UPLC SEPARATION ANALYSIS OF EMTRICITABINE, TENOFOVIR, COBICISTAT AND ELVITEGRAVIR FROM THEIR DEGRADATION PRODUCTS

Objective: A simple, rapid, accurate and precise stability-indicating UPLC analytical method has been developed and validated for the quantitative analysis of Emtricitabine, Tenofovir, Cobicistat and Elvitegravir in bulk drugs and combined dosage forms.Methods: ACE C18 (50 mm x 3 mm, 2µ). The column temperature was maintained at 30oC and run time 8 min. The mobile phase was a mixture of Mobile Phase: A–0.1% TFA in Acetonitrile, B–0.1% TFA in Milli-Q-water. The injection volume of samples was 20μl. UV detection was carried out using a UV-PDA detector at 240 nm. The validation of this method was done as per ICH guidelines.Results: The retention times were observed as 1.46, 3.59, 4.13, 4.64 min for Emtricitabine, Tenofovir disoproxyl fumarate, Cobicistat, and Elvitegravir respectively. Linearity ranges were observed 150-275 µg/ml Emtricitabine, 250-375 µg/ml Tenofovir, 100-225 µg/ml Cobicistat and 100-225 µg/ml Elvitegravir. Relative Standard Deviation did not exceed 2.Conclusion: The newly developed UPLC method for separation of different degradation products along with the pure drugs were found to be capable of giving faster retention times while still maintaining good resolution than that achieved with conventional HPLC. The decreased flow rate 0.4 ml/min, in UPLC indicate more economical. This method exhibited an excellent performance in terms of sensitivity and speed. The results of stress testing undertaken according to the ICH guidelines reveal that the method is specific and stability-indicating. The proposed method has the ability to separate these drugs from their degradation products in tablet dosage forms and hence can be applied to the analysis of routine quality control samples and samples obtained from stability studies.Keywords: Stability indicating assay, RP-UPLC, Emtricitabine, Tenofovir, Cobicistat, Elvitegravir, Forced degradation studie

AstroSat observations of eclipsing high mass X-ray binary pulsar OAO 1657-415

We present the results obtained from analysis of two AstroSat observations of the high mass X-ray binary pulsar OAO 1657-415. The observations covered 0.681-0.818 and 0.808-0.968 phases of the $\sim$10.4 day orbital period of the system, in March and July 2019, respectively. Despite being outside the eclipsing regime, the power density spectrum from the first observation lacks any signature of pulsation or quasi-periodic oscillations. However, during July observation, X-ray pulsations at a period of 37.0375 s were clearly detected in the light curves. The pulse profiles from the second observation consist of a broad single peak with a dip-like structure in the middle across the observed energy range. We explored evolution of the pulse profile in narrow time and energy segments. We detected pulsations in the light curves obtained from 0.808--0.92 orbital phase range, which is absent in the remaining part of the observation. The spectrum of OAO 1657-415 can be described by an absorbed power-law model along with an iron fluorescent emission line and a blackbody component for out-of-eclipse phase of the observation. Our findings are discussed in the frame of stellar wind accretion and accretion wake at late orbital phases of the binary.Comment: 11 pages, 8 figures and 2 tables; Accepted for publication in the Journal of Astrophysics & Astronom

Detection of X-ray pulsations at the lowest observed luminosity of Be/X-ray binary pulsar EXO 2030+375 with AstroSat

We present the results obtained from timing and spectral studies of Be/X-ray binary pulsar EXO 2030+375 using observations with the Large Area Xenon Proportional Counters and Soft X-ray Telescope of AstroSat, at various phases of its Type-I outbursts in 2016, 2018, and 2020. The pulsar was faint during these observations as compared to earlier observations with other observatories. At the lowest luminosity of 2.5$\times$10$^{35}$ erg s$^{-1}$ in 0.5--30 keV energy range, $\approx$41.3 s pulsations were clearly detected in the X-ray light curves. This finding establishes the first firm detection of pulsations in EXO 2030+375 at an extremely low mass accretion rate to date. The shape of the pulse profiles is complex due to the presence of several narrow dips. Though pulsations were detected up to $\sim$80 keV when the source was brighter, pulsations were limited up to $\sim$25 keV during the third AstroSat observation at lowest source luminosity. A search for quasi-periodic oscillations in 2$\times$10$^{-4}$ Hz to 10 Hz yielded a negative result. Spectral analysis of the AstroSat data showed that the spectrum of the pulsar was steep with a power-law index of $\sim$2. The values of photon-indices at observed low luminosities follow the known pattern in sub-critical regime of the pulsar.Comment: 12 pages, 8 figures and 2 tables; Accepted for publication in the Journal of Astrophysics & Astronom

