Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 µM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

Energy-efficient synthetic antiferromagnetic skyrmion-based artificial neuronal device.

Magnetic skyrmions offer unique characteristics such as nanoscale size, particle-like behavior, topological stability, and low depinning current density. These properties make them promising candidates for next-generation spintronics-based memory and neuromorphic computing. However, one of their distinctive features is their tendency to deviate from the direction of the applied driving force that may lead to the skyrmion annihilation at the edge of nanotrack during skyrmion motion, known as the skyrmion Hall effect (SkHE). To overcome this problem, synthetic antiferromagnetic (SAF) skyrmions that having bilayer coupling effect allows them to follow a straight path by nullifying SkHE making them alternative for ferromagnetic (FM) counterpart. This study proposes an integrate-and-fire (IF) artificial neuron model based on SAF skyrmions with asymmetric wedge-shaped nanotrack having self-sustainability of skyrmion numbers at the device window. The model leverages inter-skyrmion repulsion to replicate the IF mechanism of biological neuron. The device threshold, determined by the maximum number of pinned skyrmions at the device window, can be adjusted by tuning the current density applied to the nanotrack. Neuronal spikes occur when initial skyrmion reaches the detection unit after surpassing the device window by the accumulation of repulsive force that result in reduction of the device's contriving current results to design of high energy efficient for neuromorphic computing. Furthermore, work implements a BNN accelerator using proposed IF neuron and SAF-SOT MRAM-based synaptic devices for MNIST image classification.The presentedapproach achieves significantly higher energy efficiency compared to existing technologies like SRAM and STT-MRAM, with improvements of 2.31x and 1.36x, respectively. The presented accelerator achieves 1.42x and 1.07x higher throughput efficiency per Watt as compared to conventional SRAM and STT-MRAM based designs. .

Direct and Sequential Bioactivation of Pemigatinib to Reactive Iminium Ion Intermediates Culminates in Mechanism-Based Inactivation of Cytochrome P450 3A.

We recently established the mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data have also revealed that pemigatinib (PEM), another clinically approved FGFR1-3 inhibitor, similarly elicited time-dependent inhibition of CYP3A. This was rather unexpected, as it was previously purported that PEM did not pose any metabolism-dependent liabilities due to the absence of glutathione-related conjugates in metabolic profiling experiments conducted in human liver microsomes. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time-, concentration-, and cofactor-dependent manner consistent with MBI, with inactivator concentration at half-maximum rate constant, maximum inactivation rate constant, and partition ratio of 8.69 and 11.95 µM, 0.108 and 0.042 min-1, and approximately 44 and approximately 47 for CYP3A4 and CYP3A5, respectively. Although the rate of inactivation was diminished by coincubation with an alternative substrate or direct inhibitor of CYP3A, the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudo-irreversible metabolite-intermediate complexes. Finally, utilizing cyanide trapping and high-resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O-desmethylated metabolite at its distal morpholine moiety to reactive iminium ion hard electrophilic species that could covalently inactivate CYP3A via MBI. SIGNIFICANCE STATEMENT: This study reports for the first time the covalent MBI of CYP3A by PEM and deciphered its bioactivation pathway involving the metabolic activation of PEM and its major O-desmethylated metabolite to reactive iminium ion intermediates. Following which, a unique covalent docking methodology was harnessed to unravel the structural and molecular determinants underpinning its inactivation. Findings from this study lay the foundation for future investigation of clinically relevant drug-drug interactions between PEM and concomitant substrates of CYP3A.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Benzbromarone.

Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme-mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K I, k inact, and partition ratio of 11.61 µM, 0.10 minutes-1, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an ∼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site. SIGNIFICANCE STATEMENT: Although the bioactivation of benzbromarone (BBR) to reactive metabolites has been well characterized, its potential to cause mechanism-based inactivation (MBI) of cytochrome P450 has not been fully investigated. This study reports the MBI of CYP3A4 by BBR via irreversible covalent adduction and develops a unique covalent docking methodology to predict the structural molecular determinants underpinning the inactivation for the first time. These findings lay the groundwork for future investigation of clinically relevant drug-drug interactions implicating BBR and mechanisms of BBR-induced idiosyncratic hepatotoxicity.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Infigratinib Is a Reversible Inhibitor and Mechanism-Based Inactivator of Cytochrome P450 3A4.

