Expression and potential regulatory functions of Drosophila octopamine receptors in the female reproductive tract.

Aminergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine, is required for ovulation as well as several other female reproductive processes. Two octopamine receptors have already been shown to be expressed in the Drosophila reproductive tract and to be required for egg-laying: OAMB and Octß2R. The Drosophila genome contains 4 additional octopamine receptors-Octα2R, Octß1R, Octß3R, and Oct-TyrR-but their cellular patterns of expression in the reproductive tract and potential contribution(s) to egg-laying are not known. In addition, the mechanisms by which OAMB and Octß2R regulate reproduction are incompletely understood. Using a panel of MiMIC Gal4 lines, we show that Octα2R, Octß1R, Octß3R, and Oct-TyrR receptors are not detectable in either epithelium or muscle but are clearly expressed in neurons within the female fly reproductive tract. Optogenetic activation of neurons that express at least 3 types of octopamine receptors stimulates contractions in the lateral oviduct. We also find that octopamine stimulates calcium transients in the sperm storage organs and that its effects in spermathecal, secretory cells, can be blocked by knock-down of OAMB. These data extend our understanding of the pathways by which octopamine regulates egg-laying in Drosophila and raise the possibility that multiple octopamine receptor subtypes could play a role in this process.

Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophila vesicular monoamine transporter gene.

Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling.

Drosophila cells that express octopamine receptors can either inhibit or promote oviposition.

Adrenergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila , the ortholog of noradrenaline, octopamine (Oa), is required for ovulation as well as several other female reproductive processes. Loss of function studies using mutant alleles of receptors, transporters, and biosynthetic enzymes for Oa have led to a model in which disruption of octopaminergic pathways reduces egg laying. However, neither the complete expression pattern in the reproductive tract nor the role of most octopamine receptors in oviposition is known. We show that all six known Oa receptors are expressed in peripheral neurons at multiple sites within in the female fly reproductive tract as well as in non-neuronal cells within the sperm storage organs. The complex pattern of Oa receptor expression in the reproductive tract suggests the potential for influencing multiple regulatory pathways, including those known to inhibit egg-laying in unmated flies. Indeed, activation of some neurons that express Oa receptors inhibits oviposition, and neurons that express different subtypes of Oa receptor can affect different stages of egg laying. Stimulation of some Oa receptor expressing neurons (OaRNs) also induces contractions in lateral oviduct muscle and activation of non-neuronal cells in the sperm storage organs by Oa generates OAMB-dependent intracellular calcium release. Our results are consistent with a model in which adrenergic pathways play a variety of complex roles in the fly reproductive tract that includes both the stimulation and inhibition of oviposition.

Regulation of Drosophila oviduct muscle contractility by octopamine.

Octopamine is essential for egg-laying in Drosophila melanogaster, but the neuronal pathways and receptors by which it regulates visceral muscles in the reproductive tract are not known. We find that the two octopamine receptors that have been previously implicated in egg-laying-OAMB and Octß2R-are expressed in octopaminergic and glutamatergic neurons that project to the reproductive tract, peripheral ppk(+) neurons within the reproductive tract and epithelial cells that line the lumen of the oviducts. Further optogenetic and mutational analyses indicate that octopamine regulates both oviduct contraction and relaxation via Octß2 and OAMB respectively. Interactions with glutamatergic pathways modify the effects of octopamine. Octopaminergic activation of Octß2R on glutamatergic processes provides a possible mechanism by which octopamine initiates lateral oviduct contractions. We speculate that aminergic pathways in the oviposition circuit may be comparable to some of the mechanisms that regulate visceral muscle contractility in mammals.

Study of Feasibility of Blood Pressure Monitoring in Postpartum Women by Teleconsultation in COVID 19 Pandemic Situation.

Purpose of the Study: To study the feasibility of blood pressure monitoring and to assess the feasibility of antihypertensive dose adjustment in postpartum women by teleconsultation in COVID 19 pandemic situation. Methods: This was a descriptive longitudinal study conducted in the Department of OBGY, GMCH, Aurangabad between the study periods November 2020 to April 2021 with a sample size of 60. The feasibility of blood pressure monitoring in postpartum women by teleconsultation was measured by recruitment and retention through 12 weeks postpartum. The feasibility of anti-hypertensive dose adjustment through teleconsultation was measured by the number of women requiring hospital visit for uncontrolled blood pressure or those with warning signs and symptoms. The data were collected and analyzed. Results: The feasibility of blood pressure monitoring in postpartum women by teleconsultation was 95.23%. During follow-up, the antihypertensive drug dose was required to be increased in 3 women. Not a single woman required hospital visit or hospital readmission either due to uncontrolled hypertension or warning signs/symptoms. This indicates that the feasibility of anti-hypertensive dose adjustment by teleconsultation was good. Conclusion: We demonstrated feasibility and over all good satisfaction rate of Blood Pressure Monitoring in postpartum women by teleconsultation in COVID 19 pandemic situation and found that teleconsultation is a boon in management for postpartum hypertension to reduce readmissions and decrease maternal morbidity while ensuring social distancing and minimizing viral exposure. We recommend teleconsultation as a quality improvement initiative in maternity care.

Innovations in implementing a health systems response to violence against women in 3 tertiary hospitals of Maharashtra India: Improving provider capacity and facility readiness.