Population Preference of Net Texture prior to Bed Net Trial in Kala-Azar–Endemic Areas

Prior to a community-based efficacy trial of long-lasting insecticidal nets (LLINs) in the prevention of visceral leishmaniasis (VL; also called kala-azar), a pilot study on preference of tools was held in endemic areas of India and Nepal in September 2005

Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects

Rapidly exploring structural and dynamic properties of signaling networks using PathwayOracle

<p>Abstract</p> <p>Background</p> <p>In systems biology the experimentalist is presented with a selection of software for analyzing dynamic properties of signaling networks. These tools either assume that the network is in steady-state or require highly parameterized models of the network of interest. For biologists interested in assessing how signal propagates through a network under specific conditions, the first class of methods does not provide sufficiently detailed results and the second class requires models which may not be easily and accurately constructed. A tool that is able to characterize the dynamics of a signaling network using an unparameterized model of the network would allow biologists to quickly obtain insights into a signaling network's behavior.</p> <p>Results</p> <p>We introduce <it>PathwayOracle</it>, an integrated suite of software tools for computationally inferring and analyzing structural and dynamic properties of a signaling network. The feature which differentiates <it>PathwayOracle </it>from other tools is a method that can predict the response of a signaling network to various experimental conditions and stimuli using only the connectivity of the signaling network. Thus signaling models are relatively easy to build. The method allows for tracking signal flow in a network and comparison of signal flows under different experimental conditions. In addition, <it>PathwayOracle </it>includes tools for the enumeration and visualization of coherent and incoherent signaling paths between proteins, and for experimental analysis – loading and superimposing experimental data, such as microarray intensities, on the network model.</p> <p>Conclusion</p> <p><it>PathwayOracle </it>provides an integrated environment in which both structural and dynamic analysis of a signaling network can be quickly conducted and visualized along side experimental results. By using the signaling network connectivity, analyses and predictions can be performed quickly using relatively easily constructed signaling network models. The application has been developed in Python and is designed to be easily extensible by groups interested in adding new or extending existing features. <it>PathwayOracle </it>is freely available for download and use.</p

A cellular defense memory imprinted by early life toxic stress

Stress exposure early in life is implicated in various behavioural and somatic diseases. Experiences during the critical perinatal period form permanent, imprinted memories promoting adult survival. Although imprinting is widely recognized to dictate behaviour, whether it actuates specific transcriptional responses at the cellular level is unknown. Here we report that in response to early life stresses, Caenorhabditis elegans nematodes form an imprinted cellular defense memory. We show that exposing newly-born worms to toxic antimycin A and paraquat, respectively, stimulates the expression of toxin-specific cytoprotective reporters. Toxin exposure also induces avoidance of the toxin-containing bacterial lawn. In contrast, adult worms do not exhibit aversive behaviour towards stress-associated bacterial sensory cues. However, the mere re-encounter with the same cues reactivates the previously induced cytoprotective reporters. Learned adult defenses require memory formation during the L1 larval stage and do not appear to confer increased protection against the toxin. Thus, exposure of C. elegans to toxic stresses in the critical period elicits adaptive behavioural and cytoprotective responses, which do not form imprinted aversive behaviour, but imprint a cytoprotective memory. Our findings identify a novel form of imprinting and suggest that imprinted molecular defenses might underlie various pathophysiological alterations related to early life stress. © 2019, The Author(s)

Stress biology:Complexity and multifariousness in health and disease

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.</p

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell