Leishmania donovani mevalonate kinase regulates host actin for inducing phagocytosis.

Despite the well-established role of macrophages in phagocytosing Leishmania, the contribution of the parasite to this process is not well understood. Present study provides insights into the mechanism underlying the MVK-induced entry of L. donovani and improve our knowledge of host-pathogen interactions. We have discussed Mevalonate kinase (MVK)-induced actin reorganization, modulation of signaling pathways and host cell functions. Our results show that LdMVK gains access to macrophage cytosol and induces actin assembly modulation through the activation of actin-related proteins: VASP, Src and ERM. We have also demonstrated that LdMVK induces Ca2+ signaling and Akt pathway in macrophages, which are critical components of Leishmania survival and proliferation. Interestingly, we found that antibodies against LdMVK can kill Leishmania-infected macrophages in culture by forming extracellular traps, highlighting the potential of LdMVK in inhibiting parasite death. Overall, LdMVK is a virulent factor in Leishmania that mediates parasite internalization and host modulation by targeting host proteins phosphorylation and calcium homeostasis having significant implications in disease progression.

Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment.

The vaginal microenvironment of healthy women has a predominance of Lactobacillus crispatus, L. iners, L. gasseri, and L. jensenii. The genomic repertoire of the strains of each of the species associated with the key attributes thereby regulating a healthy vaginal environment needs a substantial understanding.We studied all available human strains of the four lactobacilli across different countries, isolated from vaginal and urinal sources through phylogenetic and pangenomic approaches. The findings showed that L. iners has the highest retention of core genes, and L. crispatus has more gene gain in the evolutionary stratum. Interestingly, L. gasseri and L. jensenii demonstrated major population-specific gene-cluster gain/loss associated with bacteriocin synthesis, iron chelating, adherence, zinc and ATP binding proteins, and hydrolase activity. Gene ontology enrichment analysis revealed that L. crispatus strains showed greater enrichment of functions related to plasma membrane integrity, biosurfactant, hydrogen peroxide synthesis, and iron sequestration as an ancestral derived core function, while bacteriocin and organic acid biosynthesis are strain-specific accessory enriched functions. L. jensenii showed greater enrichment of functions related to adherence, aggregation, and exopolysaccharide synthesis. Notably, the key functionalities are heterogeneously enriched in some specific strains of L. iners and L. gasseri.This study shed light on the genomic features and their variability that provides advantageous attributes to predominant vaginal Lactobacillus species maintaining vaginal homeostasis. These findings evoke the need to consider region-specific candidate strains of Lactobacillus to formulate prophylactic measures against vaginal dysbiosis for women's health.

Comprehensive bacterial-metabolite profiles of Hawaijar, Bekang, and Akhone: a comparative study on traditional fermented soybeans of north-east India.

Preparation of traditionally fermented soybeans varies across ethnicities with distinct tastes, flavour, and nutritional values. The fermented soybean varieties Hawaijar, Bekang, and Akhone of north-east India are associated with diverse ethnic groups from Manipur, Mizoram, and Nagaland, respectively. These varieties differ in substrate and traditional practice that exerts differential bacterial-metabolite profile, which needs an in-depth analysis i. Culture-dependent and independent techniques investigated the bacterial diversity of the fermented soybean varieties. Gas chromatography and mass spectroscopy (GC-MS) studied these varieties' metabolite profiles. The common dominant bacterial genera detected in Hawaijar, Bekang, and Akhone were Bacillus, Ignatzschinaria, and Corynebacterium, with the presence of Brevibacillus and Staphylococcus exclusively in Hawaijar and Oceanobacillus in Bekang and Akhone. The metabolite analysis identified a higher abundance of essential amino acids, amino and nucleotide sugars, and vitamins in Hawaijar, short-chain fatty acids in Bekang, polyunsaturated fatty acids in Akhone and Hawaijar, and prebiotics in Akhone. The bacteria-metabolite correlation analysis predicted four distinct bacterial clusters associated with the differential synthesis of the functional metabolites. While B. subtilis is ubiquitous, cluster-1 comprised B. thermoamylovorans/B. amyloliquefaciens, cluster-2 comprised B. tropicus, cluster-3 comprised B. megaterium/B. borstelensis, and cluster-4 comprised B. rugosus. To the best of our knowledge, this is the first comparative study on traditional fermented soybean varieties of north-east India linking bacterial-metabolite profiles which may help in designing starters for desired functionalities in the future.

Emergency obstetric referrals in public health facilities: A descriptive study from urban Maharashtra, India.

