cytotoxicity to all three cell lines with IC50 around 1μM, and downregulate FOXM1 and transcription of its downstream molecules such as CCNB1.By a combination of in silico screening coupled to cellular cytotoxicity studies, we have taken the first step towards identifying potential inhibitors of FOXM1 that can replace thiostrepton.
View details for DOI 10.1016/j.bmc.2015.06.002
View details for PubMedID 26164623
Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo, demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.
View details for DOI 10.1002/adfm.201500875
View details for Web of Science ID 000358504000001
View details for PubMedCentralID PMC4978190
The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.
View details for DOI 10.1111/ajt.13189
View details for Web of Science ID 000356494300013
View details for PubMedID 25727073
View details for DOI 10.1053/j.gastro.2015.04.002
View details for PubMedID 25863215
Systemic therapies for inflammatory bowel disease are associated with increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermo-sensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel upon reaching body temperature. Following rectal administration to mice with dextran sulphate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform.
View details for DOI 10.1053/j.gastro.2015.04.002
View details for PubMedID 25863215
View details for DOI 10.1016/j.apsusc.2015.02.120
View details for Web of Science ID 000351626700017
Introduction: Cardiovascular diseases such as coronary heart disease often necessitate the surgical repair using conduits. Although autografts still remain the gold standard, the inconvenience of harvesting and/or insufficient availability in patients with atherosclerotic disease has given impetus to look into alternative sources for vascular grafts. Areas covered: There are four main techniques to produce tissue-engineered vascular grafts (TEVGs): i) biodegradable synthetic scaffolds; ii) gel-based scaffolds; iii) decellularised scaffolds and iv) self-assembled cell-sheet-based techniques. The first three techniques can be grouped together as scaffold-guided approach as it involves the use of a construct to function as a supportive framework for the vascular graft. The most significant advantages of TEVGs are that it possesses the ability to grow, remodel and respond to environmental factors. Cell sources for TEVGs include mature somatic cells, stem cells, adult progenitor cells and pluripotent stem cells. Expert opinion: TEVG holds great promise with advances in nanotechnology, coupled with important refinements in tissue engineering and decellularisation techniques. This will undoubtedly be an important milestone for cardiovascular medicine when it is eventually translated to clinical use.
View details for DOI 10.1517/14712598.2015.980234
View details for PubMedID 25427995
View details for DOI 10.1517/14712598.2015.980234
View details for Web of Science ID 000348623200008
View details for PubMedID 25427995
There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.
View details for DOI 10.1073/pnas.1413445112
View details for PubMedID 25535360
Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics.
View details for DOI 10.1016/j.biomaterials.2014.08.045
View details for Web of Science ID 000346541100028
View details for PubMedID 25443792
Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo, demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.
View details for PubMedID 27516731
The success of a tissue engineering scaffold depends on a fine balance being achieved between the physicochemical and biological properties. This study attempts to understand the influence of silica concentration on the functional properties of collagen-silica (CS) composite scaffolds for soft tissue engineering applications. Increasing the ratio of silica to collagen (0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2.0 w/w) gave a marked advantage in terms of improving the water uptake and compressive modulus of the CS scaffolds, while also enhancing the biological stability and the turnover time. With increase in silica concentration the water uptake and compressive modulus increased concurrently, whereas it was not so for surface porous architecture and biocompatibility which are crucial for cell adhesion and infiltration. Silica:collagen ratio of ≤1 exhibits favourable surface biocompatibility, and any further increase in silica concentration has a detrimental effect.
View details for PubMedID 25922091
Magnetic resonance imaging (MRI) has come to be known as a unique radiological imaging modality because of its ability to perform tomographic imaging of body without the use of any harmful ionizing radiation. The radiologists use MRI to gain insight into the anatomy of organs, including the brain, while biomedical researchers explore the modality to gain better understanding of the brain structure and function. However, due to limited resolution and contrast, the conventional MRI fails to show the brain microstructure. Diffusion tensor imaging (DTI) harnesses the power of conventional MRI to deduce the diffusion dynamics of water molecules within the tissue and indirectly create a three-dimensional sketch of the brain anatomy. DTI enables visualization of brain tissue microstructure, which is extremely helpful in understanding various neuropathologies and neurodegenerative disorders. In this review, we briefly discuss the background and operating principles of DTI, followed by current trends in DTI applications for biomedical and clinical investigation of various brain diseases and disorders.
View details for PubMedID 27077135
Infrared (IR) imaging is a collection of non-invasive imaging techniques that utilize the IR domain of the electromagnetic spectrum for tissue assessment. A subset of these techniques construct images using back-reflected light, while other techniques rely on detection of IR radiation emitted by the tissue as a result of its temperature. Modern IR detectors sense thermal emissions and produce a heat map of surface temperature distribution in tissues. Thus, the IR spectrum offers a variety of imaging applications particularly useful in clinical diagnostic area, ranging from high-resolution, depth-resolved visualization of tissue to temperature variation assessment. These techniques have been helpful in the diagnosis of many medical conditions including skin/breast cancer, arthritis, allergy, burns, and others. In this review, we discuss current roles of IR-imaging techniques for diagnostic applications in dermatology with an emphasis on skin cancer, allergies, blisters, burns and wounds.
View details for PubMedID 26691203
Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 μg/mL (250 μM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia.
View details for DOI 10.2147/DDDT.S77063
View details for Web of Science ID 000349239100003
View details for PubMedID 25709405
Phospholipids are important membrane components involved in diverse biological activities ranging from cell signaling to infection by viral particles. A thorough understanding of protein-phospholipid interaction dynamics is thus crucial for deciphering basic cellular processes as well as for targeted drug discovery. For any specific phospholipid-protein binding experiment, various groups have reported different binding constants, which are strongly dependent on applied conditions of interactions. Here, we report a method for accurate determination of the binding affinity and specificity between proteins and phospholipids using a model interaction between PLC-δ1/PH and phosphoinositide phospholipid PtdIns(4,5)P2. We developed an accurate Force Distance Spectroscopy (FDS)-based assay and have attempted to resolve the problem of variation in the observed binding constant by directly measuring the bond force. We confirm the FDS findings of a high bond strength of ∼0.19 ± 0.04 nN by Surface Plasmon Resonance (SPR) data analysis, segregating non-specific interactions, which show a significantly lower K(D) suggesting tight binding.
View details for DOI 10.1039/c5an00498e
View details for Web of Science ID 000356171400028
View details for PubMedID 26040325
Vascularisation is often deemed the holy grail of tissue engineering because it is one of the key preconditions that determine the in vivo viability of tissue constructs. Given that a well-developed vascular network allows greater complexity in tissue design and helps regulate tissue metabolism, it appears that the overall outcome of engineered tissue implants depends on the success of microvessel formation, maturation and patterning. Current approaches to vascularising tissue include both in vivo and ex vivo techniques, where blood vessel formation is either spontaneous or guided by physical or biochemical factors. The success of these strategies can then be monitored and evaluated for clinical benefit through numerous standard and novel strategies. Despite the impressive progress in the field of tissue engineering in recent times, there are still numerous technical, immunological, surgical and ethical challenges to overcome. Future prospects in this field are likely to depend on the adoption of a wide-ranging approach incorporating a combination of salient themes such as genetic manipulation, modular assembly and bioreactor coupling. Where applicable, the potential contributions of nanobiotechnology to tissue vascularisation will be discussed as appropriate.
View details for PubMedID 26046286
A versatile approach for the design and fabrication of multilayer magnetic scaffolds with tunable magnetic gradients is described. Multilayer magnetic gelatin membrane scaffolds with intrinsic magnetic gradients were designed to encapsulate magnetized bioagents under an externally applied magnetic field for use in magnetic-field-assisted tissue engineering. The temperature of the individual membranes increased up to 43.7 °C under an applied oscillating magnetic field for 70 s by magnetic hyperthermia, enabling the possibility of inducing a thermal gradient inside the final 3D multilayer magnetic scaffolds. On the basis of finite element method simulations, magnetic gelatin membranes with different concentrations of magnetic nanoparticles were assembled into 3D multilayered scaffolds. A magnetic-gradient-controlled distribution of magnetically labeled stem cells was demonstrated in vitro. This magnetic biomaterial-magnetic cell strategy can be expanded to a number of different magnetic biomaterials for various tissue engineering applications.
View details for PubMedID 26451743
View details for PubMedCentralID PMC4867029
Improving patency rates of current cardiovascular implants remains a major challenge. It is widely accepted that regeneration of a healthy endothelium layer on biomaterials could yield the perfect blood-contacting surface. Earlier efforts in pre-seeding endothelial cells in vitro demonstrated success in enhancing patency, but translation to the clinic is largely hampered due to its impracticality. In situ endothelialization, which aims to create biomaterial surfaces capable of self-endothelializing upon implantation, appears to be an extremely promising solution, particularly with the utilization of endothelial progenitor cells (EPCs). Nevertheless, controlling cell behavior in situ using immobilized biomolecules or physical patterning can be complex, thus warranting careful consideration. This review aims to provide valuable insight into the rationale and recent developments in biomaterial strategies to enhance in situ endothelialization. In particular, a discussion on the important bio-/nanoengineering considerations and lessons learnt from clinical trials are presented to aid the future translation of this exciting paradigm.
View details for PubMedID 26460851
Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 μg/mL (250 μM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia.
View details for DOI 10.2147/DDDT.S77063
View details for PubMedID 25709405
Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.
View details for DOI 10.5966/sctm.2014-0007
View details for PubMedID 25038246
View details for PubMedCentralID PMC4149299
Pathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.In this study, we examined the impact of diabetes on the murine ASC niche, as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.Our results demonstrate that diabetes alters the ASC niche in situ, and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.Perturbations in specific cellular subpopulations, visible only on a single cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in diabetic patients may be limited, and that interventions to improve cell function before application are warranted.
View details for DOI 10.1186/scrt468
View details for Web of Science ID 000338465500001
View details for DOI 10.1016/j.clinthera.2014.04.019
View details for Web of Science ID 000338481600002
View details for DOI 10.1063/1.4879457
View details for Web of Science ID 000337140800076
Zinc protoporphyrin (ZnPP) is a promising metalloporphyrin with sufficient potency, but has poor solubility and is not absorbed well orally. Intragastric administration of ZnPP microparticles (30 μmol/kg) to 3-day-old mice resulted in a twofold increase in potency and no signs of phototoxicity.The use of polymeric particulate delivery systems can improve the stability and enhance intestinal absorption of ZnPP, while retaining HO inhibitory potency without photosensitising effects, and thus is potentially useful in treating neonatal hyperbilirubinemia.
View details for DOI 10.1111/apa.12554
View details for PubMedID 24417721
Due to the limited self-renewal capacity of cardiomyocytes, the mammalian heart exhibits impaired regeneration and insufficient ability to restore heart function after injury. Cardiovascular tissue engineering is currently considered as a promising alternative therapy to restore the structure and function of the failing heart. Recent evidences suggest that the epicardium may play critical roles in regulation of myocardial development and regeneration. One of the mechanisms has been proposed for the restorative effect of the epicardium is the specific physiomechanical cues that this layer provides to the cardiac cells. In this article we explore whether a new generation of epicardium-mimicking, acellular matrices can be utilized to enhance cardiac healing after injury. The matrix consists of a dense collagen scaffold, with optimized biomechanical properties approaching those of embryonic epicardium. Grafting the epicardial patch onto the ischemic myocardium, promptly post the incidence of infarct, resulted in preserved contractility, attenuated ventricular remodeling, diminished fibrosis, and vascularization within the injured tissue in the adult murine heart.
View details for DOI 10.4161/bioe.27751
View details for PubMedID 24637710
Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation.
View details for DOI 10.1016/j.biomaterials.2013.09.092
View details for PubMedID 24161166
Skin cancer is the leading cause of malignancy in the United States, with Basal Cell Carcinoma, Squamous Cell Carcinoma , and Melanoma being the three most common diagnoses, respectively. Squamous Cell Carcinoma (SCC) is a particular concern for patients suffering from Dystrophic Epidermolysis Bullosa (DEB), a disease that affects the production and function of collagen VII, a protein that forms the anchoring fibrils which bind the epidermis to the dermis. Patients with DEB suffer from chronic blistering and wounds that have impaired healing capabilities, often leading to the development of SCC and eventual mortality. Nanomedicine is playing an increasing role in the delivery of effective therapeutics to combat a wide range of diseases, including the imaging and treatment of SCC. In this review, we discuss the role of nanoparticles in the treatment of SCC with an emphasis on PLGA nanoparticles and SCCs found in patients suffering from DEB, and address recent patents that are pertinent to the development of novel nanomedical therapeutics.
View details for PubMedID 25506404
Pathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.In this study, we examined the impact of diabetes on the murine ASC niche, as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.Our results demonstrate that diabetes alters the ASC niche in situ, and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.Perturbations in specific cellular subpopulations, visible only on a single cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in diabetic patients may be limited, and that interventions to improve cell function before application are warranted.
View details for DOI 10.1186/scrt468
View details for PubMedID 24943716
Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.
View details for DOI 10.1016/j.biomaterials.2013.08.017
View details for PubMedID 23992980
In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34(+) cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.
View details for DOI 10.1371/journal.pone.0077112
View details for Web of Science ID 000325552200103
View details for PubMedCentralID PMC3793009
Pulmonary hypertension (PH) is a serious condition that affects mainly young and middle-aged women, and its etiology is poorly understood. A prominent pathological feature of PH is accumulation of macrophages near the arterioles of the lung. In both clinical tissue and the SU5416 (SU)/athymic rat model of severe PH, we found that the accumulated macrophages expressed high levels of leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme for leukotriene B4 (LTB4). Moreover, macrophage-derived LTB4 directly induced apoptosis in pulmonary artery endothelial cells (PAECs). Further, LTB4 induced proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We found that LTB4 acted through its receptor, BLT1, to induce PAEC apoptosis by inhibiting the protective endothelial sphingosine kinase 1 (Sphk1)-endothelial nitric oxide synthase (eNOS) pathway. Blocking LTA4H decreased in vivo LTB4 levels, prevented PAEC apoptosis, restored Sphk1-eNOS signaling, and reversed fulminant PH in the SU/athymic rat model of PH. Antagonizing BLT1 similarly reversed established PH. Inhibition of LTB4 biosynthesis or signal transduction in SU-treated athymic rats with established disease also improved cardiac function and reopened obstructed arterioles; this approach was also effective in the monocrotaline model of severe PH. Human plexiform lesions, one hallmark of PH, showed increased numbers of macrophages, which expressed LTA4H, and patients with connective tissue disease-associated pulmonary arterial hypertension exhibited significantly higher LTB4 concentrations in the systemic circulation than did healthy subjects. These results uncover a possible role for macrophage-derived LTB4 in PH pathogenesis and identify a pathway that may be amenable to therapeutic targeting.
