Chitosan reduced in-situ synthesis of gold nanoparticles on paper towards fabricating highly sensitive, stable uniform SERS substrates for sensing applications.

Surface-Enhanced Raman Spectroscopy (SERS) is a powerful surface-sensitive technique for molecular analysis. Its use is limited due to high cost, non-flexible rigid substrates such as silicon, alumina or glass and less reproducibility due to non-uniform surface. Recently, paper-based SERS substrates, a low-cost and highly flexible alternative, received significant attention. We report here a rapid, inexpensive method for chitosan-reduced, in-situ synthesis of gold nanoparticles (GNPs) on paper devices towards direct utilization as SERS substrates. GNPs have been prepared by reducing chloroauric acid with chitosan as a reducing and capping reagent on the cellulose-based paper surface at 100 °C, under the saturated humidity condition (100 % humidity). GNPs thus obtained were uniformly distributed on the surface and had fairly uniform particle size with a diameter of 10 ± 2 nm. Substrate coverage of resulting GNPs directly depended on the precursor's ratio, temperature and reaction time. Techniques such as TEM, SEM, and FE-SEM were utilized to determine the shape, size, and distribution of GNPs on paper substrate. SERS substrate produced by this simple, rapid, reproducible and robust method of chitosan-reduced, in situ synthesis of GNPs, showed exceptional performance and long-term stability, with a detection limit of up to 1 pM concentration of test analyte, R6G. Present paper-based SERS substrates are cost-effective, reproducible, flexible, and suitable for field applications.

Protein Aggregation and Cataract: Role of Age-Related Modifications and Mutations in α-Crystallins.

* The article is published as a part of the Special Issue "Protein Misfolding and Aggregation in Cataract Disorders" (Vol. 87, No. 2). ** To whom correspondence should be addressed. Cataract is a major cause of blindness. Due to the lack of protein turnover, lens proteins accumulate age-related and environmental modifications that alter their native conformation, leading to the formation of aggregation-prone intermediates, as well as insoluble and light-scattering aggregates, thus compromising lens transparency. The lens protein, α-crystallin, is a molecular chaperone that prevents protein aggregation, thereby maintaining lens transparency. However, mutations or post-translational modifications, such as oxidation, deamidation, truncation and crosslinking, can render α-crystallins ineffective and lead to the disease exacerbation. Here, we describe such mutations and alterations, as well as their consequences. Age-related modifications in α-crystallins affect their structure, oligomerization, and chaperone function. Mutations in α-crystallins can lead to the aggregation/intracellular inclusions attributable to the perturbation of structure and oligomeric assembly and resulting in the rearrangement of aggregation-prone regions. Such rearrangements can lead to the exposure of hitherto buried aggregation-prone regions, thereby populating aggregation-prone state(s) and facilitating amorphous/amyloid aggregation and/or inappropriate interactions with cellular components. Investigations of the mutation-induced changes in the structure, oligomer assembly, aggregation mechanisms, and interactomes of α-crystallins will be useful in fighting protein aggregation-related diseases.

Long term observation of ocular surface alkali burn in rabbit models: Quantitative analysis of corneal haze, vascularity and self-recovery.

Limbal Stem Cell Deficiency (LSCD), caused due to corneal injury, primarily by chemical/alkali burns, leads to compromised vision. Recently, several animal models of corneal alkali burn injury have become available. The majority of the studies with these animal models start interventions soon after the injury. However, in the clinical setting, there is a considerable delay before the intervention is initiated. Detailed knowledge of the molecular, histopathological, and clinical parameters associated with the progression of the injury leading to LSCD is highly desirable. In this context, we set out to investigate clinical, histopathological parameters of ocular surface alkali burn over a long period of time, post-injury. Limbal stem cell-deficient animal models of rabbits were created by alkali burn using sodium hydroxide, which was then assessed for their progression towards LSCD by grading the alkali burn, corneal haze, and vascularization. Additionally, cells present on the corneal surface after the burn was investigated by histology and immunophenotyping. Grading of rabbit eyes post-alkali burn had shown complete conjunctivalization in 80% (n = 12/15) of the rabbits with the alkali burn grade score of 3.88 ± 0.29 in three months and remained stable at four months (4.12 ± 0.24). However, ocular surface showed self-healing in 20% (n = 3/15) of the rabbits with a score of 1.67 ± 0.34 in four months irrespective of similar alkali injury. These self-healing corneas exhibited decreased opacity score from 2.51 ± 0.39 to 0.66 ± 0.22 (p = 0.002) and regressed vascularity from 1.66 ± 0.41 to 0.66 ± 0.33 in one to nine months, respectively. Restoration of the corneal phenotype (CK3+) was observed in central and mid-peripheral regions of the self-healing corneas, and histology revealed the localization of inflammatory cells to the peripheral cornea when compared to conjunctivalized and scarred LSCD eyes. Our study shows the essentiality to consider the time required for surgical intervention after the corneal alkali injury in rabbit models as evident from their tendency to self-heal and restore corneal phenotype without therapy. Such information on the possibility of self-healing should be useful in further studies as well as determining interventional timings and strategy during clinical presentation of corneal alkali burns.

Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin.

BACKGROUND: αA-crystallin plays an important role in eye lens development. Its N-terminal domain is implicated in several important biological functions. Mutations in certain conserved arginine residues in the N-terminal region of αA-crystallin lead to cataract with characteristic cytoplasmic/nuclear aggregation of the mutant protein. In this study, we attempt to gain mechanistic insights into the congenital cataract caused by the R54C mutation in human αA-crystallin. METHODS: We used several spectroscopic techniques to investigate the structure and function of the wild-type and R54CαA-crystallin. Immunoprecipitation, chromatin-enrichment followed by western blotting, immunofluorescence and cell-viability assay were performed to study the interaction partners, chromatin-association, stress-like response and cell-death caused by the mutant. RESULTS: Although R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone-like activity was comparable to that of wild-type. When expressed in lens epithelial cells, R54CαA-crystallin exhibited a speckled appearance in the nucleus rather than cytoplasmic localization. R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of caspase 3, leading to apoptosis. Analysis of the "interactome" revealed an increase in interaction of the mutant protein with nucleosomal histones, and its association with chromatin. CONCLUSIONS: The study shows that alteration of "interactome" and nucleosomal association, rather than loss of chaperone-like activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin. GENERAL SIGNIFICANCE: The study provides a novel mechanism of cataract caused by a mutant of αA-crystallin, and sheds light on the possible mechanism of stress and cell death caused by such nuclear inclusions.

HSPB5 (αB-crystallin) confers protection against paraquat-induced oxidative stress at the organismal level in a tissue-dependent manner.

Oxidative stress is one of the major and continuous stresses, an organism encounters during its lifetime. Tissues such as the brain, liver and muscles are more prone to damage by oxidative stress due to their metabolic activity, differences in physiological and adaptive processes. One of the defence mechanisms against continuous oxidative stress is a set of small heat shock proteins. αB-Crystallin/HSPB5, a small heat shock protein, gets upregulated under stress and acts as a molecular chaperone. In addition to acting as a molecular chaperone, HSPB5 is shown to have a role in other cytoprotective functions such as inhibition of apoptosis, prevention of oxidative stress and stabilisation of cytoskeletal system. Such protection in vivo, at the organism level, particularly in a tissue-dependent manner, has not been investigated. We have expressed HSPB5 in fat body (liver), neurons and specifically in dopaminergic and motor neurons in Drosophila and investigated its protective effect against paraquat-induced oxidative stress. We observed that expression of HSPB5 in neurons and fat body confers protection against paraquat-induced oxidative stress. Expression in dopaminergic neurons showed a higher protective effect. Our results clearly establish the protective ability of HSPB5 in vivo; the extent of protection, however, varies depending on the tissue in which it is expressed. Interestingly, neuronal expression of HSPB5 resulted in an improvement in negative geotropic behaviour, whereas specific expression in muscle tissue did not show such a beneficial effect.

Cross-linked chitosan biofunctionalized paper-based microfluidic device towards long term stabilization of blood typing antibodies.

Long term stability of antibodies at room temperature is a major challenge in the commercialization of point-of-care devices for diagnostics. Since chitosan has been proven to be an excellent biofunctionalization material, the effects of four different biofunctionalization processes were studied to improve the room temperature stability of antibodies immobilized on chitosan modified paper-based microfluidic devices using blood typing antibodies as candidates. The devices used in this work have a flower-shaped design with 4 test zones at each corner. In three zones Anti-A, Anti-B, and Anti-D (Anti-Rh) antibodies are immobilized and the fouth zone represents the control (no antibodies) after biofunctionalization. The biofunctionalization of the paper devices was done with chitosan and chitosan cross-linked with sodium triphosphate pentabasic, glutaraldehyde, and sodium hydroxide. These devices were used for blood typing assays using real blood samples. A similar assay was also performed on unmodified (non-biofunctionalized) paper devices for comparison. Chitosan based biofunctionalized paper-devices showed better stability, up to 100 days as compared to 14 days on unmodified paper, at room temperature. Such biofunctionalized paper-based devices will be suitable for on-field and remote testing without any technical expertise and requirement for the cold chain.

