Trehalose Monooleate: A Potential Antiaggregation Agent for Stabilization of Proteins.

Protein aggregation is a major problem of therapeutic proteins because aggregation decreases their therapeutic activity and shelf life and induces immunogenicity. Stabilization against aggregation is commonly attained by addition of different excipients like sugars, surfactants, buffers, salts, amino acids, polymers, etc. Generally these excipients are required in combination for stabilization. Sugars are required at a higher concentration, and commonly used surfactants like polysorbates have shortcomings due to oxidative degradation. With a view to have a multipurpose excipient to be effective at a lower concentration, we designed antiaggregation agents (AAAs) that would encompass the functionalities of two or more conventional excipients and would curtail the number of excipients to be added for stabilization. Our first designed AAA, trehalose monooleate (TMO), is a sugar-fatty acid derivative. It has been evaluated in silico by docking on aggregation prone regions of model protein bovine serum albumin (BSA), and experimentally its effectiveness has been validated as stabilizer against agitation and thermal stress. TMO has a lower CMC of 6 mg/L, is nonhemolytic, and was found to be nontoxic by sulforhodamine B (SRB) colorimetric assay in Human Hepatoma Cell Line (Hep-G2) using adriamycin as positive contol. Various spectroscopic and separation analytical techniques were employed to monitor the aggregation profile of BSA in presence and absence of TMO. CD spectroscopy showed complete retention of helical structure at concentration as low as 0.05% of TMO, while fluorescence spectroscopy provided vital insights into conformational stability rendered by TMO. Native-PAGE and SEC-HPLC studies demonstrated absence of aggregates. Molecular dynamics study on BSA-TMO docked complex further substantiated the stabilization effect. Overall, it can be said that TMO has good antiaggregation property. The present work is a preliminary attempt toward understanding protein excipient interactions and chemistry to provide rational basis for designing a single excipient for stabilization of protein formulations.

Women 'holding it' in urban India: Toilet avoidance as an under-recognized health outcome of sanitation insecurity.

Emerging research on sanitation challenges in the Global South increasingly uncovers health and social impacts by gender, particularly lack of sanitation safety. Women may employ strategies to avoid urination or defecation ('holding it') in the absence of safe sanitation, but the practice is not well understood. We quantitatively analyze survey data on women from urban slums across three cities in Maharashtra, India whose households constructed a toilet through an intervention programme. We assess relationships between household versus shared sanitation, perceptions of safety, and women's toilet avoidance behaviours, including diet restriction. At baseline, women have more than three times the odds of reporting avoidance behaviours if they perceive a community toilet to be unsafe, even after controlling for other factors. Household water insecurity is also instrumental in the relationship between avoidance and lack of safety. Finally, avoidance exhibits a significant and major drop upon provision of a household toilet. This study provides substantial support for the prevalence of habitual toilet avoidance among vulnerable urban women without access to safe sanitation. We conclude with recommendations for policy approaches and call for more attention to the health repercussions of habitual toilet avoidance among women as a consequence of sanitation insecurity.

Rational approach for design and evaluation of anti-aggregation agents for protein stabilization: A case study of trehalose phenylalaninate.

The present work introduces new anti-aggregation agent (AAA) derived through our new approach for design and evaluation of anti-aggregation agent as a multi-purpose excipient to combat protein aggregation. Therapeutic proteins undergo aggregation due to even minor changes in environmental conditions like temperature, pH, shear and stress. Excipients play a vital role in prevention of aggregation. To stabilize a protein formulation different classes of excipients are used in combination after carefully selecting through laborious and time consuming trial and error experiments. To resolve these concerns, we have developed a rational approach based on molecular docking analysis and have designed, synthesized AAAs, and validated the approach by experimental studies. Trehalose phenylalaninate (TPA) has been synthesized and evaluated for stabilization of Bovine serum albumin (BSA). TPA was found to be non-toxic with a LC50 of >80µg/ml. BSA solutions with and without TPA were subjected to thermal and agitation stress and aggregation was monitored using sophisticated analytical techniques. The helical structure of BSA was completely retained in stressed samples at 0.1% concentration of TPA. SEC-HPLC clearly demonstrated the absence of aggregates in presence of TPA. Although aggregation was not seen in fluorescence spectra but quenching due to TPA was evident. Moreover, molecular dynamics study on BSA-TPA complex showed lower RMSD.