View details for Web of Science ID 000323705100010
An unmet need exists for the development of next-generation multifunctional nanocomposite materials for biomedical applications, particularly in the field of cardiovascular regenerative biology. Herein, we describe the preparation and characterization of a novel polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer with covalently attached anti-CD34 antibodies to enhance capture of circulating endothelial progenitor cells (EPC). This material may be used as a new coating for bare metal stents used after balloon angioplasty to improve re-endothelialization. Biophysical characterization techniques were used to assess POSS-PCU and its subsequent functionalization with anti-CD34 antibodies. Results indicated successful covalent attachment of anti-CD34 antibodies on the surface of POSS-PCU leading to an increased propensity for EPC capture, whilst maintaining in vitro biocompatibility and hemocompatibility. POSS-PCU has already been used in 3 first-in-man studies, as a bypass graft, lacrimal duct and a bioartificial trachea. We therefore postulate that its superior biocompatibility and unique biophysical properties would render it an ideal candidate for coating medical devices, with stents as a prime example. Taken together, anti-CD34 functionalized POSS-PCU could form the basis of a nano-inspired polymer platform for the next generation stent coatings.
View details for DOI 10.1186/1559-4106-8-23
View details for Web of Science ID 000325084800001
View details for PubMedCentralID PMC3979469
We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.
View details for DOI 10.1016/j.actbio.2013.04.045
View details for PubMedID 23648574
View details for DOI 10.1007/s00044-012-0386-2
View details for Web of Science ID 000320673200045
The advent of optogenetics provides a new direction for the field of neuroscience and biotechnology, serving both as a refined investigative tool and as potential cure for many medical conditions via genetic manipulation. Although still in its infancy, recent advances in optogenetics has made it possible to remotely manipulate in vivo cellular functions using light. Coined Nature Methods' 'Method of the Year' in 2010, the optogenetic toolbox has the potential to control cell, tissue and even animal behaviour. This optogenetic toolbox consists of light-sensitive proteins that are able to modulate membrane potential in response to light. Channelrhodopsins (ChR) are light-gated microbial ion channels, which were first described in green algae. ChR2 (a subset of ChR) is a seven transmembrane α helix protein, which evokes membrane depolarization and mediates an action potential upon photostimulation with blue (470nm) light. By contrast to other seven-transmembrane proteins that require second messengers to open ion channels, ChR2 form ion channels themselves, allowing ultrafast depolarization (within 50milliseconds of illumination). It has been shown that integration of ChR2 into various tissues of mice can activate neural circuits, control heart muscle contractions, and even restore breathing after spinal cord injury. More compellingly, a plethora of evidence has indicated that artificial expression of ChR2 in retinal ganglion cells can reinstate visual perception in mice with retinal degeneration.
View details for DOI 10.1016/j.nbt.2013.04.007
View details for Web of Science ID 000321695200006
View details for PubMedID 23664865
Cardiovascular disease is one of the major causes of death in the world. Coronary stenting in percutaneous coronary intervention (PCI) has revolutionized the field of cardiology. Coronary stenting is seen as a less invasive procedure compared to coronary artery bypass graft (CABG) surgery. Two main types of stents currently exist in the market: bare-metal stents (BMS) and drug-eluting stents (DES). DES were developed in response to problems associated with BMS use, like neointimal hyperplasia leading to restenosis. However, the use of DES engendered other problems as well, like late stent thrombosis (ST), which is a serious and lethal complication. Gene-eluting stents (GES) have recently been proposed as a novel method of circumventing problems seen in BMS and DES. Utilizing nanotechnology, sustained and localized delivery of genes can mitigate problems of restenosis and late ST by accelerating the regenerative capacity of re-endothelialization. Therefore this review seeks to explore the realm of GES as a novel alternative to BMS and DES, and its potential implications in the field of nanotechnology and regenerative medicine.
View details for DOI 10.1021/mp3006616
View details for Web of Science ID 000317094100014
View details for PubMedID 23394068
Percutaneous coronary intervention (PCI) is used to treat blocked coronary arteries. Bare-metal stents (BMS) were first used in PCI but often necessitated repair procedures due to in-stent restenosis. Drug-eluting stents (DES) were developed to address this problem as the stent-incorporated anti-proliferative drugs prevented restenosis. However late-stent thrombosis arose with the use of DES due to polymer hypersensitivity and impaired re-endothelialization. Evidence suggests that using a combination of biofunctionalized polymers and antibody/peptide motifs can prevent thrombosis while ensuring in situ endothelialization. The advent of nanotechnology has engendered techniques like layer-by-layer self-assembly, and localized drug and gene delivery using nanoparticles. Therefore, this review seeks to explore the convergence of biotechnology and nanotechnology for the next generation coronary stent coatings, with an emphasis on its development from bench to beside.
View details for DOI 10.1016/j.jbiotec.2013.01.020
View details for Web of Science ID 000315527900022
View details for PubMedID 23376617
Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aβ, and decreased clearance of Aβ from the central nervous system (CNS). We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.
View details for DOI 10.1371/journal.pone.0058194
View details for Web of Science ID 000318334500058
View details for PubMedCentralID PMC3591408
Exosomes are biological membrane vesicles measuring 30 to 100 nm. They contain an abundance of small molecules like tetraspanins, receptors for targeting and adhesion, lipids, and RNA. They are secreted by most biological cells, and are involved in a plethora of physiological functions including, but not limited to, transport of genetic material, modulation of the immune system, and cell-to-cell communication. It has been further reported that exosomes utilize a mechanism similar to that of viruses for gaining entry into cells. Due to their viral-like transfection efficiency and inherent biological function, compelling evidence indicates that exosomes can be used as novel delivery platforms for gene therapy. Furthermore, RNA-containing exosomes derived from cells can serve as functional genetic biomarkers for diseases. This twin modality of therapeutic and diagnostic is termed theranostics in the emerging field of nanomedicine. Hence in this review, we seek to expound on the various facets of exosomes, highlighting their significance in and relevance to nano-theranostic platforms for gene therapy.
View details for DOI 10.1016/j.addr.2012.06.014
View details for Web of Science ID 000317324100006
View details for PubMedID 22820532
The soluble Abeta oligomers in brain are highly correlated with memory related synaptic dysfunctions in Alzheimer's disease (AD). However, more recent studies implicate the involvement of Abeta dimers and trimers in memory related AD pathology. Apparently, Abeta oligomers can bind with cellular prion protein at the membrane receptors, forming annular amyloid pores and membrane ion channels to induce aberrant spine cytoskeletal changes. Hence synapse targeting of Abeta oligomers involves activation of many receptors such as N-Methyl-D-aspartate (NMDA), alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nicotinic acetylcholine (nAChRs), p75 neurotrophin (p75NTR) following aberrant clustering of metabotropic glutamate receptors (mGluR5) leading to neuronal loss and LTP failure. In particular, NMDA and AMPA receptor activation by soluble amyloid oligomers involves calcium mediated mitochondrial dysfunction, decreased Ca((2+))/calmodulin-dependent protein kinase II (CaMKII) levels at the synapses accompanying dramatic loss of synaptic proteins such as postsynaptic density-95 (PSD-95), dynamin-1 and synaptophysin. This kind of receptor-Abeta oligomer interaction might eventually affect the neuronal membrane integrity by altering dielectric barrier, various synaptic proteins, spine morphology and density and P/Q calcium currents that might provoke a cascade of events leading to neuronal loss and memory failure. In this review, we try to explain in detail the various possible mechanisms that connect Abeta oligomers with synapse damage and memory failure.
View details for Web of Science ID 000317272900011
View details for PubMedID 23036017
The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu(2+) ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu(2+) supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu(2+) ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu(2+) ions, owing to binding affinity of copper towards histidine moiety present in the peptide.
View details for DOI 10.1371/journal.pone.0085160
View details for PubMedID 24386462
Peripheral nerve injury is a debilitating condition. The gold standard for treatment is surgery, requiring an autologous nerve graft. Grafts are harvested from another part of the body (a secondary site) to treat the affected primary area. However, autologous nerve graft harvesting is not without risks, with associated problems including injury to the secondary site. Research into biomaterials has engendered the use of bioartificial nerve conduits as an alternative to autologous nerve grafts. These include synthetic and artificial materials, which can be manufactured into nerve conduits using techniques inspired by nanotechnology. Recent evidence indicates that peptide amphiphiles (PAs) are promising candidates for use as materials for bioengineering nerve conduits. PAs are biocompatible and biodegradable protein-based nanomaterials, capable of self-assembly in aqueous solutions. Their self-assembly system, coupled with their intrinsic capacity for carrying bioactive epitopes for tissue regeneration, form particularly novel attributes for biochemically-engineered materials. Furthermore, PAs can function as biomimetic materials and advanced drug delivery platforms for sustained and controlled release of a plethora of therapeutic agents. Here we review the realm of nerve conduit tissue engineering and the potential for PAs as viable materials in this exciting and rapidly advancing field.
View details for DOI 10.1016/j.jconrel.2012.08.009
View details for Web of Science ID 000312266500009
View details for PubMedID 22910143
To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.
View details for DOI 10.1074/jbc.M112.371229
View details for PubMedID 22955287
The application of nanotechnology in biology and medicine represents a significant paradigm shift in the approach to the treatment of cancer. Evidence suggests that when exposed to near-infrared radiation (NIR), carbon nanotubes (CNTs) dissipate a substantial amount of heat energy. We have developed a novel nanocomposite polymer, polyhedral oligomeric silsesquioxane poly (carbonate-urea) urethane (POSS-PCU). POSS-PCU displays excellent biocompatibility and has been used in making artificial organs as well as protective coatings for medical devices.Functionalizing (or "coating") CNTs with POSS-PCU confers biocompatibility and increase the amount of heat energy generated, by enhancing dispersion. Here we demonstrate that POSS-PCU-functionalized multi-walled CNTs (MWNTs) act synergistically together when exposed to NIR to thermally ablate cancer cells.Given that POSS-PCU has already been used in human in first-in-man studies as trachea, lacrimal duct, bypass graft and other organs, our long-term goal is to take POSS-PCU coated CNTs to clinical studies to address the treatment of cancer by optimizing its therapeutic index and increasing its specificity via antibody conjugation.
View details for DOI 10.1186/1477-3155-10-34
View details for Web of Science ID 000310031500001
View details for PubMedID 22849373
Impaired wound healing in diabetes is a well-documented phenomenon. Emerging data favor the involvement of free radicals in the pathogenesis of diabetic wound healing. We investigated the beneficial role of the sustained release of reactive oxygen species (ROS) in diabetic dermal wound healing. In order to achieve the sustained delivery of ROS in the wound bed, we have incorporated glucose oxidase in the collagen matrix (GOIC), which is applied to the healing diabetic wound. Our in vitro proteolysis studies on incorporated GOIC show increased stability against the proteases in the collagen matrix. In this study, GOIC film and collagen film (CF) are used as dressing material on the wound of streptozotocin-induced diabetic rats. A significant increase in ROS (p < 0.05) was observed in the fibroblast of GOIC group during the inflammation period compared to the CF and control groups. This elevated level up regulated the antioxidant status in the granulation tissue and improved cellular proliferation in the GOIC group. Interestingly, our biochemical parameters nitric oxide, hydroxyproline, uronic acid, protein, and DNA content in the healing wound showed that there is an increase in proliferation of cells in GOIC when compared to the control and CF groups. In addition, evidence from wound contraction and histology reveals faster healing in the GOIC group. Our observations document that GOIC matrices could be effectively used for diabetic wound healing therapy.
View details for DOI 10.1177/0885328210390402
View details for Web of Science ID 000303649700002
View details for PubMedID 21363874
Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Accumulating evidence supports soluble amyloid-β (Aβ) oligomers as the leading candidate for the causative agent in AD and synapses as the primary site of Aβ oligomer action. However, the molecular and cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and cognitive impairments remain poorly understood. Using primary cultures of rat hippocampal neurons as a model system, we show that the partitioning defective-1 (PAR-1)/microtubule affinity-regulating kinase (MARK) family kinases act as critical mediators of Aβ toxicity on synapses and dendritic spines. Overexpression of MARK4 led to tau hyperphosphorylation, reduced expression of synaptic markers, and loss of dendritic spines and synapses, phenotypes also observed after Aβ treatment. Importantly, expression of a non-phosphorylatable form of tau with the PAR-1/MARK site mutated blocked the synaptic toxicity induced by MARK4 overexpression or Aβ treatment. To probe the involvement of endogenous MARK kinases in mediating the synaptic toxicity of Aβ, we employed a peptide inhibitor capable of effectively and specifically inhibiting the activities of all PAR-1/MARK family members. This inhibitor abrogated the toxic effects of Aβ oligomers on dendritic spines and synapses as assayed at the morphological and electrophysiological levels. Our results reveal a critical role for PAR-1/MARK kinases in AD pathogenesis and suggest PAR-1/MARK inhibitors as potential therapeutics for AD and possibly other tauopathies where aberrant tau hyperphosphorylation is involved.
View details for DOI 10.1093/hmg/ddr576
View details for Web of Science ID 000300721300015
View details for PubMedID 22156579
View details for PubMedCentralID PMC3284124
View details for DOI 10.1007/s10989-011-9278-4
View details for Web of Science ID 000299525400006
In this study, we examined the capacity of a biomimetic pullulan-collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.
View details for DOI 10.1016/j.biomaterials.2011.09.041
View details for Web of Science ID 000297399700009
View details for PubMedID 21963148
Substantial evidence suggests that the aggregation of amyloid-β (Aβ) peptide into fibrillar structures that is rich in β-sheets is implicated as the cause of Alzheimer's disease. Therefore, an attractive therapeutic strategy is to prevent or alter Aβ aggregation. Phenolic compounds are natural substances that are composed of one or more aromatic phenolic rings and present in wine, tea, fruits, vegetables and a wide variety of plants. In this work, we investigated the effects of ferulic acid, morin, quercetin and gossypol against Aβ aggregation. From the ThT and turbidity assays, it is observed that in addition to the fibril aggregate, another type of aggregate is formed in the presence of morin, quercetin, and gossypol. On the other hand, ferulic acid did not prevent fibril formation, but it did appear to reduce the average length of fibrils compared to Aβ alone. To study the protective effects of phenolic compounds on Aβ-induced toxicity, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism, human Aβ is expressed intracellularly in the body wall muscle. We found that exposure of Caenorhabditis elegans to ferulic acid give more protection against Aβ toxicity than morin, quercetin and gossypol.
View details for DOI 10.1007/s11064-011-0580-5
View details for Web of Science ID 000302404000007
View details for PubMedID 21858698
Stem cell-based therapies offer tremendous potential for skin regeneration following injury and disease. Functional stem cell units have been described throughout all layers of human skin and the collective physical and chemical microenvironmental cues that enable this regenerative potential are known as the stem cell niche. Stem cells in the hair follicle bulge, interfollicular epidermis, dermal papillae, and perivascular space have been closely investigated as model systems for niche-driven regeneration. These studies suggest that stem cell strategies for skin engineering must consider the intricate molecular and biologic features of these niches. Innovative biomaterial systems that successfully recapitulate these microenvironments will facilitate progenitor cell-mediated skin repair and regeneration.
View details for DOI 10.1155/2012/926059
View details for PubMedID 22701121
Amyloid diseases occur due to conformational change in the native protein. Understanding the amyloid peptide structural stability and conformational preference at the molecular level in membranous environment may lead to advancement in drug design and therapy. The conformational preferences of amyloid peptide fragments, Aβ₁₋₁₁, Aβ₁₂₋₂₂, Aβ₂₃₋₃₃ and Aβ₃₄₋₄₂ was studied in buffers, trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS) micelles using circular dichroism spectroscopy. The fragment, Aβ₁₋₁₁ in TFE adopts a mixture of random coil and turn conformations. Aβ₁₂₋₂₂ and Aβ₂₃₋₃₃ underwent transition from random coil to helix conformation, while Aβ₃₄₋₄₂ exhibited β-sheet conformation in initial stage which was unaltered on complete evaporation of TFE. Addition of SDS to Aβ₁₂₋₂₂ and Aβ₃₄₋₄₂ favors β-sheet structure, which was predominant in the case of Aβ₃₄₋₄₂. However, in Aβ₁₋₁₁ and Aβ₂₃₋₃₃, no secondary structural change was noticed even at high SDS concentrations. On aging, all the peptide fragments showed β-sheet conformational transition. The C-terminal fragment has the ability to adopt predominant β-sheet conformation even in the presence of detergent and membrane mimicking environment. Altogether, the structural information gained from the short fragments could be further used for determining their role in the organization of Aβ peptide in stable fibril form.
View details for DOI 10.1016/j.npep.2011.09.001
View details for Web of Science ID 000297875400002
View details for PubMedID 22019176
Cell-based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from the acute inflammatory environment. Here, a biomimetic hydrogel system is described that is based on the polymer pullulan, a carbohydrate glucan known to exhibit potent antioxidant capabilities. It is shown that pullulan hydrogels are an effective cell delivery system and improve mesenchymal stem cell survival and engraftment in high-oxidative-stress environments. The results suggest that glucan hydrogel systems may prove beneficial for progenitor-cell-based approaches to skin regeneration.
View details for DOI 10.1002/mabi.201100180
View details for Web of Science ID 000297555500002
View details for PubMedID 21994074
Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.
View details for DOI 10.1002/jbm.a.33113
View details for PubMedID 21630430
Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.
View details for DOI 10.1002/jbm.a.33113
View details for Web of Science ID 000293699800007
Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.
View details for DOI 10.1038/nm.2424
View details for Web of Science ID 000294605100038
View details for PubMedID 21873986
Cancer is one of the main causes of death in the world, and according to the WHO it is projected to continue rising. Current diagnostic modalities for the detection of cancer include the use of x-rays, magnetic resonance imaging and positron emission tomography, among others. The treatment of cancer often involves the use (or combination) of chemotherapeutic drugs, radiotherapy and interventional surgery (for solid and operable tumors). The application of nanotechnology in biology and medicine is advancing rapidly. Recent evidence suggests that quantum dots (QDs) can be used to image cancer cells as they display superior fluorescent properties compared with conventional chromophores and contrast agents. In addition, carbon nanotubes (CNTs) have emerged as viable candidates for novel chemotherapeutic drug delivery-platforms. The unique photothermal properties of CNTs also allow them to be used in conjunction with near infrared radiation and lasers to thermally ablate cancer cells. Furthermore, mounting evidence indicates that it is possible to conjugate QDs to CNTs, making it possible to exploit their novel attributes in the realm of cancer theranostics (diagnostics and therapy). Here we review the current literature pertaining to the applications of QDs and CNTs in oncology, and also discuss the relevance and implications of nanomedicine in a clinical setting.
View details for DOI 10.2217/NNM.11.64
View details for Web of Science ID 000295697600019
View details for PubMedID 21955079
Enhanced production of a 42-residue beta amyloid peptide (Aβ(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ(42) peptide (Aβ(42)Nle35p37) that appear to organize Aβ(42) into stable oligomers. 2D NMR on the Aβ(42)Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.
View details for DOI 10.1371/journal.pone.0021776
View details for Web of Science ID 000293097300006
View details for PubMedID 21799748
View details for PubMedCentralID PMC3142112
Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.
View details for DOI 10.1016/j.biomaterials.2011.03.016
View details for Web of Science ID 000291193700019
View details for PubMedID 21477858
View details for PubMedCentralID PMC3090516
New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.
View details for DOI 10.1089/ten.tea.2010.0298
View details for Web of Science ID 000287801600005
View details for PubMedID 20919949
To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.
View details for DOI 10.1016/j.actbio.2010.06.033
View details for Web of Science ID 000284385300018
View details for PubMedID 20601236
View details for PubMedCentralID PMC2957527
Conformational transition of soluble monomeric amyloid beta-peptide (Abeta) into oligomeric and protofibrillar aggregates plays a key role in the pathogenesis of Alzheimer's disease (AD). One of the central questions surrounding the molecular pathophysiology of AD is how the soluble Abeta is converted into its aggregated toxic form. A more detailed understanding of the conformational transitions involved in the self-assembly of Abeta may facilitate the design of inhibitors of aggregation. In this study, we evaluated the wild-type (WT) Abeta 16-28 peptide (KLVFFAEDVGSNK) and its associated mutants, including A21G (Flemish), E22K (Italian), E22Q (Dutch), and E22G (Arctic) mutants, by examining, in particular, their aggregation kinetics in the presence and in the absence of negatively charged and zwitterionic lipids. Circular dichroic and thioflavin T fluorescence studies indicated that the WT peptide undergoes a rapid conformational transition into beta-sheet structure in solution, whereas the Arctic and Dutch variants show a markedly rapid transition into beta-sheet structure in the presence of negatively charged lipids. These results provide strong evidence suggesting that the reduction in net charge, with a concurrent increase in the net hydrophobicity of the peptide alone or when complexed with lipid in solution, determines the rate of aggregate formation.
View details for DOI 10.1007/s12031-010-9380-7
View details for Web of Science ID 000278155000005
View details for PubMedID 20480256
Folding and self-assembly of amyloid beta (Abeta) peptide are linked to Alzheimer's disease. To understand the initial stage of amyloid-beta peptide aggregation, conformational characteristics of monomers of wild-type (WT) Abeta(28-35) and its mutant peptides A30G and A30I were investigated using density functional theory calculations and experimental studies. Monomeric structures and their relative stabilities were obtained on the basis of systematic structural optimization in the gas-phase and in the aqueous medium. Computations were performed by hybrid Hartree-Fock-Density Functional Theory (HF-DFT) at B3LYP/6-31G * level. Experimentally, the conformational transitions in the early stages of the octapeptide Abeta(28-35) and its mutants A30G and A30I in solution were characterized by CD, Thioflavin assay and FRET spectroscopy. Examination of the secondary structures revealed that Abeta(28-35) and its mutant monomers exist in random coil conformation in the aqueous medium in agreement with the theoretical predictions, which upon aging is transformed to sheet with different kinetics. This study deals with the structurally important intermediates and it may help to understand the mechanism of amyloid fibril aggregation leading to the onset of Alzheimer's disease.
View details for DOI 10.1016/j.jsb.2010.02.017
View details for Web of Science ID 000277946200002
View details for PubMedID 20188180
Brain-derived neurotrophic factor (BDNF) activates the receptor tropomyosin-related kinase B (TrkB) with high potency and specificity, promoting neuronal survival, differentiation, and synaptic function. Correlations between altered BDNF expression and/or function and mechanism(s) underlying numerous neurodegenerative conditions, including Alzheimer disease and traumatic brain injury, suggest that TrkB agonists might have therapeutic potential. Using in silico screening with a BDNF loop-domain pharmacophore, followed by low-throughput in vitro screening in mouse fetal hippocampal neurons, we have efficiently identified small molecules with nanomolar neurotrophic activity specific to TrkB versus other Trk family members. Neurotrophic activity was dependent on TrkB and its downstream targets, although compound-induced signaling activation kinetics differed from those triggered by BDNF. A selected prototype compound demonstrated binding specificity to the extracellular domain of TrkB. In in vitro models of neurodegenerative disease, it prevented neuronal degeneration with efficacy equal to that of BDNF, and when administered in vivo, it caused hippocampal and striatal TrkB activation in mice and improved motor learning after traumatic brain injury in rats. These studies demonstrate the utility of loop modeling in drug discovery and reveal what we believe to be the first reported small molecules derived from a targeted BDNF domain that specifically activate TrkB.We propose that these compounds constitute a novel group of tools for the study of TrkB signaling and may provide leads for developing new therapeutic agents for neurodegenerative diseases.
View details for DOI 10.1172/JCI41356
View details for Web of Science ID 000277248000041
View details for PubMedID 20407211
View details for PubMedCentralID PMC2860903
A new method for creating substrates made out of ordered collagen fibers, on which cells in culture can align, is proposed. The substrates can be used for research in cell culture, and this research presents a significant advance in the technology to coat implants in order to improve cell adhesion. In the procedure presented here, a molecular solution of collagen is spread at the interface of a saline solution and air to induce fiber formation, compressed at a high speed to induce orientation and deposited on solid substrates via Langmuir-Blodgett transfer. Several interfacial techniques are employed to investigate the behavior of collagen, which is shown to be dependent on the salt concentration of the subphase as well as the temperature. After Langmuir-Blodgett transfer, primary human fibroblasts and adipose-derived stem cells are cultured on the collagen substrates. Both types of cells respond favorably to the collagen orientation and align with the deposited fibers. The technique presented here provides a simple method to produce well-controlled, oriented collagen substrates that can be used in tissue culture research or scaffolding applications without the use of additives and/or bioincompatible materials.
View details for DOI 10.1021/la9031317
View details for Web of Science ID 000274636900079
View details for PubMedID 20000428
Microglia clear amyloid beta (Abeta) after immunization. The interaction of Abeta with the microglial cell surface also results in cytokine expression. Soluble oligomers and protofibrils of Abeta may be more neurotoxic than Abeta fibrils. We investigated the effects of oligomeric, protofibrillar and fibrillar Abeta40 and Abeta42 peptides on uptake and IL-1alpha expression by primary microglia. Abeta peptide assemblies were extensively characterized. Primary microglial cells were exposed to different Abeta40 and Abeta42 assemblies and IL-1alpha expression was quantified. To study uptake, microglial cells were exposed to different assemblies of Cy3-labeled Abeta. We found that Abeta42 and Abeta40 oligomers and fibrils induced IL-1alpha expression, but protofibrils did not. We also observed that all forms of Abeta42 (oligomer, protofibril and fibril) and Abeta40 fibrils were taken up by the microglial cells. These results demonstrate that microglial cells can take up non-fibrillar Abeta and that oligomeric peptide induces an inflammatory response. The uptake of oligomeric and protofibrillar Abeta by microglia merits further investigation as a potential means for removing these neurotoxic species from the brain.
View details for DOI 10.1016/j.neurobiolaging.2008.01.011
View details for PubMedID 18339452
View details for Web of Science ID 000269755300171
Oligomeric forms of amyloid-beta (Abeta) are thought to play a causal role in Alzheimer's disease (AD), and the p75 neurotrophin receptor (p75(NTR)) has been implicated in Abeta-induced neurodegeneration. To further define the functions of p75(NTR) in AD, we examined the interaction of oligomeric Abeta(1-42) with p75(NTR), and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Abeta, and fluorescence resonance energy transfer analysis revealed that Abeta oligomers interact with the extracellular domain of p75(NTR). In vitro studies of Abeta-induced death in neuron cultures isolated from wild-type and p75(NTR-/-) mice, in which the p75(NTR) extracellular domain is deleted, showed reduced sensitivity of mutant cells to Abeta-induced cell death. Interestingly, Abeta-induced neuritic dystrophy and activation of c-Jun, a known mediator of Abeta-induced deleterious signaling, were completely prevented in p75(NTR-/-) neuron cultures. Thy1-hAPP(Lond/Swe) x p75(NTR-/-) mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild-type p75(NTR). Abeta levels were not affected, suggesting that removal of p75(NTR) extracellular domain reduced the ability of excess Abeta to promote neuritic degeneration. These findings indicate that although p75(NTR) likely does not mediate all Abeta effects, it does play a significant role in enabling Abeta-induced neurodegeneration in vitro and in vivo, establishing p75(NTR) as an important therapeutic target for AD.
View details for DOI 10.1523/JNEUROSCI.0620-09.2009
View details for PubMedID 19710315
During the course of cancer radiation treatment, normal skin invariably suffers from the cytotoxic effects of gamma-radiation and reactive oxygen species (ROS), which are generated from the interaction between radiation and the water molecules in cells. The present study was designed to investigate the radioprotective role of alpha-lipoic acid (LA), an antioxidant on murine skin fibroblasts exposed to a single dose of 2, 4, 6, or 8Gy gamma-radiation. Irradiation of fibroblasts significantly increased ROS, nitric oxide, and lipid peroxidation (P < 0.001); all of these factors substantially decreased with 100 microM LA treatment. Hydroxyl radical (OH(.)) production from 8Gy irradiated fibroblasts was measured directly by electron spin resonance using spin-trapping techniques. LA was found to inhibit OH(.) production at 100-microM concentrations. Dose-dependent depletion of antioxidants, such as catalase and glutathione reductase, was observed in irradiated fibroblasts (P < 0.001), along with increased superoxide dismutase (P < 0.001). LA treatment restored antioxidant levels. Concentration of the pro-inflammatory cytokine IL-1beta was significantly reduced in irradiated fibroblasts when treated with LA. MTT and lactate dehydrogenase assays demonstrated that LA treatment reduced cell injury and protected cells against irradiation-induced cytotoxicity. Thus, we conclude that results are encouraging and need further experiments to demonstrate a possible benefit in cancer patients and the reduction of harmful effects of radiation therapy.
View details for DOI 10.1007/s10565-008-9087-5
View details for Web of Science ID 000267293600003
View details for PubMedID 18553143
A number of distinct beta-amyloid (Abeta) variants or multimers have been implicated in Alzheimer's disease (AD), and antibodies recognizing such peptides are in clinical trials. Humans have natural Abeta-specific antibodies, but their diversity, abundance, and function in the general population remain largely unknown. Here, we demonstrate with peptide microarrays the presence of natural antibodies against known toxic Abeta and amyloidogenic non-Abeta species in plasma samples and cerebrospinal fluid of AD patients and healthy controls aged 21-89 years. Antibody reactivity was most prominent against oligomeric assemblies of Abeta and pyroglutamate or oxidized residues, and IgGs specific for oligomeric preparations of Abeta1-42 in particular declined with age and advancing AD. Most individuals showed unexpected antibody reactivities against peptides unique to autosomal dominant forms of dementia (mutant Abeta, ABri, ADan) and IgGs isolated from plasma of AD patients or healthy controls protected primary neurons from Abeta toxicity. Aged vervets showed similar patterns of plasma IgG antibodies against amyloid peptides, and after immunization with Abeta the monkeys developed high titers not only against Abeta peptides but also against ABri and ADan peptides. Our findings support the concept of conformation-specific, cross-reactive antibodies that may protect against amyloidogenic toxic peptides. If a therapeutic benefit of Abeta antibodies can be confirmed in AD patients, stimulating the production of such neuroprotective antibodies or passively administering them to the elderly population may provide a preventive measure toward AD.