HspB5 protects mouse neural stem/progenitor cells from paraquat toxicity.

INTRODUCTION: HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress. OBJECTIVE: The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (mNSPCs) against paraquat-induced toxicity. METHODS: The recombinant human HspB5 was expressed in E.coli and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting. RESULTS: The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of mNSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively. CONCLUSION: Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the mNSPCs and confers protection against paraquat toxicity.

Inflammation, vascularization and goblet cell differences in LSCD: Validating animal models of corneal alkali burns.

Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid-Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.

Glycosylation differentially modulates membranolytic and chaperone-like activities of PDC-109, the major protein of bovine seminal plasma.

The major bovine seminal plasma protein, PDC-109, is a mixture of glycosylated (BSP-A1) and non-glycosylated (BSP-A2) isoforms of a 109-residue long polypeptide. It binds to spermatozoa by specifically recognizing choline phospholipids on the plasma membrane and destabilizes it by penetrating the hydrophobic interior, resulting in lipid efflux, which is necessary for sperm capacitation and successful fertilization. PDC-109 also acts as a molecular chaperone and protects target proteins from denaturation and aggregation induced by various types of stress. In order to investigate the role of glycosylation in these activities, we have separated BSP-A1 and BSP-A2 from PDC-109, and also cloned and expressed BSP-A2 in E. coli and purified the recombinant BSP-A2 (rBSP-A2) to homogeneity. Employing biophysical and biochemical approaches we have investigated the membrane-perturbing and chaperone-like activities (CLA) of PDC-109, BSP-A1, BSP-A2 and recombinant BSP-A2 (rBSP-A2). The results obtained demonstrate that glycan-lacking wild-type BSP-A2 and rBSP-A2 exhibit higher membrane-perturbing activity but decreased CLA as compared to PDC-109. In contrast, BSP-A1 exhibits significantly higher CLA than PDC-109, but its ability to destabilize membranes is considerably lower. This differential modulation of the membrane-perturbing and chaperone-like activities has been explained on the basis of higher membrane-penetrating ability and lower solubility of glycan-lacking BSP-A2 as compared to the glycosylated BSP-A1.

Drug Repurposing for Retinoblastoma: Recent Advances.

Retinoblastoma is the intraocular malignancy that occurs during early childhood. The current standard of care includes chemotherapy followed by focal consolidative therapies, and enucleation. Unfortunately, these are associated with many side and late effects. New drugs and/or drug combinations need to be developed for safe and effective treatment. This compelling need stimulated efforts to explore drug repurposing for retinoblastoma. While conventional drug development is a lengthy and expensive process, drug repurposing is a faster, alternate approach, where an existing drug, not meant for treating cancer, can be repurposed to treat retinoblastoma. The present article reviews various attempts to test drugs approved for different purposes such as calcium channels blockers, non-steroidal antiinflammatory drugs, cardenolides, antidiabetic, antibiotics and antimalarial for treating retinoblastoma. It also discusses other promising candidates that could be explored for repurposing for retinoblastoma.

Fabrication of cost-effective and efficient paper-based device for viscosity measurement.

Use of paper-based devices for affordable diagnostics is gaining interest due to unique advantages such as affordability, portability, easy disposability and inherent capillarity. As capillary transportation is an integral component of paper-based devices, low sample volume with faster measurement becomes an additional advantage. We have developed a simple, paper-based microfluidic device suitable for measuring the viscosity of Newtonian fluids as well as a few non-Newtonian fluids with sample volume as little as 12-20 µL. The results could be obtained much faster than the conventional methods. A comparative analysis of the results obtained with our paper-based viscometer and with that of the conventional Ostwald viscometer shows a correlation coefficient greater than 0.99. Apart from viscosity measurement, the paper-based devices were tested for protein denaturation and polymer molecular weight determination. Our results show that the paper-based viscometer could be a potential alternative for the conventional viscometers in the viscosity range from 0.9 cP up till ∼40 cP, with added benefits in terms of time, cost and low sample volume requirement.

In Vitro and In Vivo Demonstration of Human-Ovarian-Cancer Necrosis through a Water-Soluble and Near-Infrared-Absorbing Chlorin.