Thymol, a monoterpene, inhibits aldose reductase and high-glucose-induced cataract on isolated goat lens.

BACKGROUND: Overactivation of aldose reductase (AR) enzyme has been implicated in the development of various diabetic complications. In the present study, the inhibitory effect of thymol was investigated on AR enzyme and its anti-cataract activity was also examined on isolated goat lens. MATERIALS AND METHODS: Various concentrations of thymol were incubated with AR enzyme prepared from isolated goat lens. Molecular docking studies were carried out using Schrodinger software to verify the binding of thymol with AR as well as to understand their binding pattern. Further, thymol was evaluated for its anti-cataract activity in high-glucose-induced cataract in isolated goat lens in vitro. Quercetin was maintained as standard (positive control) throughout the study. RESULTS: Thymol showed potent inhibitory activity against goat lens AR enzyme with an IC50 value of 0.65 µg/ml. Docking studies revealed that thymol binds with AR in similar binding pattern as that of quercetin. The high-glucose-induced cataract in isolated goat lens was also improved by thymol treatment. Thymol was also able to significantly (P < 0.001) reduce the oxidative stress associated with cataract. CONCLUSION: The results suggest that thymol may be a potential therapeutic approach in the prevention of diabetic complications through its AR inhibitory and antioxidant activities.

Osteopontin as a therapeutic target for cancer.

INTRODUCTION: Cancer is a complex pathological disorder, established as a result of accumulation of genetic and epigenetic changes, which lead to adverse alterations in the cellular phenotype. Tumor progression involves intricate signaling mediated through crosstalk between various growth factors, cytokines and chemokines. Osteopontin (OPN), a chemokine-like protein, is involved in promotion of neoplastic cancer into higher grade malignancies by regulating various facets of tumor progression such as cell proliferation, angiogenesis and metastasis. AREAS COVERED: Tumors as well as stroma-derived OPN play key roles in various signaling pathways involved in tumor growth, angiogenesis and metastasis. OPN derived from tumor-activated macrophages modulates the tumor microenvironment and thereby regulate melanoma growth and angiogenesis. OPN also regulates hypoxia-inducible factor-1α-dependent VEGF expression leading to breast tumor growth and angiogenesis in response to hypoxia. Thus, a clear understanding of the molecular mechanism underlying OPN-mediated regulation will shed light on exciting avenues for further investigation of targeted therapies. Silencing of OPN using RNAi technology, blocking OPN activity using specific antibodies and small-molecule inhibitors might provide novel strategies, which would aid in developing effective therapeutics for the treatment of various types of cancer. EXPERT OPINION: This review focuses on new possibilities to exploit OPN as a tumor and stroma-derived therapeutic target to combat cancer.

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin α(v)ß(3) interaction.

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α(v)ß(3) integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α(v)ß(3) integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.

Osteopontin: a potentially important therapeutic target in cancer.

INTRODUCTION: Cancer is an extremely complex disease and most cancer treatments are limited to chemotherapy, radiation and surgery. The progression of tumours towards malignancy requires the interaction of various cytokines, growth factors, transcription factors and effector molecules. Osteopontin is a cytokine-like, calcium-binding, extracelular-matrix- associated member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of proteins. It plays an important role in determining the oncogenic potential of various cancers. The role of osteopontin in various pathophysiological conditions suggests that the alteration in post-translational modification result in different functional forms that might change its normal physiological functions. AREAS COVERED: Osteopontin -based anticancer therapy, which may provide a new insight for the effective management of cancer. EXPERT OPINION: A better understanding of the signalling mechanism by which osteopontin promotes tumourigenesis may be useful in crafting novel osteopontin -based anticancer therapy. The role of osteopontin in promoting cancer progression is the subject of in depth investigation and thus targeting osteopontin might be a suitable therapeutic approach for the treatment of cancer.

Vitreous humor: biochemical constituents in estimation of postmortem interval.