View details for DOI 10.1073/pnas.0904866106
View details for Web of Science ID 000268178400059
View details for PubMedID 19581601
View details for PubMedCentralID PMC2715538
Alzheimer's disease (AD) is caused by the deposition of beta-amyloid (Abeta) protein in brain. The current AD immunotherapy aims to prevent Abeta plaque deposition and enhance its degradation in the brain. In this work, the peptides B-cell epitope Abeta(1-12), T-cell epitope Abeta(29-40) and full-length Abeta(1-42) were loaded separately to the poly (D,L-lactide co-glycolide) (PLG) microparticles by using W/O/W double emulsion solvent evaporation method with entrapment efficacy of 70.46%, 60.93%, and 65.98%, respectively. The prepared Abeta PLG microparticles were smooth, spherical, individual, and nonporous in nature with diameters ranging from 2 to 12 microm. The cumulative in vitro release profiles of Abeta(1-12), Abeta(29-40), and Abeta(1-42) from PLG microparticles sustained for long periods and progressively reached to 73.89%, 69.29%, and 70.08% by week 15. In vitro degradation studies showed that the PLG microparticles maintained the surface integrity up to week 8 and eroded completely by week 16. Oral immunization of Abeta peptides loaded microparticles in mice elicited stronger immune response by inducing anti-Abeta antibodies for prolonged time (24 weeks). The physicochemical characterization and immunogenic potency of Abeta peptides incorporated PLG microparticles suggest that the microparticles formulation of Abeta can be a potential oral AD vaccine.
View details for DOI 10.1002/jps.21600
View details for Web of Science ID 000266572600012
View details for PubMedID 18980172
The principal component of the amyloid deposits in Alzheimer's disease is the beta-amyloid polypeptide, while in type II diabetes the deposits consist primarily of Islet amyloid polypeptide. These amyloid forming polypeptides consist of highly polymorphic domains, which take different conformations including random coil, helical and beta strand depending upon the microenvironment. We have studied major fibril-forming components of IAPP and beta AP and demonstrated that conformational polymorphism of these peptides in different microenvironments correlate with cellular toxicity and proteasomal inhibitory activity. On treating with trifluoroethanol (TFE) the peptide fragments undergo structural transition from a random coil to a helical conformation. Even though these domains share the same gross amyloid structural characteristic, their proteasomal activities differ. We found that even the tetrapeptides have significant proteasomal inhibitory activity indicating that the amyloid formation is involved in the enhanced life of the smaller aggregates of full-length and fragment peptides, which could explain the toxicity of these sequences.
View details for DOI 10.1016/j.jsb.2008.12.011
View details for Web of Science ID 000265560900002
View details for PubMedID 19374013
A novel series of N-Mannich bases of benzimidazole derivatives were synthesized and characterized by (1)H NMR, IR spectral studies and elemental analysis. The compounds were screened for analgesic and anti-inflammatory activity. 1-((Diethylamino)-methyl)-2-styryl benzimidazole 4 at 40mg/kg was found to be equipotent to paracetamol. 1-((Piperidin-1-yl) methyl)-2-styryl-benzimidazole 6 at 40mg/kg was found to be more potent than Diclofenac. Corneal permeability and quantum chemical calculations were performed to correlate the hydrogen bonding ability with permeability and activity. The energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were correlated with pharmacological activity. The semi-empirical PM3 calculations (quantum chemical calculations) revealed that E(LUMO) and energy gap DeltaE were capable of accounting for the high in vitro bovine corneal permeability and activity of the compounds.
View details for DOI 10.1016/j.ejmech.2008.03.043
View details for Web of Science ID 000265339900062
View details for PubMedID 18486995
Accumulating evidence suggests that Abeta(1-42)-membrane interactions may play an important role in the pathogenesis of Alzheimer's disease. However, the mechanism of this structural transition remains unknown. In this work, we have shown that submicellar concentrations of sodium dodecyl sulfate (SDS) can provide a minimal platform for Abeta(1-42) self-assembly. To further investigate the relation between Abeta(1-42) structure and function, we analyzed peptide conformation and aggregation at various SDS concentrations using circular dichroism (CD), Fourier transform infrared spectroscopy, and gel electrophoresis. These aggregates, as observed via atomic force microscopy, appeared as globular particles in submicellar SDS with diameters of 35-60 nm. Upon sonication, these particles increased in disc diameter to 100 nm. Pyrene I (3)/I (1) ratios and 1-anilinonaphthalene-8-sulfonic acid binding studies indicated that the peptide interior is more hydrophobic than the SDS micelle interior. We have also used Forster resonance energy transfer between N-terminal labeled pyrene and tyrosine (10) of Abeta(1-42) in various SDS concentrations for conformational analysis. The results demonstrate that SDS at submicellar concentrations accelerates the formation of spherical aggregates, which act as niduses to form large spherical aggregates upon sonication.
View details for DOI 10.1007/s00249-008-0379-8
View details for Web of Science ID 000264263100002
View details for PubMedID 19005650
The p75 neurotrophin receptor (p75(NTR)) is expressed by neurons particularly vulnerable in Alzheimer's disease (AD). We tested the hypothesis that non-peptide, small molecule p75(NTR) ligands found to promote survival signaling might prevent Abeta-induced degeneration and synaptic dysfunction. These ligands inhibited Abeta-induced neuritic dystrophy, death of cultured neurons and Abeta-induced death of pyramidal neurons in hippocampal slice cultures. Moreover, ligands inhibited Abeta-induced activation of molecules involved in AD pathology including calpain/cdk5, GSK3beta and c-Jun, and tau phosphorylation, and prevented Abeta-induced inactivation of AKT and CREB. Finally, a p75(NTR) ligand blocked Abeta-induced hippocampal LTP impairment. These studies support an extensive intersection between p75(NTR) signaling and Abeta pathogenic mechanisms, and introduce a class of specific small molecule ligands with the unique ability to block multiple fundamental AD-related signaling pathways, reverse synaptic impairment and inhibit Abeta-induced neuronal dystrophy and death.
View details for DOI 10.1371/journal.pone.0003604
View details for Web of Science ID 000265134200003
View details for PubMedID 18978948
View details for PubMedCentralID PMC2575383
Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.
View details for DOI 10.1016/j.bbapap.2008.07.015
View details for Web of Science ID 000261019000022
View details for PubMedID 18775521
AA (amyloid protein A) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an intravenous injection of protein extracted from AA-laden mouse tissue. Previous findings affirm that AA fibrils can enhance the in vivo amyloidogenic process by a nucleation seeding mechanism. Accumulating evidence suggests that globular aggregates rather than fibrils are the toxic entities responsible for cell death. In the present study we report on structural and morphological features of AEF (amyloid-enhancing factor), a compound extracted and partially purified from amyloid-laden spleen. Surprisingly, the chief amyloidogenic material identified in the active AEF was diffusible globular oligomers. This partially purified active extract triggered amyloid deposition in vital organs when injected intravenously into mice. This implies that such a phenomenon could have been inflicted through the nucleation seeding potential of toxic oligomers in association with altered cytokine induction. In the present study we report an apparent relationship between altered cytokine expression and AA accumulation in systemically inflamed tissues. The prevalence of serum AA monomers and proteolytic oligomers in spleen AEF is consistent to suggest that extrahepatic serum AA processing might lead to local accumulation of amyloidogenic proteins at the serum AA production site.
View details for DOI 10.1042/BJ20071696
View details for Web of Science ID 000260158400004
View details for PubMedID 18564059
Oxidative stress is one of the hypothesized pathogenic mechanisms for neurodegenerative diseases, including Alzheimer's disease (AD); numerous studies suggest that Abeta is toxic to neurons by free radical mediated mechanism. A constant feature in AD brain is selective neuronal loss, accompanied by dysfunction of several neurotransmitter systems, such as cholinergic, serotoninergic and noradrenergic systems. In the present study, we studied the neuroprotective role of melatonin against amyloid protofibrils and the toxicity of protofibrils on serotoninergic and noradrenergic systems. Mice were divided into four groups (n=8 each), control, Scrambles Abeta(35-25) treated, Abeta(25-35) injected, and melatonin treated. A single dose of Abeta(25-35) (25 microg) was administered to mice via intraperitoneal injection. Melatonin (50 mg/kg body weight) was administered intraperitoneally for 3 days to the Abeta(25-35) injected mice. Control mice received only physiological saline and Scrambles receives Abeta(35-25) single intraperitoneal injection of 25 microg of Abeta(35-25). Our study showed that melatonin significantly reduces reactive oxygen species (ROS) production in the astrocytes, lymphocytes and hepatocytes of Abeta injected mice by increasing the levels of scavenging enzymes, SOD, catalase and GSH when compared to the untreated group. Immunohistochemistry study reveals that melatonin prevents the activation of GFAP in neocortex and transcription factor NF-kappaB in liver and neocortex of Abeta injected mice. It also prevents the elevation of dopamine depletion and its degradation products. Thus, while melatonin may be a potential therapeutic agent in the prevention of oxidative stress associated with Abeta and AD, it can also prevent dopamine turnover induced by Abeta.
View details for DOI 10.1016/j.lfs.2008.05.011
View details for Web of Science ID 000257936100003
View details for PubMedID 18590917
Inflammation can activate macrophages or monocytes and sequentially release several inflammatory cytokines and reactive oxygen species (ROS). Oxidative stress-induced acute inflammatory response plays an important role in several diseases. This study was designed to investigate the prophylactic effect of the antioxidant lipoic acid (LA) during inflammation-induced mice. Mice were divided in to three groups (n = 8 in each): control, systemic inflammation, and LA treated mice with systemic inflammation. Results show that ROS was significantly higher in lymphocytes, hepatocytes, and astrocytes (P < 0.05) of inflammation induced mice when compared with control but no significant changes were observed in the LA treated group. Increased levels of lipid peroxidation (LPO) and decreased activities of oxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione, and ATPase were observed in the inflammation-induced mice, which returned to near normalcy following LA therapy. In vitro study has shown that LA treatment not only suppresses the increased LPO levels but also inhibits the lipid break down resulting from autoxidation. In addition, increased immunoreactivity of the astrocyte marker glial fibrillary acidic protein (GFAP) was observed in the neocortex region of inflammation-induced mice, whereas nuclear factor kappa B p65 (NFkappaB) immunoreactivity was observed in both the neocortex and liver of the same group which were effectively controlled by LA therapy suggesting that LA can efficiently manage systemic inflammation.
View details for DOI 10.1007/s11010-008-9748-y
View details for Web of Science ID 000256091000013
View details for PubMedID 18401559
Skeletal muscle disorder, inclusion-body myositis (IBM) has been known for accumulation of amyloid characteristic proteins in muscle. To understand the biophysical basis of IBM, the interaction of amyloid fibrils with skeletal myoblast cells (SMC) has been studied in vitro. Synthetic insulin fibrils and Abeta(25-35) fibrils were used for this investigation. From the saturation binding analysis, the calculated dissociation constant (K(d)) for insulin fibril and Abeta(25-35) fibrils were 69.37+/-11.17nM and 115.60+/-12.17nM, respectively. The fibrillar insulin comparatively has higher affinity binding to SMC than Abeta fibrils. The competitive binding studies with native insulin showed that the amount of bound insulin fibril was significantly decreased due to displacement of native insulin. However, the presence of native insulin is not altered the binding of beta-amyloid fibril. The cytotoxicity of insulin amyloid intermediates was measured. The pre-fibrillar intermediates of insulin showed significant toxicity (35%) as compared to matured fibrils. Myoblast treated with beta-amyloid fibrils showed more oxidative damage than the insulin fibril. Cell differentiating action of amyloidic insulin was assayed by creatine kinase activity. The insulin fibril treated cells differentiated more slowly compared to native insulin. However, beta-amyloid fibrils do not show cell differentiation property. These findings reinforce the hypothesis that accumulation of amyloid related proteins is significant for the pathological events that could lead to muscle degeneration and weakness in IBM.
View details for DOI 10.1016/j.abb.2008.03.021
View details for Web of Science ID 000256459800003
View details for PubMedID 18397759
The interaction of the beta-amyloid peptide (Abeta) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of Abeta oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble Abeta(28-35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant beta-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/P(i), but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form beta-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease.
View details for DOI 10.1111/j.1742-4658.2008.06378.x
View details for Web of Science ID 000255285700007
View details for PubMedID 18422968
Abeta amyloid peptide is believed to induce oxidative stress leading to inflammation, which is postulated to play a significant role in the toxicity of Alzheimer's disease (AD). This study was designed to investigate the inhibitory effects of DL-alpha lipoic acid (LA), a potential free radical scavenger, on oxidative vulnerability induced by intraperitoneal injection of Abeta25-35 amyloid fibrils in mice. Mice were divided into three groups: control, Abeta amyloid toxicity induced (AT), and LA treated (ATL). Blood Plasma was separated, liver, spleen and brain were dissected and analysis of oxidants, antioxidants, ATPases, glial fibrillary acidic protein (GFAP) and nuclear factor kappa-B (NFkappaB) were carried out. Results show biochemical parameters such as reactive oxygen species (ROS) and lipid peroxidation (LPO) were significantly lowered (P < 0.05) and levels of antioxidants and ATPase (P < 0.05) were significantly increased (P < 0.05) in hepatocytes, splenocytes and astrocytes of the ATL group. Moreover, our histological results revealed a decreased GFAP immunoreactivity in the neocortical region and NFkappaB immunoreactivity in neocortex, liver and spleen. This study reiterates LA as a potent free radical scavenger to combat oxidative vulnerability in the treatment for Abeta amyloid toxicity.
View details for DOI 10.1007/s11010-008-9705-9
View details for Web of Science ID 000254204500018
View details for PubMedID 18224425
View details for Web of Science ID 000253761000128
View details for Web of Science ID 000253761000043
Widespread cerebral deposition of a 40-42 amino acid peptide called amyloid beta peptide (A beta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease (AD). The clinical study provides evidence that accumulation of protofibrils due to the Arctic mutation (E22G) causes early AD onset. Melatonin showed beneficial effects in an AD mouse model. Mice were divided into four different groups (n=8 per group): (i) control group, (ii) scrambled A beta-injected group, (iii) A beta protofibril-injected group and (iv) melatonin-treated group. A single dose of (5 microg) A beta protofibril was administered to the A beta protofibril-injected and melatonin-treated groups via intracerebroventricular injections. The results demonstrate that melatonin treatment significantly reduces A beta protofibril-induced reactive oxygen species (ROS) production, intracellular calcium levels and acetylcholinesterase activity in the neocortex and hippocampus regions. Based on these findings it is suggested that melatonin therapy might be a useful treatment for AD patients.