Infigratinib (INF) is a promising selective inhibitor of fibroblast growth factor receptors 1-3 that has recently been accorded both orphan drug designation and priority review status by the US Food and Drug Administration for the treatment of advanced cholangiocarcinoma. Its propensity to undergo bioactivation to electrophilic species was recently expounded upon. However, other than causing aberrant idiosyncratic toxicities, these reactive intermediates may elicit mechanism-based inactivation of cytochrome P450 enzymes. In this study, we investigated the interactions between INF and the most abundant hepatic CYP3A. Our findings revealed that, apart from being a potent noncompetitive reversible inhibitor of CYP3A4, INF inactivated CYP3A4 in a time-, concentration- and NADPH-dependent manner with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratio of 4.17 µM, 0.068 minute-1, and 41, respectively, when rivaroxaban was employed as the probe substrate. Coincubation with testosterone (alternative CYP3A substrate) or ketoconazole (direct CYP3A inhibitor) attenuated the rate of inactivation, whereas the inclusion of glutathione and catalase did not confer such protection. The lack of enzyme activity recovery after dialysis for 4 hours and oxidation with potassium ferricyanide, coupled with the absence of the characteristic Soret peak signature collectively substantiated that inactivation of CYP3A4 by INF was not mediated by the formation of quasi-irreversible metabolite-intermediate complexes but rather through irreversible covalent adduction to the prosthetic heme and/or apoprotein. Finally, glutathione trapping and high-resolution mass spectrometry experimental results unraveled two plausible bioactivation mechanisms of INF arising from the generation of a p-benzoquinonediimine and epoxide reactive intermediate. SIGNIFICANCE STATEMENT: The potential of INF to cause MBI of CYP3A4 was unknown. This study reports the reversible noncompetitive inhibition and irreversible covalent MBI of CYP3A4 by INF and proposes two potential bioactivation pathways implicating p-benzoquinonediimine and epoxide reactive intermediates, following which a unique covalent docking methodology was harnessed to elucidate the structural and molecular determinants underscoring its inactivation. Findings from this study lay the groundwork for future investigation of clinically relevant drug-drug interactions between INF and concomitant substrates of CYP3A4.

Locus-Specific Reversible DNA Methylation Regulates Transient IL-10 Expression in Th1 Cells.

IL-10 is a pleiotropic cytokine with multifaceted functions in establishing immune homeostasis. Although expressed by Th1 and Th2 cells, conventional Th1 cells produce marginal levels of IL-10 compared with their Th2 counterparts. In this study, we investigated the epigenetic mechanisms of Il-10 gene expression in Th1 cells. Bioinformatics EMBOSS CpG plot analysis and bisulfite pyrosequencing revealed three CpG DNA methylation sites in the Il-10 gene locus. Progressive DNA methylation at all of the CpG regions of interest (ROIs) established a repressive program of Il-10 gene expression in Th1 cells. Interestingly, Th1 cells treated with IL-12 and IL-27 cytokines, thereby mimicking a chronic inflammatory condition in vivo, displayed a significant increase in IL-10 production that was accompanied by selective DNA demethylation at ROI 3 located in intron 3. IL-10-producing T cells isolated from lymphocytic choriomeningitis virus-infected mice also showed enhanced DNA demethylation at ROI 3. Binding of STAT1 and STAT3 to demethylated ROI 3 enhanced IL-10 expression in an IL-12/IL-27-dependent manner. Accordingly, CD4+ T cells isolated from STAT1- or STAT3-knockout mice were significantly defective in IL-10 production. Our data suggest that, although stably maintained DNA methylation at the promoter may repress IL-10 expression in Th1 cells, locus-specific reversible DNA demethylation may serve as a threshold platform to control transient Il-10 gene expression.

Widening cracks in patriarchy: mothers and daughters navigating gender norms in a Mumbai slum.

Inequitable gender norms can be harmful to girls' and boys' health and sexuality. Programmatic approaches that help renegotiate gendered power relationships are sorely needed. This qualitative study reveals how Parivartan, a sport-based intervention in a Mumbai informal settlement, helped families resist inequitable gender norms that limited girls' mobility in public spaces. Fifteen girl athletes were interviewed in two rounds of face-to-face in-depth interviews. Results identify the strategies girls' mothers used to support their daughters' participation in the programme when they feared their husbands' disapproval. Rather than openly confronting their husbands, mothers worked from within the patriarchal gender order, through its 'cracks', for instance initially hiding their daughters' participation from their husbands. At an appropriate moment, girls' mothers revealed to their husbands about their daughters playing sports, convincing them of the usefulness of the programme. Girls' participation profoundly and positively affected relationships between daughters, mothers and fathers. Over time, parents' trust that girls would not compromise family honour increased, eventually changing the acceptability of girls' playing sport in public in spite of the patriarchal gender order. Concluding remarks offer key implications for effective interventions, highlighting the historical nature of gender transformation processes.