Background: An effective referral system is key to access timely emergency obstetric care. The criticality of referrals makes it necessary to understand its pattern at the health system level. This study aims to document the patterns and primary reasons of obstetric case referral and the maternal and perinatal outcome of the cases in public health institutions in select areas of urban Maharashtra, India. Methods: The study is based on the health records of public health facilities in Mumbai and its adjoining three municipal corporations. The information on pregnant women referred for obstetric emergencies was collected from patient referral forms of municipal maternity homes and peripheral health facilities between 2016 and 2019. Maternal and child outcome data was obtained from "Received-In" peripheral and tertiary health facilities to track whether the referred woman reached the referral facility for delivery. Descriptive statistics were used to analyze demographic details, referral patterns, reasons of referrals, referral communication and documentation, time and mode of transfer and delivery outcomes. Results: 14% (28,020) women were referred to higher health facilities. The most common reasons for referral were pregnancy-induced hypertension or eclampsia (17%), previous caesarean section (12%), fetal distress (11%) and Oligohydramnios (11%). 19% of all referrals were entirely due to unavailability of human resources or health infrastructure. Non-availability of emergency Operation Theatre (47%) and Neonatal Intensive Care Unit (45%) were the major non-medical reasons for referrals. Absence of health personnel such as anaesthetist (24%), paediatrician (22%), physician (20%) or obstetrician (12%) was another non-medical reason for referrals. Referring facility had a phone-based communication about the referral with the receiving facility in less than half of the cases (47%). 60% of the referred women could be tracked in higher health facilities. Of the tracked cases, 45% women delivered via caesarean section. Most of the deliveries (96%) resulted in live birth outcomes. 34% of the newborns weighed less than 2,500 grams. Conclusion: Improving referral processes are critical to enhance the overall performance of emergency obstetric care. Our findings emphasize the need for a formal communication and feedback system between referring and receiving facilities. Simultaneously, ensuring EmOC at different levels of health facilities by upgradation of health infrastructure is recommended.

To use or not to use: Exploring factors influencing the uptake of modern contraceptives in urban informal settlements of Mumbai.

Rapid urbanization and a high unmet need for family planning in urban informal settlements point to the significance of identifying gaps that exist in the path of voluntary uptake of contraceptives. We undertook this study to better understand the perspectives related to family planning among women living in informal settlements of Mumbai. We used a mixed-methods approach, including a cross-sectional survey with 1407 married women of reproductive age and face-to-face in-depth interviews with 22 women, both users and non-users of modern contraceptives. 1070 (76%) of the participants were using modern contraceptives and women's age, education, parity, socioeconomic status and exposure to family planning interventions were the main determinants of contraceptive use. Poor contraceptive awareness before marriage coupled with social norms of early childbearing and completing family resulted in unplanned and less spaced pregnancies even among current users. In such cases, women either continued with the pregnancy or opted for abortion which sometimes could be unsafe. The decision to use contraceptives was taken in most cases after achieving the desired family size and was also influenced by belief in traditional methods, fear of side effects, spousal/family awareness and counselling by frontline workers. We recommend strengthening of sexual and reproductive health component of adolescent health programs. It is pertinent to inform women about their reproductive rights and most importantly empower them to practice these rights. This can be achieved by increasing women's age at marriage and continued promotion of formal education. Widespread misconceptions related to the side effects of modern methods need to be mitigated via counselling. Referral, follow-up, and suggestions on available choices of contraceptives should be given in case women face any side effects from the use of contraceptives. At the same time, improving spousal awareness and communication regarding family planning will allow couples to make informed decisions. Finally, roping in role models in the community will create an environment conducive to operationalizing rights-based family planning.

Recent advances in understanding of multifaceted changes in the vaginal microenvironment: implications in vaginal health and therapeutics.

The vagina endures multifaceted changes from neonatal to menopausal phases due to hormonal flux, metabolite deposition, and microbial colonization. These features have important implications in women's health. Several pre-factors show dynamic characteristics according to the phases that shift the vaginal microbiota from anaerobes to aerobes which is a hallmark of healthy vaginal environment. These factors include oestrogen levels, glycogen deposition, and vaginal microstructure. In the adult phase, Lactobacillus is highly dominant and regulates pH, adherence, aggregation, immune modulation, synthesis of bacteriocins, and biosurfactants (BSs) which are antagonistic to pathogens. Maternal factors are protective by favouring the colonization of lactobacilli in the vagina in the neonatal phase, which diminishes with age. The dominance of lactobacilli and dysbiosis in the adult phase depends on intrinsic and extrinsic factors in women, which vary between ethnicities. Recent developments in probiotics used against vaginal microbiome dysbiosis have shown great promise in restoring the normal microbiota including preventing the loss of beneficial bacteria. However, further in-depth studies are warranted to ensure long-term protection by probiotics. This review highlights various aspects of the vaginal microenvironment in different phases of growth and diverse ethnicities. Furthermore, it discusses future trends for formulating more effective population-specific probiotics and implications of paraprobiotics and postbiotics as effective therapeutics.