View details for DOI 10.1080/10715760802277388
View details for Web of Science ID 000257894700007
View details for PubMedID 18654881
A beta vaccination as a therapeutic intervention of Alzheimer's has many challenges, key among them is the regulation of inflammatory processes concomitant with excessive generation of free radicals seen during such interventions. Here we report the beneficial effects of melatonin on inflammation associated with A beta vaccination in the central and peripheral nervous system of mice. Mice were divided into three groups (n=8 in each): control, inflammation (IA), and melatonin-treated (IAM). The brain, liver, and spleen samples were collected after 5 days for quantitative assessment of plasma lipid peroxides (LPO), an oxidative stress marker, and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (Gpx). IA group mice have shown the elevated concentration of LPO significantly while there was a reduction at antioxidant enzyme levels. In addition, a significant (P<0.05) reduction in neurotransmitters like dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) was also observed in the IA group mice. Nevertheless, their metabolites, such as homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P<0.05) as compared to control. Samples were further evaluated at microscopic level to examine the neuropathological changes by immunohistochemical methods. Melatonin treatment effectively reversed these above changes and normalized the LPO and antioxidant enzyme levels (P<0.05). Furthermore, melatonin salvaged the brain cells from inflammation. Our Immunohistochemical findings in the samples of melatonin-treated animals (IAM group) indicated diminished expression of glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (Nf kappa B) than those observed in the IA group samples. Our results suggest that administration of melatonin protects inflammation associated with A beta vaccination, through its direct and indirect actions and it can be an effective adjuvant in the development of vaccination in immunotherapy for Alzheimer's disease (AD).
View details for DOI 10.1007/s11010-006-9353-x
View details for Web of Science ID 000246262200005
View details for PubMedID 17136482
Chronically elevated blood glucose levels result in reduced leukocyte function and cell malnutrition, which contribute to a high rate of wound infection and associated healing problems in diabetic patients. In the present study, the role of biotinylated GHK peptide (BioGHK) incorporated collagen biomaterial was tested for wound healing in diabetic rats. The rate of wound contraction and the levels of collagen, uronic acid, protein and DNA in the granulation tissue were determined. Further, the concentration of nitric oxide and other skin antioxidants was also monitored during the study. In diabetic rats treated with BioGHK incorporated collagen (Peptide Incorporated Collagen--PIC), the healing process was hastened with an increased rate of wound contraction. Glutathione (GSH) and ascorbic acid levels in the skin of streptozotocin-induced diabetic rats were higher in the PIC group as compared to control (Untreated) and collagen (Collagen Film--CF) treated groups. Superoxide dismutase (SOD) and catalase (CAT) activity was altered in all the groups. In vitro fibroblast cell culture studies suggest that PIC promotes fibroblast growth. Histological evaluation by haematoxylin-eosin and Masson's trichrome method revealed epithelialization, increased synthesis of collagen and activation of fibroblasts and mast cells in the PIC group. This study provides a rationale for the topical application of BioGHK incorporated collagen as a feasible and productive approach to support diabetic wound healing.
View details for DOI 10.1016/j.lfs.2006.09.018
View details for Web of Science ID 000243576500002
View details for PubMedID 17049946
Alzheimer's disease is the most common cause of dementia and is widely believed to be due to the accumulation of beta-amyloid peptides (Abeta) and their interaction with the cell membrane. Abetas are hydrophobic peptides derived from the amyloid precursor proteins by proteolytic cleavage. After cleavage, these peptides are involved in a self-assembly-triggered conformational change. They are transformed into structures that bind to the cell membrane, causing cellular degeneration. However, it is not clear how these peptide assemblages disrupt the structural and functional integrity of the membrane. Membrane fluidity is one of the important parameters involved in pathophysiology of disease-affected cells. Probing the Abeta aggregate-lipid interactions will help us understand these processes with structural detail. Here we show that a fluid lipid monolayer develop immobile domains upon interaction with Abeta aggregates. Atomic force microscopy and transmission electron microscopy data indicate that peptide fibrils are fragmented into smaller nano-assemblages when interacting with the membrane lipids. Our findings could initiate reappraisal of the interactions between lipid assemblages and Abeta aggregates involved in Alzheimer's disease.
View details for DOI 10.1529/biophysj.106.085944
View details for Web of Science ID 000241945600016
View details for PubMedID 17098805
View details for PubMedCentralID PMC1635663
A controlled-release drug delivery of contraceptive steroids levonorgestrel (LNG) and ethinyl estradiol (EE) has been developed by successful encapsulation of LNG and EE in poly (lactide-co-glycolide) (PLG) microspheres.Smooth, spherical, steroid-loaded PLG microspheres with a mean size of 10-25 microm were prepared by using the water/oil/water double-emulsion solvent evaporation method.In vitro release profiles showed an increased burst release of LNG/EE on Week 1; thereafter, the release was sustained. At the end of Week 7, the release of LNG/EE from 1:5 and 1:10 PLG microspheres was 75.64% and 62.55%. respectively. In vitro degradation studies showed that the PLG microspheres maintained surface integrity up to Week 8 and then eroded completely by Week 20. In an in vivo study, the serum concentration of LNG/EE in rats showed a triphasic release response, with an initial burst release of 8 ng/mL LNG and 14 pg/mL EE on Day 1; thereafter, a controlled release of the drugs to the systemic circulation was maintained until Week 15, maintaining constant drug levels of 2 ng/mL LNG and 3-4 pg/mL EE in the blood. Histological examination of steroid-loaded PLG microspheres injected intramuscularly into the thigh muscle of Wistar rats showed minimal inflammatory reaction, demonstrating that contraceptive-steroid-loaded microspheres were biocompatible.This controlled-release and biocompatible nature of the PLG microspheres may have potential application in contraceptive therapy.
View details for DOI 10.1016/j.contraception.2006.01.015
View details for Web of Science ID 000239484700010
View details for PubMedID 16860053
Oral hepatitis B vaccine formulation was prepared by successful encapsulation of immunogenic peptide representing residues 127-145 of the immunodominant B-cell epitope of hepatitis B surface antigen (HBsAg) in poly(D,L-lactide co-glycolide) (PLG) microparticles. The smooth, spherical PLG microparticles with a diameter of around 10 microm was prepared by using W/O/W double emulsion solvent evaporation method. The entrapment efficiency of B-cell epitope peptide (BCEP) into PLG microparticles was 64%. In vitro studies showed B-cell epitope loaded PLG microparticles (BCEM) released the peptide in sustained profile and reached 64.9% efficiency by Day 25. Single oral immunization of mice with BCEM led to the significant induction of specific serum IgG and IgM anti-HB antibodies. After the termination of antibody induction, the orally immunized mice were infected with HBsAg, which resulted in the rapid production of antibodies against HBsAg as a result of secondary immune response. PLG microparticles formulation approach may have potential in increasing the efficacy of microparticulate systems for the oral administration of hepatitis B vaccine.
View details for DOI 10.1016/j.vaccine.2006.04.011
View details for Web of Science ID 000238638200007
View details for PubMedID 16713035
Silver nitrate administration stimulates immune activation, inflammation and deterioration in cell function. It is well established that hippocampal and cortical tissue are susceptible to degeneration in responses to insult such as oxidative stress or infection. This study was designed to investigate the prophylactic effect of alpha-crystallin, a major chaperone lens protein comprising of alpha-A and alpha-B subunits in inflammation induced mice. Mice were divided into three groups (n=6 in each), control, inflammation and alpha-crystallin treated. Our result shows that alpha-crystallin pretreatment effectively diminished systemic inflammation induced glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (NFkappaB) expression in the mice neocortex, reversed elevated intracellular calcium levels, acetylcholine esterase activity and depletion of glucose. Furthermore it also significantly prevented nitric oxide (P<0.05) and lipid peroxide production in the plasma, liver, neocortex and hippocampus of the inflammation-induced mice. In order to demonstrate the direct *OH and nitric oxide radical scavenging ability of alpha-crystallin, an In vitro experiment using primary astrocyte culture subjected to lipopolysaccharide (LPS), a well-known inflammatory stimuli were also carried out. This study reiterates that alpha-crystallin therapy may serve as a potent pharmacological agent in neuroinflammation.
View details for DOI 10.1016/j.bbadis.2005.11.007
View details for Web of Science ID 000235439000003
View details for PubMedID 16443350
Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects.
View details for DOI 10.1093/jb/mvj038
View details for Web of Science ID 000237355200003
View details for PubMedID 16567397
Contraceptive steroids levonorgestrel (LNG) and ethinyl estradiol (EE) have been encapsulated with poly(epsilon-caprolactone) (PCL) microspheres using a w / o /w double emulsion method. The microspheres prepared were smooth and spherical, with a mean size from 8-25 microm. In vitro release profiles of microspheres showed a trend of increasing initially at the first week, and thereafter the release was sustained. At the end of the seventh week LNG/EE from 1:5 and 1:10 PCL microspheres were 60 and 48%, 52 and 46%, respectively. An in vitro degradation study shows that at the 20th week the microspheres maintained the surface integrity. The PCL microspheres showed a triphasic in vivo release profile with an initial burst effect due to the release of the steroid adsorbed on the microsphere surface, a second sustained release phase due to the steroid diffusion through the pores or channels formed in the polymer matrix, and third phase due to polymer bioerodible. Histological examination of PCL microspheres injected intramuscularly into thigh muscle of a rat showed a minimal inflammatory reaction demonstrating that contraceptive steroid-loaded microspheres were biocompatible. The level of inflammatory cytokines determined by immunostaining for IL-1alpha, the tissue response to formulations at the first week was considered mild, whereas at the end of the 20th week the inflammatory response ceased. Thus, this study helped us to evaluate the feasibility of using these microspheres as a long-acting biodegradable drug delivery system for contraceptive steroids.
View details for PubMedID 16108044
View details for Web of Science ID 000233267600010
View details for PubMedID 16279992
Acute inflammation activates macrophages or monocytes and subsequently releases several inflammatory cytokines and reactive oxygen and nitrogen species. These proinflammatory cytokines activate astrocytes and trigger neurodegenerative diseases. In this work, we chose to address the mechanistic aspects of alpha-crystallin's protective function in inflammation-triggered neurotoxicity in mice. Alpha-crystallin, a lens structural protein, comprising alpha-A and alpha-B subunits is an ubiquitous molecular chaperone, which have been shown to reduce reactive oxygen species (ROS) production and enhance cellular glutathione level in the acute inflammation-induced mice. Results show that the proinflammatory cytokines such as interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) were significantly high (P<0.05) in the plasma, liver, cortex and hippocampus of inflammation-induced mice when compared to control. Alpha-crystallin pretreatment prevents inflammation-induced cytokines and NO production. In addition, a significant (P<0.05) reduction of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) was also observed in the inflammation-induced mice. Nevertheless, their metabolites, such as 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P<0.05) as compared to control. The results indicate that alpha-crystallin pretreatment controls the inflammation-induced DA, 5-HT and NE catabolism and suggest that alpha-crystallin has the potential to act as an anti-inflammatory agent in the neuroprotective processes.
View details for DOI 10.1016/j.brainresbull.2005.07.002
View details for Web of Science ID 000232172200010
View details for PubMedID 16144660
View details for DOI 10.1016/j.procbio.2005.03.060
View details for Web of Science ID 000231833100014
Acute inflammation can activate macrophages or monocytes and subsequently release several inflammatory cytokines and reactive oxygen species (ROS). Oxidative stress triggered by the production of ROS plays deleterious role leading to multiple organ failure. This study was designed to investigate the prophylactic effect of alpha-crystallin, a major chaperone lens protein comprising of alpha-A and alpha-B subunits in inflammation-induced mice. Mice were divided into three groups (n=6 in each): control, inflammation and alpha-crystallin-treated. Results show that ROS was significantly higher in the lymphocytes, hepatocytes and astrocytes (P<0.05) of inflammation-induced mice when compared to control, but no significant changes were observed in the alpha-crystallin-treated group. Increased level of lipid peroxidation (LPO) and decreased activities of antioxidant such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione were observed in the inflammation-induced mice when compared to control, whereas the activities of these were found to be normal followed by alpha-crystallin treatment. We also observed a reduction in reduced glutathione levels in hepatocytes of inflammation-induced mice, which were normalized on alpha-crystallin treatment. The in vitro study has shown that alpha-crystallin treatment not only suppresses the increase in LPO levels but also inhibits the lipid breakdown resulting from autooxidation in mouse cerebral cortex homogenate, and strongly suggests that alpha-crystallin therapy may serve as a potent pharmacological agent in systemic inflammation.
View details for DOI 10.1016/j.bbadis.2004.11.002
View details for Web of Science ID 000229948100012
View details for PubMedID 15949709
Collagen is one of the best materials used for nerve guide preparation due to its biocompatibility and desirable tensile strength. In this work, we have compared regeneration and functional reinnervation after sciatic nerve resection with bioresorbable crosslinked collagen guides in 10 mm gap. The crosslinking was carried out either with glutaraldehyde (GTA) or microwave irradiation (MWI). The multilayered collagen membrane used for nerve guides are prepared by lamellar evaporation technique. Functional evaluations of the regenerated nerves were performed by measuring the sciatic functional index (SFI), nerve conduction velocity (NCV), and electromyography (EMG). Transmission electron microscopic studies showed growth of axonal cable with fewer myelinated axons, Schwann cells and more unmyelinated axons present in the case of group treated with uncrosslinked collagen tubes after 1 month of implantation. However, we have observed more myelinated axons in the case of autograft, GTA, and MWI crosslinked collagen tube implants across the gap of 1 cm after the same period of implantation. Smaller myelinated fiber diameter was observed in the case of GTA crosslinked collagen tube group when compared with the autograft and MWI collagen tube groups. There were more myelinated axons during the 3rd and 6th months postoperatively using these conduits as substantiated by light microscopic studies of the regenerated nerve. The conduction velocity and recovery index improved significantly after 5 months reaching the normal values in the autograft and MWI crosslinked collagen groups compared to GTA and uncrosslinked collagen tubes.
View details for DOI 10.1016/j.brainres.2005.03.022
View details for Web of Science ID 000229898600007
View details for PubMedID 15927550
The use of peptide-based drugs is limited by their rapid degradability and toxicity at high concentration during their therapeutic application. These problems could be managed by the use of a peptide delivery agent for sustained release in the site of action. Collagen is one of the most proven biomaterials of good biocompatibility with an exceptional ligand encapsulating property. In this work, we have shown that pexiganan, an antimicrobial, 22-amino-acid peptide could be incorporated and delivered to the wound-healing site against bacterial strains Pseudomonas aeruginosa and Staphylococcus aureus. The release profiles of pexiganan collagen films with different collagen concentration were studied. The release of pexiganan from 2.5% w/w of collagen film showed a sustainable activity over 72 h with effective antimicrobial concentrations. Pexiganan-incorporated collagen (PIC)-treated groups were compared with open wound (OW)- and collagen film (CF)-treated rats. PIC-treated animals showed a diminishing level of bacterial growth as compared with OW- and CF-treated animals. The biochemical parameters such as hydroxyproline, protein, DNA, uronic acid, hexosamine, SOD, and catalase content in the granulation tissue of the healing wound revealed increased proliferation of cells involved in tissue reconstruction in PIC-treated groups when compared with OW- and CF-treated groups. Furthermore, spectroscopic studies suggested that collagen structure is not perturbed by pexiganan incorporation. This study provides rationale for application of collagen membrane for antimicrobial peptide delivery in infected wounds.