Analysis of biochemical constituents of the vitreous humor can be useful in determining the postmortem interval as there is proportionate postmortem rise of potassium and fall in sodium concentration. We studied 120 autopsy cases to determine the utility of potassium, sodium, calcium, and chloride levels, and sodium/potassium ratio in estimating the postmortem interval. There was a linear relationship between vitreous potassium concentration and postmortem interval, whereas an inverse relationship between vitreous sodium/potassium ratio and postmortem interval was noted. Other factors like age, sex, cause of death, season of death, and refrigeration of sample did not influence the vitreous humor potassium values. Using the statistical tools, a new formula was derived to determine the postmortem interval based on the potassium concentration and a review of previous literature is presented. Hence, the findings of this study supported a central role of vitreous humor biochemistry in many postmortem forensic and pathological evaluations.

Anaesthetic Considerations in a Patient with Klippelfeil Syndrome Undergoing Occipito Cervical Fixation

A rare case with Klippel-Feil syndrome with a classic triad having short neck, low posterior hair line and restricted motion of neck because of fused cervical vertebrae was scheduled for occipito cervical fixation, foramen magnum decompression, fusion with local bones and cervical traction. We present the anaesthetic management of this patient highlighting the various anomalies associated with Klippel-Feil syndrome and the presence of a difficult airway.

Prostaglandin E2 regulates tumor angiogenesis in prostate cancer.

In cancer management, the cyclooxygenase (COX)-targeted approach has shown great promise in anticancer therapeutics. However, the use of COX-2 inhibitors has side effects and health hazards; thus, targeting its major metabolite prostaglandin E(2) (PGE(2))-mediated signaling pathway might be a rational approach for the next generation of cancer management. Recent studies on several in vitro and in vivo models have revealed that elevated expression of COX-2 correlates with prostate tumor growth and angiogenesis. In this study, we have shown the in-depth molecular mechanism and the PGE(2) activation of the epidermal growth factor receptor and beta3 integrin through E prostanoid 2 (EP2)-mediated and EP4-mediated pathways, which lead to activator protein-1 (AP-1) activation. Moreover, PGE(2) also induces activating transcription factor-4 (ATF-4) activation and stimulates cross-talk between ATF-4 and AP-1, which is unidirectional toward AP-1, which leads to the increased expressions of urokinase-type plasminogen activator and vascular endothelial growth factor and, eventually, regulates prostate tumor cell motility. In vivo Matrigel angiogenesis assay data revealed that PGE(2) induces angiogenesis through EP2 and EP4. Human prostate cancer specimen analysis also supported our in vitro and in vivo studies. Our data suggest that targeting PGE(2) signaling pathway (i.e., blocking EP2 and EP4 receptors) might be a rational therapeutic approach for overcoming the side effects of COX-2 inhibitors and that this might be a novel strategy for the next generation of prostate cancer management.

Curcumin suppresses breast tumor angiogenesis by abrogating osteopontin-induced VEGF expression.

The development and progression of malignant tumors depends on the formation of new blood vessels inside the tumor. This phenomenon is termed tumor angiogenesis. Angiogenesis is one of the fundamental processes that occur during cancer progression, and depends on the expression and activation of various angiogenic molecules, cytokines, growth factors, kinases and transcription factors. We recently demonstrated that the chemokine-like ECM-associated protein osteopontin (OPN) turns on the angiogenic switch by upregulating expression of vascular endothelial growth factor (VEGF) in a human breast cancer model. Furthermore, we proposed that targeting OPN-induced VEGF expression could be a potential therapeutic approach for the treatment of breast cancer. In this study, we demonstrate that curcumin (diferuloylmethane) abrogates OPN-induced VEGF expression and curbs OPN-induced VEGF-dependent breast tumor angiogenesis in vivo. We also explore the fact that curcumin in combination with anti-VEGF or anti-neuropilin (NRP)-1 antibody exhibits enhanced anti-angiogenic activity compared to curcumin alone. Our results indicate that curcumin suppresses OPN-induced VEGF expression and tumor angiogenesis, and suggest that this study may aid in the development of a curcumin-based OPN-targeted therapeutic approach to the control of breast tumor angiogenesis.