Is unintended birth associated with physical intimate partner violence? Evidence from India.

A growing number of studies have tested the association between intimate partner violence (IPV) and the unintendedness of pregnancy or birth, and most have suggested that unintendedness of pregnancy is a cause of IPV. However, about nine in every ten women face violence after delivering their first baby. This study examined the effects of the intendedness of births on physical IPV using data from the National Family Health Survey (2015-16). The multivariate logistic regression model analysis found that, compared with women with no unwanted births (2.9%), physical IPV was higher among those women who had unwanted births (6.9%, p<0.001), followed by those who had mistimed births (4.4 %, p<0.001), even after adjusting for several women's individual and socioeconomic characteristics. Thus, the reduction of women with mistimed and unwanted births could reduce physical IPV in India. The study highlights the unfinished agenda of family planning in the country and argues for the need to integrate family planning and Reproductive, Maternal and Child Health Care (RMNCH) services to yield multi-sectoral outcomes, including the elimination of IPV.

Presence of Intra-helical Salt-Bridge in Loop E Half-Helix Can Influence the Transport Properties of AQP1 and GlpF Channels: Molecular Dynamics Simulations of In Silico Mutants.

Major intrinsic protein (MIP) superfamily contains water-transporting AQP1 and glycerol-specific GlpF belonging to two major phylogenetic groups, namely aquaporins (AQPs) and aquaglyceroporins (AQGPs). MIP channels have six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). LE region contributes two residues to the aromatic/arginine (Ar/R) selectivity filter (SF) within the MIP channel. Bioinformatics analyses have shown that all AQGPs have an intra-helical salt-bridge (IHSB) in LE half-helix and all AQGPs and majority of AQPs have helix destabilizing Gly and/or Pro in the same region. In this paper, we mutated in silico the acidic and basic residues in GlpF to Ser and introduced salt-bridge interaction in AQP1 LE half-helix by substituting Ser residues at the equivalent positions with acidic and basic residues. We investigated the influence of IHSB in LE half-helix on the transport properties of GlpF and AQP1 mutant channels using molecular dynamics simulations. With IHSB abolished in LE half-helix, the GlpF mutant exhibited a significantly reduced water transport. In contrast, the introduction of IHSB in the two AQP1 mutants has increased water transport. Absence of salt-bridge in LE half-helix alters the SF geometry and results in a higher energy barrier for the solutes in the Ar/R selectivity filter. Presence/absence of IHSB in LE half-helix influences the channel transport properties and it is evident especially for the AQGPs. By modulating its helical flexibility, LE half-helix can perhaps play a regulatory role in transport either on its own or in conjunction with other extracellular regions.

The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors.

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 µs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.

NFAT1 Regulates Systemic Autoimmunity through the Modulation of a Dendritic Cell Property.

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Choice of contraceptive methods in public and private facilities in rural India.

BACKGROUND: Client-centric quality of care (QoC) in family planning (FP) services are imperative for contraceptive method adoption and continuation. Less is known about the choice of contraceptive method in India beyond responses to the three common questions regarding method information, asked in demographic and health surveys. This study argues for appropriate measurement of method choice and assesses its levels and correlates in rural India. METHODS: A cross-sectional study was conducted with new acceptors of family planning method (N = 454) recruited from public and private health facilities in rural Bihar and Uttar Pradesh, the two most populous states in India. The key quality of care indicator 'method choice' was assessed using four key questions from client-provider interactions that help in making a choice about a particular method: (1) whether the provider asked the client about their preferred method, (2) whether the provider told the client about at least one additional method, (3) whether the client received information without any single method being promoted by the provider, and (4) client's perception about receipt of method choice. The definition of method choice in this study included women who responded "yes" to all four questions in the survey. The relationship between contraceptive communication and receipt of method choice was assessed using logistic regression analyses, after adjusting for socio-demographic characteristics of the respondents. RESULTS: Although 62% of clients responded to a global question and reported that they received the method of their choice, only 28% received it based on responses about client-provider interactions. Receipt of the information on side-effects of the selected method (Adjusted Odds Ratio [AOR]: 7.4, 95% Confidence Interval [CI]: 3.96-13.86) and facility readiness to provide a range of contraceptive choice (AOR: 2.67, 95% CI: 1.48-4.83) were significantly associated with receipt of method choice. CONCLUSIONS: Findings demonstrated that women's choice of contraceptive could be improved in rural India if providers give full information prior to and during the acceptance of a method and if facilities are equipped to provide a range of choice of contraceptive methods.