View details for PubMedID 15800884
The major pathological ramification of Alzheimer's disease (AD) is accumulation of beta-Amyloid (Abeta) peptides in the brain. An emerging therapeutic approach for AD is elimination of excessive Ass peptides and preventing its re-accumulation. Immunization is the most effective strategy in removing preexisting cerebral Abetas and improving the cognitive capacity as shown in transgenic mice model of AD. However, active immunization is associated with adverse effect such as encephalitis with perivascular inflammation and hemorrhage. Details about the mechanistic aspects of propagation of these toxic effects are matter of intense enquiry as this knowledge is essential for the understanding of the AD pathophysiology. The present work aimed to study the oxidative vulnerability in the plasma, liver and brain of the inflammation-induced rats subjected to Ass immunization. Induction of inflammation was performed by subcutaneous injection of 0.5?ml of 2% silver nitrate. Our present result shows that the proinflammatory cytokines such as IL1alpha and TNFalpha are increased significantly in the inflammation-induced, Abeta1-42, Abeta25-35 treated groups and inflammation with Abeta25-35 treated group when compared to control, complete Freund's adjuvant and Abeta35-25 treated groups. These increased proinflammatory cytokines concurrently releases significant amount of free radicals in the astrocytes of respected groups. The present result shows that nitric oxide (NO) level was significantly higher (P<0.001) in plasma, liver and brain of the rat subjected to inflammation, Abeta1-42, Abeta25-35 and inflammation with Abeta25-35 injected groups when compared to control. The increased level of lipid peroxides (LPO) (P<0.001) and decreased antioxidant status (P<0.05) were observed in the plasma, liver and brain of inflammation-induced group when compared to control. Our result shows that significant oxidative vulnerability was observed in the inflammation with Ass treated rats when compared to other groups. Based on our results, we suggest that immunization of AD patients with Ass should be done with caution as the increase in Ass could trigger the brain inflammation in uncontrollable level.
View details for DOI 10.1080/10715760500117373
View details for Web of Science ID 000229707900006
View details for PubMedID 16036338
Vascular amyloidosis in Alzheimer's disease (AD) results in the exposure of red blood cells to beta-amyloid fibrils (A beta). The potential in vivo ramifications of this exposure have been investigated by injecting A beta(1-40) alone or A beta-bound mouse red blood cells into the circulation of C57BL/6 mice. Results indicate that when A beta(1-40) is injected alone, a transient uptake of the fibrils by red blood cells occurs in vivo. When A beta-bound red blood cells were injected, beta-amyloid is rapidly removed from these cells in vivo. Double-labeling experiments indicate that while some of the red blood cells bound to A beta(1-40) are removed from circulation, a major fraction of these cells remain in circulation even after A beta is removed. Immunohistochemistry of murine tissue samples obtained after sacrificing the animals suggests that within 1 h after injection of A beta(1-40) or A beta-bound red blood cells, A beta is found in spleen phagocytes and liver Kupffer cells. Heme staining further indicates that the binding of A beta(1-40) to red blood cells enhances red cell phagocytosis by the spleen.
View details for DOI 10.1016/j.nbd.2004.11.004
View details for Web of Science ID 000228672900004
View details for PubMedID 15837558
Matrikines are small peptide fragments of extracellular matrix proteins that display potent tissue repair activities. Difficulties in achieving sustained delivery of bioactive concentration of matrikines in the affected area limits their therapeutic use. The present study evaluates the effects biotinylated matrikine peptide (bio-glycyl-histidyl-lysine) incorporated collagen membrane for dermal wound healing processes in rats. Biotinylated peptide incorporated collagen matrix (PIC) showed better healing when compared to wounds treated with collagen matrix [CF (collagen film)] and without collagen [CR (control)]. Binding studies indicate that biotinylated GHK (Bio-GHK) binds effectively to the collagen matrix and red blood cell (RBC) membrane when compared with t-butyloxycarbonyl substituted GHK (Boc-GHK). Wound contraction, increased cell proliferation, and high expression of antioxidant enzymes in PIC treated group indicate enhanced wound healing activity when compared to CF and CR groups. Interestingly Bio-GHK incorporated collagen increases the copper concentration by ninefold at the wound site indicating the wound healing property of Bio-GHK can also be linked with both copper localization and matrikine activities. These results demonstrate the possibility of using Bio-GHK incorporated collagen film as a therapeutic agent in the wound healing process.
View details for PubMedID 15803494
The structure of insulin in amyloid fibrillar form has been recently shown as a well folded conformation using cryoelectron microscopy [Jimenez, J.L., Nettleton, E.J., Bouchard, M., Robinson, C.V., Dobson, C.M., Saibil H.R., 2002. The protofilament structure of insulin amyloid fibrils. Proc. Natl. Acad. Sci. USA. 99 9196-9201.]. Most of the amyloid aggregates elicit maximum toxicity in the protofibrillar (PF) intermediate state. Here, we describe PF intermediates of insulin are made-up monomers with flexible conformers. We also show protofibrils have three-dimensionally extended hydrophobic cavity to bind with 1-anilinonaphthalene-8-sulphonate (ANS) molecules. Energy transfer measurement revealed that ANS dye binding site of PF is within the range of FRET distance of insulin tyrosine residues. Significant proportion of beta-sheet, helical, and turn structures in the PF form indicate conformational dynamics in the folded chain of insulin in the PF assembled form. Though the conformational flexibility is noticeably present in the assembly, addition of GdnHCl could completely unfold PF into disordered structure suggesting structural "zipping" in the PF form. We have also shown that helical conformer inducing solvent 2,2,2-trifluoroethanol (TFE) could dissociate the PF aggregate indicating possible involvement of beta-sheets in contributing to PF stability.
View details for DOI 10.1016/j.jsb.2005.02.009
View details for Web of Science ID 000229157600006
View details for PubMedID 15866741
The major pathological consequence of Alzheimer disease (AD) is accumulation of beta-amyloid (Abeta) peptide fibrillar plaque in the brain and subsequent inflammatory reaction associated with the surrounding cells due to the presence of these aggregates. Inflammation is the major complication associated with Abeta peptide vaccination. Abeta peptide activated T-helper cells are shown to enhance the existing-inflammatory conditions in the brain and other organs of AD patients. Hence systematic studies on potential approaches that will prevent inflammation during the vaccination are highly desired. DL-alpha-lipoic acid (LA), an antioxidant with known function as cofactor in mitochondrial dehydrogenase reactions, will be a good candidate to annul the oxidative damage due to vaccination triggered inflammation. For the first time, levels of principal neurotransmitters and their major metabolites in hippocampus and neocortex regions of brain are quantified to find out the level of inflammation. We have used high performance liquid chromatography with electro chemical detection (HPLC-EC) for monitoring neurotransmitter levels. We have shown a significant (p<0.05) reduction of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) in the systemic inflammation induced (SI), vaccinated (VA) and inflammation induced vaccinated (IV) mice. Nevertheless their metabolites such as 5-hydroxyindole acetic acid (5-HIAA) and homovanillic acid (HVA) are significantly (p<0.05) increased when compared with control. Interestingly, antioxidant LA treated mice with systemic inflammation (IL), vaccinated (VL) and inflammation induced vaccinated (IVL) mice exhibited enhanced level of 5-HT, DA and NE and the concentration of 5-HIAA and HVA gradually returned to normal. These results suggest a possible new way for monitoring and modifying the inflammation and thereby preventing Abeta vaccination mediated tissue damage.
View details for DOI 10.1016/j.brainresbull.2005.01.010
View details for Web of Science ID 000228678500011
View details for PubMedID 15811602
Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.
View details for Web of Science ID 000227317100004
View details for PubMedID 15703015
The highly conserved ubiquitin-proteasome system (UPS) controls the stability of most nuclear and cytoplasmic proteins and is therefore essential for virtually all aspects of cellular function. We have previously shown that the UPS is impaired in the presence of aggregated proteins that become deposited into cytoplasmic inclusion bodies (IBs). Here, we report that production of protein aggregates specifically targeted to either the nucleus or cytosol leads to global impairment of UPS function in both cellular compartments and is independent of sequestration of aggregates into IBs. The observation of severe UPS impairment in compartments lacking detectable aggregates or aggregation-prone protein, together with the lack of interference of protein aggregates on 26S proteasome function in vitro, suggests that UPS impairment is unlikely to be a consequence of direct choking of proteasomes by protein aggregates. These data suggest a common proteotoxic mechanism for nuclear and cytoplasmic protein aggregates in the pathogenesis of neurodegenerative disease.
View details for DOI 10.1016/j.molcel.2004.12.021
View details for Web of Science ID 000226905200005
View details for PubMedID 15694337
Recent reports indicate that beta-amyloid peptide (Abeta) vaccine based therapy for Alzheimer's disease (AD) may be on the horizon. There are however, concerns about the safety of this approach. Immunization with Abeta has several disadvantages, because it crosses the blood brain barrier and cause inflammation and neurotoxicity. The present work is aimed to study the protective effective of alpha-lipoic acid (LA) in the oxidative vulnerability of beta-amyloid in plasma, liver, spleen and brain, when Abeta fibrils are given intraperitoneally in inflammation induced mice. Result shows that reactive oxygen species (ROS) in the astrocytes of inflammation induced mice along with Abeta (IA) has shown 2.5-fold increase when compared with LA treated mice. The increased level of lipid peroxidase (LPO) (p < 0.05) and decreased antioxidant status (p < 0.05) were observed in the plasma, liver, spleen and brain of LA induced mice when compared with LA treated mice. Data shows that there were no significant changes observed between the control and LA treated mice. Our biochemical and histological results highlight that significant oxidative vulnerability was observed in IA treated mice, which was prevented by LA therapy. Our findings suggest that the antioxidant effect of LA when induced with Abeta may serve as a potent therapeutic tool for inflammatory AD models.
View details for DOI 10.1007/s11010-005-3301-z
View details for Web of Science ID 000227736000004
View details for PubMedID 15792351
Pexiganan (Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys), a 22 amino acid peptide, is an analogue of the magainin family of antimicrobial peptides present in the skin of the African clawed frog. Conformational analysis of pexiganan was carried out in different solvent environments for the first time. Organic solvents, trifluoroethanol (TFE) and methanol, were used to study the secondary structural preferences of this peptide in the membrane-mimicking environments. In addition, aqueous (D2O) and dimethyl sulfoxide (DMSO) solutions were also investigated to study the role of hydrogen bonding involved in the secondary structure formation. Fourier transform infrared absorption, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) measurements were carried out under the same conditions to ascertain the conformational assignments in different solvents. All these spectroscopic measurements suggest that the pexiganan peptide has the tendency to adopt different structures in different environments. Pexiganan appears to adopt an alpha-helical conformation in TFE, a sheet-stabilized beta-turn structure in methanol, a random coil with beta-turn structure in D2O, and a solvated beta-turn structure in DMSO.
View details for DOI 10.1002/bip.20132
View details for Web of Science ID 000232334800003
View details for PubMedID 15657879
View details for DOI 10.1080/154214090892401
View details for Web of Science ID 000228901700006
We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119-126)) and VVGGLGG (PrP(121-127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119-126) showed a helical conformational propensity, whereas PrP(121-127) adopted a more random coil structure. In 5% SDS, PrP(119-126) showed more alpha-helical content than in TFE solution, and PrP(121-127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a beta-sheet structure with a significant percentage of helical conformation and beta-turn structure in PrP(119-126) and PrP(121-127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119-126) were insoluble in SDS, and PrP(121-127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119-126) and PrP(121-127) formed stable monolayer's consisting of multimeric assemblages at the air-water interface. Monomeric PrP(119-126) was more toxic to astrocytes than the control Abeta peptide; however, the fibrillar form of PrP(119-126) was less toxic to astrocytes. PrP(121-127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119-126) and PrP(121-127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119-126) and PrP(121-127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119-126) and PrP(121-127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119-126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121-127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide.
View details for DOI 10.1016/j.jsb.2004.05.006
View details for Web of Science ID 000224759000004
View details for PubMedID 15477098
In the present study we have elucidated the toxicity of a novel amyloid forming model peptide, Poly (leucine-glutamic acid). The toxicity of the fibrils prepared from this peptide was analyzed in peripheral blood lymphocytes (PBL). The MTT reduction assay revealed that the viability of PBL decreases significantly upon treatment with Poly(leucine-glutamic acid) (Poly [LE]). Enhanced DCFH-DA fluorescence in treated cells suggests that peptide toxicity is probably mediated by the formation of free radicals. In vivo and in vitro biochemical studies indicated that Poly [LE] inactivates the antioxidant system of cells. Perturbation of Poly [LE] in a membrane lipid environment was assessed by circular dichroism (CD) using phosphotidyl choline-cholesterol bilayers. The CD results revealed that LE enhances its beta sheet content in a bilayer environment. Sequestration of Poly [LE] in lipid rafts demonstrates that it has a binding cleft similar to Abeta in lymphocyte raft domains. Nuclear membrane binding studies showed that Poly [LE] binds to nuclear membranes and may cause genotoxicity.
View details for DOI 10.1093/jb/mvh156
View details for Web of Science ID 000226416900008
View details for PubMedID 15625314
The main objective of this work was to develop a system consisting of polymeric microspheres loaded with steroid drugs. The drugs were encapsulated using biodegradable poly(lactide-co-glycolide) (PLG) and poly(epsilon-caprolactone) (PCL) by double emulsion solvent evaporation method. The lipophilic drugs, levonorgestrel and ethinylestradiol were made soluble by adding ethanol/water mixture. The effects of parameters like polymer concentration and stabilizer concentration were studied on the size, size distribution, surface properties and loading efficiencies of microspheres. The formulated microspheres were smooth, spherical and uniform in shape and size. Fourier transformed infrared spectroscopy and differential scanning calorimetry studies seemed to confirm the absence of chemical interaction between the drugs and the polymers, while the drugs were dispersed in the polymer. The increase in polymer concentrations increased the size as well as the loading efficiency of microspheres. Data obtained in this study demonstrated that the PLG/PCL microspheres may be a suitable polymeric carrier for long acting injectable drug delivery.
View details for Web of Science ID 000223208200015
View details for PubMedID 15304994
Alzheimer's disease is associated with vascular amyloidosis. As blood flows through the microcirculation, red blood cells (RBCs) come in contact with the vasculature. RBCs as well as endothelial cells (ECs) are known to bind beta amyloid fibrils. This suggests that a potential effect of amyloidosis may involve the interactions of RBCs with ECs lining the wall of the blood vessels mediated by amyloid fibrils. We have studied the effect of beta-amyloid peptide[1-40] (Abeta1-40) fibrils on the interactions of murine RBCs with ECs derived from bovine lung microvascular endothelium (BLMVEC) as well as bovine pulmonary arterial endothelium (BPAEC) in culture. We show that the initial incorporation of Abeta fibrils onto either RBCs or ECs cause RBCs to adhere to the ECs with greater affinity for the microvascular cells than the arterial cells. In addition, there is a transfer of Abeta fibrils between the RBCs and the ECs. Both the transfer and adhesion occurs when the amyloid fibrils are on the ECs or on the RBCs. However, with the amyloid fibrils on the RBCs, the adhesion and the transfer are greater than with the fibrils on the ECs. These results suggest that amyloidosis may affect the flow of RBCs through the microcirculation and that RBCs may play a role in propagating amyloidosis through the vasculature.
View details for DOI 10.1179/016164104225016227
View details for Web of Science ID 000222545100018
View details for PubMedID 15265278
Successful nerve regeneration process was achieved with improved mechanical strength by crosslinking tubular nerve guides made up of collagen. The multilayered collagen sheets were prepared from laminar evaporation of collagen solution. Scanning electron micrograph of the collagen tubes crosslinked with glutaraldehyde (GTA), microwave irradiation showed porous, fibrillar structures of collagen filaments in these matrices. The mechanical property of the crosslinked collagen tubes was carried out by tensile strength measurements. Fourier transform infrared spectra of the collagen films show that the native triple helicity was unaltered during multilayered preparation. It was observed that the structural integrity is unaltered during the multilayer preparation. Microscopic analysis indicates that the tubule surface acts as a surface of adherence and proliferation for the sprouting axons from the cut proximal nerve stumps. Solute diffusion studies on these tubes indicate that they are highly porous to wide range of molecular sizes during regeneration. Among the two types of crosslinking, the microwave irradiated collagen conduits results in ample myelinated axons compared with GTA group, where we observed more unmyelinated axons.
View details for DOI 10.1016/j.biomaterials.2003.09.075
View details for Web of Science ID 000188881500016
View details for PubMedID 14751744
This study evaluated the effects of noncovalently incorporated crystallin into the collagen matrix for dermal wound-healing processes in rats. Crystallin-incorporated collagen matrix (CIC) showed better healing when compared to wounds treated with collagen matrix (CS) and without collagen (CR). Biochemical parameters and histological analysis revealed that increased wound contraction enhanced cell proliferation and efficient radical scavenging in the CIC group. The higher shrinkage temperature of CIC films when compared to CS groups suggested increased hydrothermal stability for the former material. An in vitro release study of CIC has showed sustained and time-dependent release of crystallin from the collagen matrix. These results demonstrate the possibility of using crystallin as therapeutic protein in the wound-healing process.
View details for PubMedID 15116414
The wound healing process involves extensive oxidative stress to the system, which generally inhibits tissue remodeling. In the present study, an improvement in the quality of wound healing was attempted by slow delivery of antioxidants like curcumin from collagen, which also acts as a supportive matrix for the regenerative tissue. Curcumin incorporated collagen matrix (CICM) treated groups were compared with control and collagen treated rats. Biochemical parameters and histological analysis revealed that increased wound reduction, enhanced cell proliferation and efficient free radical scavenging in CICM group. The higher shrinkage temperature of CICM films suggests increased hydrothermal stability when compared to normal collagen films. Spectroscopic studies revealed that curcumin was bound to the collagen without affecting its triple helicity. Further we adopted the antioxidant assay using 2,2'-azobisisobutyronitrile to assess in vitro antioxidant activity of CICM. The antioxidant studies indicated that CICM quenches free radicals more efficiently. This study provides a rationale for the topical application of CICM as a feasible and productive approach to support dermal wound healing.
View details for DOI 10.1016/S0142-9612(03)00625-2
View details for Web of Science ID 000188706000020
View details for PubMedID 14738855
Conformational analysis of peptide 1, H-Leu-Leu-Ile-Leu-OMe on complexing with macro cycle calix[8]arene has been carried out using (1)H-NMR and FTIR spectroscopic techniques. Stoichiometry of the complex formed in the 1:8 ratio was evidenced by a Job plot. NMR studies of the above peptide show a marked downfield shift and an increase in (3)J values for NH resonances on complexing with calix[8]arene. The characteristic NOE connectivity between N(i+1)H and C(ialpha)H confirm beta-sheet conformation in the complexed state. Both (1)H-NMR and FTIR results indicate that the alpha-amino group of Leu I is proximal to the macrocycle and is involved in hydrogen bond formation with phenolic hydrogen atom of the calix[8]arene. This suggests that calix[8]arene provides a suitable platform for peptide 1 to self-assemble in a parallel beta-sheet conformation. The nature of calix[8]arene interaction with peptide 1 has been studied using dynamic NMR studies, which concludes that a bifurcated hydrogen bonding interaction exists in the molecular interfaces of the assembly.
View details for DOI 10.1002/jmr.629
View details for Web of Science ID 000188851600007
View details for PubMedID 14872539
View details for DOI 10.1080/15421400490427485
View details for Web of Science ID 000223534900004
Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.
View details for DOI 10.1002/bip.20131
View details for Web of Science ID 000224937700005
View details for PubMedID 15468066
This paper describes the regeneration of lesioned sciatic nerve with collagen tubes incorporated with RGD cell-adhesive peptide. Collagen implants of 14 mm were grafted to bridge a gap length of 10 mm nerve defect in a rat model. The regenerated tissues were analyzed histomorphologically. The number of myelinated axons in the regenerated mid-graft of the RGD peptide incorporated groups was statistically significant (p<0.05) than control collagen tube and autograft control after 30 days postoperatively. After 90 days of implantation, the mean counts were still statistically significant in the case of RGD peptide group than control collagen and autograft groups. Immunofluorescence studies demonstrated the staining of S100 proteins in the peripherally located cells indicating the proliferation of Schwann cells in the early days of regeneration. The staining pattern of integrin-alphaV was observed mostly in the perineurial regions in close proximity to the RGD peptide incorporated collagen tubes. Other studies like sciatic functional index, conduction velocity at 90 days postoperatively suggest complete regeneration of lesioned nerves with RGD incorporated collagen implants.
View details for DOI 10.1016/j.brainres.2003.08.057
View details for Web of Science ID 000187249400024
View details for PubMedID 14642848
Present study describes the development of a new formulation of levonorgestrel and ethinylestradiol based on double emulsion-solvent evaporation technique using poly(epsilon-caprolactone) (PCL) as biodegradable polymer. The effect of polymer concentration on microspheres and entrapment of drug into microspheres were studied. PCL was selected because of its hydrophobicity and advantages over other biodegradable polymers. Characterization of biodegradable polymer used for controlled drug delivery is essential to ensure reproducibility of in vitro and in vivo performances. The selected characterisation techniques established for PCL microspheres include its loading and entrapment efficiencies, DSC to analyse thermal behaviour, SEM to observe surface morphology, drug content of microspheres and in vitro release of drugs from microspheres. The SEM reports showed that microspheres were with smooth surface and DSC thermograms revealed no interaction between drug and polymer. The entrapment was found to be 58 and 47% for 1:10 and 1:5 batches and in vitro release studies showed that about 69.7% of LNG and 66.7% of EE from 1:10 batch and about 80% of LNG and 75.5% of EE from 1:5 batch for 150 days.
View details for DOI 10.1016/j.ijpharm.2003.08.011
View details for Web of Science ID 000187235100003
View details for PubMedID 14643973
View details for DOI 10.1021/la034403z
View details for Web of Science ID 000186177200068
View details for DOI 10.1016/j.cplett.2003.09.053
View details for Web of Science ID 000186265500029
Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.
View details for DOI 10.1002/bip.10474
View details for Web of Science ID 000186435800005
View details for PubMedID 14579306
The conformational studies of peptide derivatives A and B in a gel state were studied by using circular dichroism (CD), Fourier transformed infrared (FTIR), and fluorescence spectroscopic techniques. Birefringence and electron microscopic studies were carried out to characterize the morphological aspects of the fibrils in the gel. The FTIR spectra of the peptides show the absence of free NH in the gel state, implying that the intermolecular hydrogen-bond formation is the driving force for the aggregation. The CD spectrum of the peptide gels shows the presence of antiparallel and parallel beta-sheet conformation for peptide derivatives A and B, respectively. Electron microscopic studies (EM) of the peptide derivatives A and B reveal that peptide A formed rigid, rod-like structures without cross-linking and peptide B formed loose fibrils organized into highly noncovalently cross-linked mesh-like structural aggregates. Peptide A was much more soluble in alcoholic solvents than peptide B, and no birefringence was observed with Congo red (CR) staining in the temperature range of 0-80 degrees C. The spectroscopic studies indicate that peptide B consists of domains having a significant amount of beta-sheet structure and exhibiting golden yellow birefringence between 53 and 56 degrees C when stained with Congo red. On the other hand, peptide A gives no evidence of birefringence under polarized light. Fluorescence probe binding studies with pyrene in gel state with peptides A and B indicates the polarity in the interior of the aggregates. The data presented in the present work indicate that peptide B forms fibrils, which is similar to amyloid aggregates that are present in biological systems.
View details for DOI 10.1002/bip.10493
View details for Web of Science ID 000186435800006
View details for PubMedID 14579307
View details for DOI 10.1007/s00396-002-0843-4
View details for Web of Science ID 000184352800009
Increased neuronal nitric oxide synthase (nNOS) activity was observed during the prodromal period of seizures in various rat brain regions following administration of GABA(A) receptor antagonist, picrotoxin (PCT). Pretreatment with the selective nNOS inhibitor 7-nitroindazole (7-NI), dose- and time-dependently delayed the onset of clonus with a corresponding decrease in nNOS activity. The threshold dose of antiepileptic drugs (AEDs; diazepam, phenobarbitone and gabapentin) have potentiated the anticonvulsant action by pretreatment with graded doses of 7-NI. The increase in efficacy of anticonvulsant action correlated with a corresponding decrease of PCT-evoked increase in nNOS activity. The present data support a role for abnormal nNOS activity in mechanisms that trigger seizures and suggest a possible NO-mediated interplay between GABA(A) and glutamate receptors. The results of the present study provide evidence for a trigger role of neuronally produced NO in epileptogenesis induced by PCT and the participation of nNOS inhibitory mechanisms in the action of AEDs.
View details for DOI 10.1016/S0006-8993(03)02878-6
View details for Web of Science ID 000184397100012
View details for PubMedID 12850575
Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).
View details for Web of Science ID 000183820600045
View details for PubMedID 12829502
We have been developing antioxidants incorporated collagen matrix as a novel biomaterial for various biomedical applications. In this study we made use of quercetin incorporated collagenous matrix for dermal wound healing in rat. Quercetin incorporated collagen (QIC) treated groups were compared with control and collagen (CS) treated animals. QIC treated animal showed a better healing when compared to control and CS treated wound. The biochemical parameters like hydroxyproline, protein, uronic acid content in the healing wound, revealed that there is an increase in proliferation of cells in quercetin treated groups when compared to CS group and there is considerable increase in wound contraction when compared to CS treated group. In addition we adapted the antioxidant assay using 2,2'-azobisisobutryonitrile (AIBN) to assess in vitro antioxidant activity of QIC. The antioxidant studies indicate QIC quench the radicals more efficiently. These results suggested that quercetin incorporated collagen matrix could be a novel dressing material for dermal wound healing.
View details for DOI 10.1016/S0142-9612(03)00059-0
View details for Web of Science ID 000182913700011
View details for PubMedID 12711523
Amyloid beta-protein (A beta) accumulation in brain is thought to be important in causing the neuropathology of Alzheimer's disease (AD). A beta interactions with both neurons and microglial cells play key roles in AD. Since vascular deposition of A beta is also implicated in AD, the interaction of red cells with these toxic aggregates gains importance. However, the effects of A beta interactions with red blood cells are less well understood. Synthetic amyloid beta-protein (1-40) was labeled with biotin and preincubated at 37 degrees C for 4, 14 and 72 h to produce fibrils. Flow cytometry was used to study the binding of these fibrils to red cells. The amyloid fibrils had a high affinity for the red cell with increased binding for the larger fibrils produced by longer preincubation. Bovine serum albumin (BSA) did not reverse the binding, but actually resulted in a more efficient binding of the A beta fibrils to the red cells. The interaction of A beta with red cells increased the mean cell volume and caused the cells to become more spherical. This effect was greater for the longer fibrils. At the same time the interaction of A beta with red cells produced an increase in their fluorescence measured after 16-h incubation at 37 degrees C. This increase in fluorescence is attributed to the formation of fluorescent heme degradation products. The effect of prior hemoglobin oxidation, catalase inhibition and glutathione peroxidase inhibition indicated that the amyloid-induced oxidative damage to the red cell involved hydrogen peroxide-induced heme degradation. These results suggest that amyloid interactions with the red cell may contribute to the pathology of AD.
View details for DOI 10.1016/S0304-4165(03)00101-6
View details for Web of Science ID 000185709300004
View details for PubMedID 12829257
View details for DOI 10.1021/la026661m
View details for Web of Science ID 000182389100049
View details for DOI 10.1002/app.11651
View details for Web of Science ID 000180466900020
View details for Web of Science ID 000181647600014
Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.
View details for Web of Science ID 000181058200003
View details for PubMedID 12558947
Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.
View details for DOI 10.1139/O03-009
View details for Web of Science ID 000181210800007
View details for PubMedID 12683636
View details for Web of Science ID 000188666700004
View details for DOI 10.1007/s00396-002-0749-1
View details for Web of Science ID 000179938100012
View details for Web of Science ID 000177917100028
We have studied the binding of hemoglobin to the red cell membrane by centrifugation and fluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specifically reacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band 3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobin bound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding was quantitatively compared with the binding determined by centrifugation, which measures binding to band 3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 association constants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpreted in terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interaction associated with the conformational change produced when hemoglobin binds results in a 2.8-fold decrease in the intrinsic constant of (1.54 +/- 0.25) x 10(7) M(-1) for the binding of the second hemoglobin molecule. From the changes in lifetime produced by binding the first and second hemoglobin molecules, it was possible to show that the conformational change associated with binding the second hemoglobin molecule results in a decrease of the heme-eosin distance from 47.90 to 44.78 A. Reaction of cyanate with the alpha-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding of hemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobin molecule to band 3 decreases by a factor of 29 to (5.34 +/- 0.15) x 10(5) M(-1). The anticooperative interaction is greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobin tetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobin is very different with the second HbOCN increasing the heme-eosin distance to 55.99 A. The utilization of eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directly study the binding to band 3. At the same time a sensitive probe of the conformational changes, which occur when hemoglobin binds to band 3, is provided.
View details for DOI 10.1021/bi02007e
View details for Web of Science ID 000176674100012
View details for PubMedID 12093280
Collagen, a unique connective tissue protein finds extensive application as biocompatible biomaterial in wound healing, as drug carriers, cosmetics, etc. A study has been undertaken to stabilise Type-I collagen of rat-tail tendon using plant polyphenol (Acacia Mollissima) in the presence of an acrylic polymer. It has been found that collagen fibres pre-treated with acrylic polymer followed by the treatment with Acacia Mollissima exhibited an increase in hydrothermal stability by 25 degrees C. Infrared spectroscopic studies display the changes in the spectral characteristics of native and treated collagen films. Transmission electron microscopic and circular dichroic studies provide an insight into the understanding of the improved stabilisation of collagen, due to treatment with acrylic polymer and plant polyphenols. The study is expected to enhance the biomaterial applications of collagen tissues.
View details for Web of Science ID 000176015400003
View details for PubMedID 12069323
Protecting groups in N- and C-terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1, Boc-Ala-Ile-Ile-Gly-OMe (Boc: tert-butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a beta-sheet conformation. In contrast, peptide 2, Ac-Ala-Ile-Ile-Gly-OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.
View details for Web of Science ID 000175775600002
View details for PubMedID 12010515
View details for Web of Science ID 000173665300010
View details for Web of Science ID 000183440300002
A hydrophobic pentadecapeptide, AGAAAA-GAVVGGLGG (1), part of the prion sequence PrP (106-127), on fresh aqueous dissolution takes a mixture of random and sheet conformations which forms a stable monolayer with a high beta-sheet content when compressed at the air-water interface. This also develops into a kinetically stabilized beta-sheet structure on sonication.
View details for DOI 10.1039/b206886a
View details for Web of Science ID 000178192300027
View details for PubMedID 12397998
9-O-Acetyl neuraminic acid specific lectin (AchatininH) was isolated from the hemolymph of the land snail Achatina fulica by affinity chromatography on sheep submaxillary mucin (SSM) coupled cyanogen bromide activated Sepharose 4B. The molecular weight of the native protein was 2.42 kDa. UV-Vis absorption, fluorescence and circular dichroism spectroscopic studies on AchatininH revealed the importance of divalent metal ions (Ca2 +, Mg2+ and Mn2+) on lectin conformational change associated with activity of lectins. The binding of these cations changes lambdamax to shorter wavelength in the far UV region (blue shift) and longer wavelength in UV region (red shift), indicating substantial contribution of aromatic side chain in the far UV region on binding with metal ions. The results infer that divalent cations cause conformational changes in lectin which may be responsible for affinity with their carbohydrate moiety.
View details for Web of Science ID 000089334800007
View details for PubMedID 11083028
Concentration dependent morphological characteristics of a novel dipeptide derivative Lys-Asp-Lauryl.HBr (1) has been presented. Evidence for "onion" like vesicle formation at higher concentration (>8.2 x 10(-3) M) of peptide (1) in aqueous medium was obtained from conductance and 90 degrees light scattering measurements, and cryo-transmission electron microscopic studies.
View details for Web of Science ID 000088148700013
View details for PubMedID 10915047
The tetrapeptide derivative Tyr-Gly-Phe-Ala-OBz (1) forms monolayers as confirmed by compressibility studies carried out at various temperatures. Peptide 1 monolayer exhibits an anomalous structural transition at 40 degrees C as evidenced by pi-A isotherms recorded at different temperatures. The structural transition is also observed in aqueous solution of trifluoroacetate of peptide 1 as evidenced by fluorescence and Raman scattering intensity measurements.
View details for Web of Science ID 000087219200014
View details for PubMedID 10843215
View details for Web of Science ID 000086225200009
This study investigates the age associated changes in hemorheological properties and cerebral blood flow. Partial correlations indicate that part of the age-dependent decrease in flow velocities can be attributed to a hemorheological decrement resulting in part from enhanced oxidative stress in the aged. A possible link with Alzheimer's pathology is suggested by the augmented hemorheological impairment resulting from in vitro incubation of red cells with amyloids. These results suggest that in aging, oxidative stress as well as amyloids may influence the fluid properties of blood, resulting in a potential decrement in blood flow and oxygen delivery to the brain. Animal intervention studies further demonstrate that altered hemorheological properties of blood can actually influence cognitive function. The relationships shown to exist between hemorheology, blood flow, amyloids, oxidative stress, and cognitive function suggest that these factors may be one of the mechanisms operating in the complex etiology of Alzheimer's disease.
View details for Web of Science ID 000087830000012
View details for PubMedID 10867210
View details for Web of Science ID 000085412600007
View details for Web of Science ID 000082124900009
The results of the present study clearly shows that a correlation exists between nitric oxide (NO) and gamma-aminobutyric acid transaminase (GABAT-T) activity as well as gamma-aminobutyric acid (GABA), glutamic acid and the activity of glutamic acid decarboxylase (GAD). Supporting of this 10 min after the administration of L-Arginine (L-Arg) increased GABA concentration and diminished the activity of GABA-T. There was no change in GAD activity and glutamic acid level. Administration of convulsion inducing agent Picrotoxin (PCT) decreased the NO concentration in the brain and enhanced the activity of GABA-T, and the fact that the NOS inhibitor (N(G)-nitro-L-Arg methyl ester (L-NAME) diminished the activity of NOS and increased the activity of GABA-T provide another support for the involvement of NO on GABA-T activity. The present study clearly showed that high concentrations of NO in the brain suppresses the activity of GABA-T.
View details for Web of Science ID 000082009500027
View details for PubMedID 10434007
In the present study, an investigation was undertaken to assess the protective efficacy in cadmium toxicity of vitamins administered simultaneously as well as post-treatment. Rats were treated with cadmium 1 mg/kg body weight (bw) powdered vitamin A chewable tablet 500 IU/kg bw/day and injectable vitamin D(3) (made into a suspension with gum tragacanth in 100 ml distilled water) 100 IU/kg bw/day, mixed with powdered pellet feed and fed to experimental animals. Spontaneous motor activity and Rota Rod Endurance time was recorded after both simultaneous (for 21 days) and post-treatment (42 days). Vitamin treated animals by themselves behaved like controls but attenuated the cadmium effect when given Cd simultaneously or as post-treatment. While the biochemical changes were assayed, vitamins which did not have any influence on their own, given simultaneously and as post-treatment, antagonized the cadmium effect on heart, liver tissues and serum. Both simultaneous and post-cadmium and -vitamin treatments significantly increased the activities of the enzymes aspartic amino transferase, alanine aminotransferase, acid phosphatase and alkaline phosphatase in tissues and serum. Simultaneous and post-vitamin treatment with Cd had an effect of bringing back the activity of the enzymes closer to control values. These data suggest that treatment with vitamin A and D can minimise the Cd effect when given to the population exposed to Cd.
View details for Web of Science ID 000082489200005
View details for PubMedID 21781925
The anticonvulsant drug Diazepam (DIA-2 mg/kg b. wt), the nitric oxide (NO) donor L-Arginine (L-Arg-2000 mg/kg b. wt) and the putative nitric oxide synthase (NOS) inhibitor N(G)-Nitro-L-Arginine methyl ester (L-NAME-50 mg/kg b. wt) were used to determine the role of endogenous NO on convulsions induced by picrotoxin (PCT-5 mg/kg b. wt) in rats. Rats given a convulsant dose of PCT (5 mg/kg b. wt) had convulsion and it suppresses the NOS activity and NO concentration in brain regions. The anticonvulsant L-Arg alone significantly increases the NO concentration and NOS activity in brain regions, but not diazepam. Whereas DIA, along with L-Arg, enhances the NO and NOS activity when compared to L-Arg alone. The combination of both OIA and L-Arg completely suppressed the convulsions. L-NAME alone had no effect to produce convulsions but it completely decreased NO concentration and NOS activity and potentiated the PCT convulsions. This was reverted by pre- and post treatment of DIA plus L-Arg indicating, the increased NO concentration and NOS activity in brain regions suppresses convulsions.
View details for Web of Science ID 000080317900005
View details for PubMedID 10357070
The impact of monocrotophos on protein and carbohydrate metabolism in different tissues of albino rats was investigated. The monocrotophos (0.25 mg/ml) was orally intubated into an experimental group of rats. In another group, the same amount of water was orally intubated (control group) for 29 days. The protein content was increased in liver, serum and spleen of albino rats after treatment with monocrotophos. The protein content decreased in muscle and kidney, and overall the free sugar level decreased in all tissues. The glycogen content increased in muscle, serum and kidney after treatment with monocrotophos, and the glycogen content and reducing sugar level decreased in liver and spleen. The significance of these results is discussed.
View details for Web of Science ID 000082814400001
View details for PubMedID 10533267
View details for Web of Science ID 000077388800003
Isoproterenol hydrochloride (ISO), a beta adrenergic agonist, is known to cause ischemic necrosis in rats. Cardiotoxicity of three different doses of ISO were studied using physiological, biochemical and histopathological parameters. The effects of single and double dose of ISO were analysed, which illustrated that single ISO dose was more cardiotoxic than double ISO dose due to ischemic preconditioning. The tetrapeptide derivatives L-lysine-L-arginine-L-aspartic acid-L-serine (tetrapeptide A) and di-tert.butyloxycarbonyl-L-lysine-L-arginine-L-aspartic acid-tert.butyl O-tert.butyl-L-serinate (tetrapeptide B) along with acetylsalicylic acid as positive control were analysed at different time points for their cardioprotective effect. The results demonstrated that optimal protective effects were observed by pretreatment with 5 mg/kg of tetrapeptide B and this was found to be slightly better than that of acetylsalicylic acid. A lesser degree of cardioprotection was noticed when low doses of tetrapeptide B were administered. This study clearly showed that single dose of ISO (50 mg/kg, s.c.) induced myocardial necrosis could be used as a model to assess cardiovascular drugs and in this model, it was demonstrated that the tetrapeptide B could exhibit optimal cardioprotective effect.
View details for Web of Science ID 000076281500019
View details for PubMedID 9788755
View details for Web of Science ID 000073914600004
A tetrapeptide corresponding to a region of the N-terminal portion of lactotransferrin with hydrophobic alkyl groups at the terminal ends was synthesized and its physicochemical properties as well as its effect on thrombin-stimulated platelet aggregation were examined. The tetrapeptide derivative, in the aggregated state, produced inhibitory effect on platelet aggregation. The concentration dependent activity of the peptide was analyzed in the light of micelle formation, with the micellar aggregate comprising four tetrapeptide units. The unique action of this peptide derivative on the inhibition of platelet aggregation might be useful in the development of potent antithrombotic drugs.
View details for Web of Science ID 000077691900008
View details for PubMedID 9880074
View details for Web of Science ID 000071329500009
View details for Web of Science ID A1997YB26100014
View details for Web of Science ID A1996VH06600012
View details for Web of Science ID A1996VD94500004
Surface active pentapeptide [2(HCOO-). Lys-Ala-Ala-Lys(Z)-Tyr-OCH3] has been synthesized and its micelle formation investigated using conductometric, pH metric, and UV spectroscopic techniques. The double head double tail peptide molecules are shown to interact with water soluble meso-tetrakis (4-sulfonatophenyl)-porphyrin [TPPS]H2 to form characteristic H-type aggregate at low concentrations, as evidenced by UV-Vis and fluorescence spectroscopic techniques. Spectroscopic analysis reveals that the aggregate contains 1:2 porphyrin-peptide combination. The equilibrium constant for the formation of peptide-porphyrin complex has been obtained by using absorption spectral data. The present studies provide new insight into the peptide-porphyrin interaction.
View details for Web of Science ID A1996UR69600033
View details for PubMedID 8670292
View details for Web of Science ID A1996UD79400011
A novel peptide, tert-butyloxycarbonyl-L-arginine-L-proline lauryl ester laurate, synthesised by a solution-phase method, formed micelles in aqueous solutions and was observed to exhibit anticoagulant activity as shown by clotting assays such as the thrombin time (TT) test, the prothrombin time (PT) test and the activated partial thromboplastin time (APTT) test. TT and PT were found to be normal up to a particular concentration of peptide, and above that level they increased with increasing concentration of peptide, while APTT did not exhibit much significance. Conductometric and potentiometric studies showed that the peptide formed a stable micelle, and the anticoagulant activity of this peptide was also compared with a non-arginine-containing peptide (control) known to form micelles. The anticoagulant action in the micellar form could be due to the inhibition of thrombin, as seen from the decrease in amidolytic activity. The inhibitory activity of the peptide was explained in the light of micelle formation.
View details for Web of Science ID A1995QQ76900012
View details for PubMedID 7601613
Ordered aggregates of Val-Leu-Pro-Phe, tetrapeptide 1, have been found in aqueous solutions. Evidence for the formation of aggregates for the above peptide was obtained by conductometric, pH metric, UV and fluorescence spectroscopic techniques. Values of critical micelle concentration (CMC) for the above peptide obtained by these methods are in good agreement with each other. The formation of organized aggregates of the peptide is favoured upon increasing the temperature (viz. the process of aggregation is endothermic). The aggregation number has been determined at different temperatures. Values of delta G0m, delta H0m, delta S0m and delta C0p have also been estimated. Binding studies with the 8-anilinonaphthyl sulfonic acid (ANS) and pyrene indicate that the interior of the aggregate is nonpolar. There are two processes with regard to the change of thermodynamical parameters like delta G0m, delta H0m, delta S0m, delta C0p and aggregation number (N). In the first process (from 5 degrees C to 40 degrees C) the driving force for aggregation seems to be the positive entropy because of water release due to intermolecular association of ionic moieties. The second process (from 40 degrees C and above) is due to intramolecular ionic interaction. The chemical shifts of the amide protons of the peptide have been presented in the light of inter- and intramolecular hydrogen-bond formation, and forces implicated in aggregation for both the first and second processes.
View details for Web of Science ID A1995QJ77400006
View details for PubMedID 7782160
View details for Web of Science ID A1994PJ67600019
View details for Web of Science ID A1994PA53700036
View details for Web of Science ID A1994MT63500024
Activity of the dipeptidyl hydrolase angiotensin converting enzyme (ACE) has been observed to be altered by treatment with adriamycin (ADR). We used an animal model of ADR nephrotoxicity to study the effects on ACE in serum, urine and tissues on days 5, 10, 15, 20, 25 and 30 after ADR administration. Both glomerular and tubular injury occurred as evidenced by heavy proteinuria, albuminuria and increased urine N-acetyl glucosaminidase (NAG) excretion. Serum ACE was significantly elevated on days 20, 25 and 30. Of great interest was the excretion of ACE in urine of treated rats which ran parallel with the total protein excretion above the barely detectable levels found in controls. ACE activity increased in kidney, adrenal gland and liver on days 15, 20, 25 and 30. Heart and brain ACE levels increased on days 25 and 30. Increased ACE activity in aorta and lungs occurred on days 20, 25 and 30. ACE activity decreased in kidney, aorta, heart and brain on days 5 and 10. These observations strongly suggest a contribution of various tissues to elevate the serum ACE level. Urinary ACE may be of potential use as an index for renal glomerular and tubular damage.
View details for Web of Science ID A1993MU47600005
View details for PubMedID 8303709
View details for Web of Science ID A1993MH35800001
View details for Web of Science ID A1993LT45500023
View details for Web of Science ID A1993LP98600040
View details for Web of Science ID A1993LD66800022
View details for Web of Science ID A1993LW54400001
View details for Web of Science ID A1993KL90400014
View details for Web of Science ID A1993KM28000003
An X-ray diffraction study was carried out on a single crystal of N-(N alpha-[(tert.-butyloxy)-carbonyl]-L-alanyl)-N,N'-dicyclohexylur ea belonging to the tetragonal space group P4(1)2(1)2, having cell dimensions a = b = 10.102(3) A, c = 46.067(7) A, V = 4701.2 A3, Z = 8. The crystal structure was solved by direct methods and refined to an R value of 0.056 for 1602 unique reflections with I greater than 2.5 sigma(I). Crystal structure analysis shows the presence of an intramolecular N-H ... O=C H-bond stabilizing the molecule in a folded form similar to that of a beta turn, forming a nine-membered ring. IR and 1H-NMR studies in CDCl3 solution confirm the stable folded conformation found in the crystalline state, as well as the existence of N-H ... O=C H-bonds in the title compound, as in peptides.
View details for Web of Science ID A1992HW20600001
View details for PubMedID 1341904