With the objective of developing efficient sensitizers for therapeutic applications, we synthesized a water-soluble 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin (TDC) and investigated its in vitro and in vivo biological efficacy, comparing it with the commercially available sensitizers. TDC showed high water solubility (6-fold) when compared with that of Foscan and exhibited excellent triplet-excited-state (84%) and singlet-oxygen (80%) yields. In vitro photobiological investigations in human-ovarian-cancer cell lines SKOV-3 showed high photocytotoxicity, negligible dark toxicity, rapid cellular uptake, and specific localization of TDC in neoplastic cells as assessed by flow-cytometric cell-cycle and propidium iodide staining analysis. The photodynamic effects of TDC include confirmed reactive-oxygen-species-induced mitochondrial damage leading to necrosis in SKOV-3 cell lines. The in vivo photodynamic activity in nude-mouse models demonstrated abrogation of tumor growth without any detectable pathology in the skin, liver, spleen, or kidney, thereby demonstrating TDC application as an efficient and safe photosensitizer.

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity.

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn's disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.

Condition responsive nanoparticles for managing infection and inflammation in keratitis.

Keratitis is a major cause of avoidable visual impairment. About 30% of patients with fungal keratitis eventually become permanently blind in the developing world. Proteases, secreted by the pathogen and the host, damage the cornea before the infection is resolved. Treating keratitis is a challenge because both infection and inflammation need to be addressed. An additional challenge is to maintain a therapeutic dose at the corneal surface as blinking and tear film wash away the drugs, administered as eye drops. We have developed a nanoparticle-based drug delivery system that enhances the drug residence time by anchoring to the cornea, down-regulates inflammation and releases the antifungal drug: all in a condition-responsive manner. The expression of Toll-Like Receptors (TLR4) on the corneal epithelial cells increases in response to infection. We have conjugated anti-TLR4 antibodies on the surface of ketoconazole-encapsulated gelatin nanoparticles. The anti-TLR4 antibody not only facilitates binding of nanoparticles to the cornea, enhancing their residence time, but also reduces the levels of inflammatory cytokines. Host and fungal proteases degrade the gelatin nanoparticle, an alternative substrate for proteases, thereby reducing corneal damage and releasing the encapsulated drug, ketoconazole, proportional to the severity of infection. After testing the efficacy of the system with human corneal epithelial cells, we have extended our studies to a rat model of keratitis. The results show a significantly increased corneal retention, suppressed inflammation and resolution of infection in the infected eyes. We believe that this will be an excellent approach to manage keratitis as well as other topical ocular infections.

Structural studies on aqueous gelatin solutions: Implications in designing a thermo-responsive nanoparticulate formulation.

Gelatin as a polymer has found extensive application in the pharmaceutical industry. It is also being used, as a matrix molecule, for nanoparticle based drug delivery applications. Gelatin nanoparticles synthesised, keeping the native structure intact, show interesting properties. Synthesizing such nanoparticles requires an understanding of the structural features of gelatin under conditions of nanoparticle synthesis and preserving them during the process. To address this we have carried out an extensive characterization of gelatin using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) under various reaction conditions that are utilized in the desolvation method for gelatin nanoparticle synthesis. We investigated the gel-sol transition, hysteresis and gelatin fibre morphology under different pH and temperature conditions. We also investigated the temperature and pH dependence of triple-helix to random-coil transition in gelatin. We finally demonstrate the synthesis of gelatin nanoparticles with native gelatin. These nanoparticles show shrinkage in size (∼90nm) with increase in temperature from 30°C (369.4 ±19.8) to 40°C (282.3±9.8). Our results suggest that by carefully selecting the reaction conditions, it is possible to synthesise nanoparticles having partially folded structures and with a varying degree of sensitivity towards temperature and pH.

Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

BACKGROUND: αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. SCOPE OF REVIEW: The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. MAJOR CONCLUSIONS: Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. GENERAL SIGNIFICANCE: Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Small heat shock proteins: Role in cellular functions and pathology.