Infection prevention preparedness and practices for female sterilization services within primary care facilities in Northern India.

BACKGROUND: In 2014, 16 women died following female sterilization operations in Bilaspur, a district in central India. In addition to those 16 deaths, 70 women were hospitalized for critical conditions (Sharma, Lancet 384,2014). Although the government of India's guidelines for female sterilization mandate infection prevention practices, little is known about the extent of infection prevention preparedness and practice during sterilization procedures that are part of the country's primary health care services. This study assesses facility readiness for infection prevention and adherence to infection prevention practices during female sterilization procedures in rural northern India. METHOD: The data for this study were collected in 2016-2017 as part of a family planning quality of care survey in selected public health facilities in Bihar (n = 100), and public (n = 120) and private health facilities (n = 97) in Uttar Pradesh. Descriptive analysis examined the extent of facility readiness for infection prevention (availability of handwashing facilities, new or sterilized gloves, antiseptic lotion, and equipment for sterilization). Correlation and multivariate statistical methods were used to examine the role of facility readiness and provider behaviors on infection prevention practices during female sterilization. RESULT: Across the three health sectors, 62% of facilities featured all four infection prevention components. Sterilized equipment was lacking in all three health sectors. In facilities with all four components, provider adherence to infection prevention practices occurred in only 68% of female sterilization procedures. In Bihar, 76% of public health facilities evinced all four components of infection prevention, and in those facilities provider's adherence to infection prevention practices was almost universal. In Uttar Pradesh, where only 55% of public health facilities had all four components, provider adherence to infection prevention practices occurred in only 43% of female sterilization procedures. CONCLUSION: The findings suggest that facility preparedness for infection prevention does play an important role in provider adherence to infection prevention practices. This phenomenon is not universal, however. Not all doctors from facilities prepared for infection prevention adhere to the practices, highlighting the need to change provider attitudes. Unprepared facilities need to procure required equipment and supplies to ensure the universal practice of infection prevention.

A randomized controlled trial comparing gastro-laryngeal tube with endotracheal intubation for airway management in patients undergoing ERCP under general anaesthesia.

BACKGROUND: Gastro laryngeal tube (GLT) is a newly introduced device. It is an advanced purpose specific design (essentially a modified laryngeal tube) which especially provides a separate wide channel specifically designed for the introduction of a gastroscope for endoscopic retrograde cholangio-pancreatography (ERCP), simultaneously functioning as a supra-glottic airway device for ventilation. METHODS: In a randomized controlled trial on 100 patients undergoing ERCP under GA, GLT was compared with endotracheal tube as an alternative airway device. Device insertion conditions, oxygenation and ventilation parameters were recorded. RESULTS: GLT was found to be comparable with ETT. Success rate of insertion of GLT was high (92%) and the insertion time of GLT was significantly shorter 42 (20-210) s vs. 206 (176-320) s - median (range). Both the devices were equally effective in normal oxygenation and ventilation. The recovery time was significantly shorter and postoperative complications such as hoarseness and dysphonia were less common in GLT group. Inserting conditions for the duodenoscope were better in GLT group. CONCLUSION: In this study, likely to be first of its kind, it is concluded that the GLT is a suitable and better alternative to ETT as it allows adequate ventilation and is associated with faster recovery times and minimal extubation-related complications while enhancing operative conditions for gastroenterologists. Its regular use in patients undergoing ERCP is strongly recommended.

Optical guiding-based cell focusing for Raman flow cell cytometer.