Corona was scary, lockdown was worse: A mixed-methods study of community perceptions on COVID-19 from urban informal settlements of Mumbai.

The COVID-19 pandemic has magnified the multiple vulnerabilities of people living in urban informal settlements globally. To bring community voices from such settlements to the center of COVID-19 response strategies, we undertook a study in the urban informal settlements of Dharavi, Mumbai, from September 2020-April 2021. In this study, we have examined the awareness, attitudes, reported practices, and some broader experiences of the community in Dharavi with respect to COVID-19. We have used a mixed-methods approach, that included a cross-sectional survey of 468 people, and in-depth interviews and focus group discussions with 49 people living in this area. Data was collected via a mix of phone and face-to-face interviews. We have presented here the descriptive statistics from the survey and the key themes that emerged from our qualitative data. People reported high levels of knowledge about COVID-19, with television (90%), family and friends (56%), and social media (47%) being the main sources of information. The knowledge people had, however, was not free of misconceptions and fear; people were scared of being forcefully quarantined and dying alone during the early days of COVID-19. These fears had negative repercussions in the form of patient-related stigma and hesitancy in seeking healthcare. A year into the pandemic, however, people reported a shift in attitudes from 'extreme fear to low fear' (67% reported perceiving low/no COVID risk in October 2020), contributing to a general laxity in following COVID-appropriate behaviors. Currently, the community is immensely concerned about the revival of livelihoods, that have been adversely impacted due to the lockdown in 2020 as well as the continued 'othering' of Dharavi for being a COVID hotspot. These findings suggest that urban informal settlements like Dharavi need community-level messaging that counters misinformation and denial of the outbreak; local reinforcement of COVID-appropriate behaviours; and long-term social protection measures.

Impact of intensified control on visceral leishmaniasis in a highly-endemic district of Bihar, India: an interrupted time series analysis.

Visceral leishmaniasis (VL) is declining in India and the World Health Organization's (WHO) 2020 'elimination as a public health problem' target has nearly been achieved. Intensified combined interventions might help reach elimination, but their impact has not been assessed. WHO's Neglected Tropical Diseases 2021-2030 roadmap provides an opportunity to revisit VL control strategies. We estimated the combined effect of a district-wide pilot of intensified interventions in the highly-endemic Vaishali district, where cases fell from 3,598 in 2012-2014 to 762 in 2015-2017. The intensified control approach comprised indoor residual spraying with improved supervision; VL-specific training for accredited social health activists to reduce onset-to-diagnosis time; and increased Information Education & Communication activities in the community. We compared the rate of incidence decrease in Vaishali to other districts in Bihar state via an interrupted time series analysis with a spatiotemporal model informed by previous VL epidemiological estimates. Changes in Vaishali's rank among Bihar's endemic districts in terms of monthly incidence showed a change pre-pilot (3rd highest out of 33 reporting districts) vs. during the pilot (9th) (p<1e-10). The rate of decline in Vaishali's incidence saw no change in rank at 11th highest, both pre-pilot & during the pilot. Counterfactual model simulations suggest an estimated median of 352 cases (IQR 234-477) were averted by the Vaishali pilot between January 2015 and December 2017, which was robust to modest changes in the onset-to-diagnosis distribution. Strengthening control strategies may have precipitated a substantial change in VL incidence in Vaishali and suggests this approach should be piloted in other highly-endemic districts.

Factors associated with women's healthcare decision-making during and after pregnancy in urban slums in Mumbai, India: a cross-sectional analysis.

BACKGROUND: Understanding factors associated with women's healthcare decision-making during and after pregnancy is important. While there is considerable evidence related to general determinants of women's decision-making abilities or agency, there is little evidence on factors associated with women's decision-making abilities or agency with regards to health care (henceforth, health agency), especially for antenatal and postnatal care. We assessed women's health agency during and after pregnancy in slums in Mumbai, India, and examined factors associated with increased participation in healthcare decisions. METHODS: Cross-sectional data were collected from 2,630 women who gave birth and lived in 48 slums in Mumbai. A health agency module was developed to assess participation in healthcare decision-making during and after pregnancy. Linear regression analysis was used to examine factors associated with increased health agency. RESULTS: Around two-thirds of women made decisions about perinatal care by themselves or jointly with their husband, leaving about one-third outside the decision-making process. Participation increased with age, secondary and higher education, and paid employment, but decreased with age at marriage and household size. The strongest associations were with age and household size, each accounting for about a 0.2 standard deviation difference in health agency score for each one standard deviation change (although in different directions). Similar differences were observed for those in paid employment compared to those who were not, and for those with higher education compared to those with no schooling. CONCLUSION: Exclusion of women from maternal healthcare decision-making threatens the effectiveness of health interventions. Factors such as age, employment, education, and household size need to be considered when designing health interventions targeting new mothers living in challenging conditions, such as urban slums in low- and middle-income countries.

Differential translational regulation of host exosomal proteins play key role in immunomodulation in antimony resistance in Visceral Leishmaniasis: A proteomic profiling study.

In host-pathogen interactions, exosomal secretions are crucial for cell to cell communication and have an established role in immunomodulation. Protozoans, including Leishmania, modulates their host vesicular secretions for better survival; although the role of exosomal secretions in unresponsive against sodium antimony gluconate (SAG) has never been documented. In this study, the exosomal proteome of RAW macrophages infected with either SAG responsive (SAGS) or SAG unresponsive (SAGR) L. donovani parasites has been compared with uninfected RAW macrophages. Proteins isolated from exosomes were labelled with iTRAQ reagents; followed by subsequent LC-TOF/-MS analysis. In total, 394 proteins (p < 0.05) were identified which were shared common among all sets. Highly differentially expressed proteins were sorted by log2 value -1 and +1 as down regulated and up regulated respectively which yielded 58 proteins in SAGR and 41 proteins during SAGS infection. Out of the 58 proteins identified during SAGR infection, 17 proteins were of immune modulatory function. Network visualization model and pathway analysis revealed the interactions among these proteins via different immunological pathways with reported involvement of some proteins in SAG resistance and host immune modulation. Hence, the differential abundance of immune pathway related proteins in exosomes of infected host during SAGR infection supports the immune modulatory strategy adopted by SAG resistant parasites for enhanced survival .

Evaluation of the Hindi version of the London Measure of Unplanned Pregnancy among pregnant and postnatal women in urban India.

BACKGROUND: Valid and reliable measures such as London Measure of Unplanned Pregnancy (LMUP) are imperative for understanding fertility-related behaviors and estimating unintended pregnancy. The aim of this study was to validate the LMUP in the Hindi language for a wider reach in India. METHODS: An interviewer administered version of the LMUP was translated and pretested in Hindi. The LMUP was field tested with married women in the reproductive age group across forty informal settlements in Mumbai in the post intervention census of a cluster randomized control trial to improve the health of women and children. Analyses involved the full sample and sub-groups according to time-from-conception. Reliability (internal consistency) was assessed using Cronbach's alpha, inter-item correlations, and item-rest correlations. Construct validity was assessed by hypothesis testing and confirmatory factor analysis. RESULTS: 4991 women were included in the study (1180 were pregnant, 2126 in their first- and 1685 in their second postnatal year). LMUP item completion rates were 100 % and the full range of LMUP scores was captured. Reliability: the scale was internally consistent (Cronbach's α = 0.84), inter-item correlations were positive, and item-rest correlations were above 0.2 for all items except item six (0.07). Construct validity: hypotheses were met, and confirmatory factor analysis showed that a one-factor model was a good fit for the data, confirming unidimensional measurement. The sub-group analysis (by pregnant, first-, and second postnatal year) showed that the psychometric properties of the LMUP were similar across the groups. In terms of LMUP scores, the women in the postnatal groups were very slightly, but significantly, more likely to have an LMUP score of 10 + compared to pregnant women; the difference between the first and second postnatal year was not significant. CONCLUSIONS: The Hindi LMUP is valid and reliable measure of pregnancy intention that may be used in India. TRIAL REGISTRATION: This study is registered with ISRCTN, number ISRCTN56183183, and Clinical Trials Registry of India, number CTRI/2012/09/003004.

Mevalonate kinase of Leishmania donovani protects parasite against oxidative stress by modulating ergosterol biosynthesis.

Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated ∼2.1 fold and ∼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.