Small heat shock proteins (sHsps) are conserved across species and are important in stress tolerance. Many sHsps exhibit chaperone-like activity in preventing aggregation of target proteins, keeping them in a folding-competent state and refolding them by themselves or in concert with other ATP-dependent chaperones. Mutations in human sHsps result in myopathies, neuropathies and cataract. Their expression is modulated in diseases such as Alzheimer's, Parkinson's and cancer. Their ability to bind Cu2+, and suppress generation of reactive oxygen species (ROS) may have implications in Cu2+-homeostasis and neurodegenerative diseases. Circulating αB-crystallin and Hsp27 in the plasma may exhibit immunomodulatory and anti-inflammatory functions. αB-crystallin and Hsp20 exhitbit anti-platelet aggregation: these beneficial effects indicate their use as potential therapeutic agents. sHsps have roles in differentiation, proteasomal degradation, autophagy and development. sHsps exhibit a robust anti-apoptotic property, involving several stages of mitochondrial-mediated, extrinsic apoptotic as well as pro-survival pathways. Dynamic N- and C-termini and oligomeric assemblies of αB-crystallin and Hsp27 are important factors for their functions. We propose a "dynamic partitioning hypothesis" for the promiscuous interactions and pleotropic functions exhibited by sHsps. Stress tolerance and anti-apoptotic properties of sHsps have both beneficial and deleterious consequences in human health and diseases. Conditional and targeted modulation of their expression and/or activity could be used as strategies in treating several human disorders. The review attempts to provide a critical overview of sHsps and their divergent roles in cellular processes particularly in the context of human health and disease.

Safety, sterility and stability of direct-from-vial multiple dosing intravitreal injection of bevacizumab.

BACKGROUND: This study aims to determine the stability, sterility and safety of bevacizumab multiple dosing from a single vial without prior aliquoting. METHODS: In-vitro and human study. Six bevacizumab vials, used in multiple patients on a single day by direct withdrawal from the vial, and stored in 4°C up to a variable period, were tested for stability (high-performance liquid chromatography; [HPLC]), sterility (culture), conformational stability by circular dichroism and fluorescence spectroscopy and the rubber cork structural integrity (electron microscopy [EM]). RESULTS: HPLC of all six samples of used bevacizumab and the control bevacizumab sample were similar; culture was negative; and the EM of rubber corks did not show an open communication. Spectroscopic studies indicated drug conformational stability. Further, there was no infection or inflammation in 221 consecutive patients (973 injections) when bevacizumab was stored at 4°C and used for one week. CONCLUSION: Bevacizumab does not lose stability when stored at 4°C. It may be used for a week by direct withdrawal from the vial without fear of infection or inflammation if all standard precautions related to intravitreal injection are adhered to.

Foil assisted replica molding for fabrication of microfluidic devices and their application in vitro.

We present a simple, rapid, benchtop, Foil Assisted Rapid Molding (FARM) method for the fabrication of microfluidic devices. This novel technique involves the use of aluminium foil, pen and an X-Y plotter to create semi-circular or plano-concave, shallow microchannels. It is an easy do-it-yourself (DIY) technique for creating a microfluidic device in three simple steps: (1) create a channel design using the CAD software, (2) plot the patterns on aluminium foil and (3) use the reverse of the engraved foil as a mold to create microfluidic devices. In this report, we present a detailed study of the proposed method by varying a range of parameters such as foil thickness, tip material, and tip sizes and by investigating their effect on the creation of channels with varying geometry. Furthermore, we demonstrated the cytocompatibility of these devices in vitro.

Modulation of stem cell differentiation by the influence of nanobiomaterials/carriers.

Stem cells, either neural [NSCs] or mesenchymal [MSCs], possess tremendous untapped potential for cell therapy. Unlike the NSCs, MSCs are multi-potent and they have high self-renewal capability and broad tissue distribution. Since they do not produce significant immune rejection on post-transplantation; they are better suited for cell-based therapies. However, several critical issues need to be addressed to maximize stem cell-derived therapeutic effects. The key factor affecting the therapeutic application of stem cells is exposure to hostile conditions in vivo such as oxidative stress, which results in considerably low survival rate of these cells at transplanted sites, thereby reducing the therapeutic efficiency. Such limitation has led scientists to design clinically relevant, innovative and multifaceted solutions including the use of nanobiomaterials. Use of cytocompatible nanobiomaterials holds great promise and has gained attention of researchers, worldwide. Various nanobiomaterials are being explored to increase the survival efficiency and direct differentiation of stem cells to generate tissue-specific cells for biomedical research and futuristic therapies. These materials have superior cytocompatability, mechanical, electrical, optical, catalytic and magnetic properties. Non-invasive visualization of the biological system has been developed using magnetic nanoparticles and magnetic resonance imaging [MRI] approaches. Apart from viral vectors, non-viral carriers such as DNA nano carriers, single stranded RNA nanoparticles, liposomes and carbon nanotubes/wires are being exploited for gene delivery into stem cells. This article reviews potential application of various biocompatible nanomaterials in stem cell research and development.