We report the use of an optical guiding arrangement generated in a microfluidic channel to produce a stream of single cells in a line for single-cell Raman spectroscopic analysis. The optical guiding arrangement consisted of dual-line optical tweezers, generated using a 1064 nm laser, aligned in the shape of a '' symbol. By controlling the laser power in the tweezers and the flow rate in the microfluidic channel, a single line flow of cells could be produced in the tail of the guiding arrangement, where the 514.5 nm Raman excitation beam was also located. Furthermore, by resonantly exciting the Raman spectrum, a good-quality Raman spectrum could be recorded from the flowing single cells as they passed through the Raman excitation focal spot without the need to trap the cells. As a proof of concept, it was shown that red blood cells (RBCs) could be guided to the tail of the optical guide and the Raman spectra of the resonantly excited cells could be recorded in a continuous manner without trapping the cells at a cell flow rate of ∼500 cells per h. From the recorded spectra, we were able to distinguish between RBCs containing hemoglobin in the normal form (normal-RBCs) and the met form (met-RBCs) from a mixture of RBCs comprising met-RBCs and normal-RBCs in a ratio of 1 : 9.

Contesting restrictive mobility norms among female mentors implementing a sport based programme for young girls in a Mumbai slum.

BACKGROUND: Harmful gender norms are known structural barriers to many public health and development interventions involving adolescent girls. In India, restrictions on girls' liberty to move freely in public spaces contribute to school dropout and early marriage, and negatively affect girls' health and wellbeing, from adolescence into adulthood. We report on mechanisms of change among female mentors 18 to 24 years old who contested discriminatory norms while implementing a sports-based programme for adolescent girls in a Mumbai slum. METHODS: We adopted a prospective qualitative research design. Our analysis is based on case studies derived from two rounds of face to face, in -depth interviews with 10 young women recruited to serve as mentors for the project's young female athletes. We combined both thematic and narrative analysis. RESULTS: The programme created opportunities for collective action, increasing mentors' ability to think and relate in a collectivized manner, and challenged the traditional female identity constructed for young women, which centres on domestic duties. The mentors themselves negotiated freedoms both in and outside their homes, which required careful and strategic bargaining. They changed the nature of key day-to-day social interactions with parents and brothers, as well as with neighbours, parents of their groups of athletes and men on the streets. They formed a new reference group for each other in terms of what was possible and acceptable. Demonstrating greater negotiation skills within the family helped win parents' trust in the mentor's ability to be safe in public spaces. Parents became active supporters by not giving into social sanctions of neighbours and relatives thus co-producing a new identity for their daughters as respectable young women doing 'good work'. They effectively side stepped reputational risk with their presence in public spaces becoming de-sexualised. CONCLUSIONS: Mentors contested mobility restrictions by taking risks as a group first, with collective agency an important step towards greater individual agency. This research provides important insights into addressing embedded social norms that perpetuate gender discriminatory practices and the social patterning of health inequalities.

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins.

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10-15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.

Most overweight and obese Indian children have nonalcoholic fatty liver disease.

 Background and rationale. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of pediatric liver disease in western countries. Its prevalence in Indian subcontinent is not well studied. MATERIAL AND METHODS: In a school based cross sectional study we have screened overweight and obese children in the age group of 11 to 15 years for NAFLD. Ultrasonography, elevated serum transaminases, fibroscan were used for defining NAFLD. Dietary habits, blood pressure, serum lipid profile, blood counts and insulin resistance were recorded. The relation of fibrosis 4 score, pediatric NAFLD fibrosis index, aspartate transaminases to platelet ratio index (APRI) with fibroscan was evaluated. RESULTS: Out of 616 students screened 198 were overweight and obese. Hundred students and their parents gave informed consent for the further evaluation. The prevalence of NAFLD was 62% in overweight and obese children. Fatty liver was found in 50 % students on ultrasonography, liver stiffness (≥ 6.1 Kilopascals) in 23% and raised alanine transaminase in 30%. Hypertension, dyslipidemia, diabetes mellitus and insulin resistance were seen in 6%, 18%, 2% and 66% students respectively. Systolic hypertension, serum triglyceride, aspartate transaminase, APRI was significantly higher in the NAFLD group. On binary logistic regression only systolic hypertension was an independent risk factor for NAFLD. CONCLUSION: In conclusion NAFLD is common in asymptomatic overweight and obese Indian children. Systolic hypertension is the only independent factor associated with NAFLD. Fibroscan has limited role for screening. We recommend screening for NAFLD in this high risk group with alanine transaminases and ultrasonography.