Background: Violence against women [VAW] is an urgent public health issue and health care providers [HCPs] are in a unique position to respond to such violence within a multi-sectoral health system response. In 2013, the World Health Organization (WHO) published clinical and policy guidelines (henceforth - the Guidelines) for responding to intimate partner violence and sexual violence against women. In this practical implementation report, we describe the adaptation of the Guidelines to train HCPs to respond to violence against women in tertiary health facilities in Maharashtra, India. Methods: We describe the strategies employed to adapt and implement the Guidelines, including participatory methods to identify and address HCPs' motivations and the barriers they face in providing care for women subjected to violence. The adaptation is built on querying health-systems level enablers and obstacles, as well as individual HCPs' perspectives on content and delivery of training and service delivery. Results: The training component of the intervention was delivered in a manner that included creating ownership among health managers who became champions for other health care providers; joint training across cadres to have clear roles, responsibilities and division of labour; and generating critical reflections about how gender power dynamics influence women's experience of violence and their health. The health systems strengthening activities included establishment of standard operating procedures [SOPs] for management of VAW and strengthening referrals to other services. Conclusions: In this intervention, standard training delivery was enhanced through participatory, joint and reflexive methods to generate critical reflection about gender, power and its influence on health outcomes. Training was combined with health system readiness activities to create an enabling environment. The lessons learned from this case study can be utilized to scale-up response in other levels of health facilities and states in India, as well as other LMIC contexts. Plain language summary: Violence against women affects millions of women globally. Health care providers may be able to support women in various ways, and finding ways to train and support health care providers in low and middle-income countries to provide high-quality care to women affected by violence is an urgent need. The WHO developed Clinical and Policy Guidelines in 2013, which provide guidance on how to improve health systems response to violence against women. We developed and implemented a series of interventions, including training of health care providers and innovations in service delivery, to implement the WHO guidelines for responding to violence against women in 3 tertiary hospitals of Maharashtra, India. The nascent published literature on health systems approaches to addressing violence against women in low and middle-income countries focuses on the impact of these interventions. This practical implementation report focuses on the interventions themselves, describes the processes of developing and adapting the intervention, and thus provides important insights for donors, policy-makers and researchers.

A Quasi-Experimental Study to Compare the Effect of Respectful Maternity Care Using Intrapartum Birth Companion of Her Choice on Maternal and Newborn Outcome in Tertiary Care Centre.

PURPOSE OF THE STUDY: Traditionally during labour woman is supported by another woman. However, in hospitals, continuous support during labour has often become the exception rather than the routine. Worldwide, there is a growing concern about the disrespect and abuse of women seeking maternity care. This prompted us to decide to change the obstetric care practices by providing a birth companion of her choice to women during labour and compare their maternal and newborn outcomes with the data from the same institute before intervention. METHODS: This was a quasi-experimental study conducted in the Department of OBGY, GMCH, Aurangabad for 20 months and compared with the previous data from the same institute before implementation of the birth companion policy. The impact of this intervention was evaluated by caesarean section rate, episiotomy rates and admission to neonatal intensive care units, and data was analysed by an appropriate statistical test. RESULTS: The rate of caesarean Section (20%) and episiotomy (8.57%) was significantly lower in the intervention group than in the control group. The rate of NICU admission and time required for initiation of breastfeeding was significantly lower in the intervention than in the control group. Around 86.6% of women from the intervention group were fully satisfied with the role of birth companion and 13.4% were partially satisfied. CONCLUSION: Our study demonstrated that the use of an intrapartum birth companion of her choice helped us improve maternal and newborn outcomes without any harm. We recommend generalizing the policy of use of the trained birth companion of her choice in the private as well as the public sector.

Strengthening Health Systems' Response to Violence Against Women in Three Tertiary Health Facilities of Maharashtra.

BACKGROUND: Domestic violence is known to have a significant impact on the health of women. Despite this, the health system in India is not equipped to respond to women facing violence. This can be attributed to limited information on how the evidence-based guidelines can be implemented in resource-constrained settings. To fill this gap, implementation research was carried out in three tertiary medical teaching hospitals in Maharashtra. METHODS: The project was implemented in the OBGY, Medicine and Emergency department of a medical college and a district hospital in the state of Maharashtra. The intervention included consultation with key providers of three departments and a 5 day training of trainers on VAW. The trainers conducted 2 day onsite training for the health care providers. System-level interventions included the development of SOPs, IEC material, documentation format and identifying places for a private consultation. The research involved a pre- and post-test to assess change in KAP of providers after training, analysis of documentation register and interviews with trained providers and survivors. RESULTS: Findings indicate a significant change in knowledge, attitude and practice of the providers. Documentation registers introduced in the facility departments showed 531 women facing violence were responded by providers in 9 months. In 59% of cases, the provider suspected violence based on presenting health complaints, indicating the success of the capacity building programmes in the development of skills to identify VAW signs and symptoms, as well as provide psychological support to women/girls. There was a high acceptability of intervention among providers. Survivors also recognised the usefulness of health care facility-based support services for violence. CONCLUSION: A multi-component intervention comprising of building capacity of providers and facility readiness is feasible to implement in low- and middle-income countries (LMIC) and can strengthen health systems' response to VAW.

Vesicular neurotransmitter transporters in Drosophila melanogaster.

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation.

Integrating gender perspectives in gynecology and obstetrics: Engaging medical colleges in Maharashtra, India.

Failure to acknowledge the impact of sex and gender differences affects the quality of health care provision, and is an impediment to reducing health inequities. Systematic efforts were initiated in Maharashtra, India for reducing these disparities by developing gender-integrated curricula in undergraduate (UG) medical education between 2015 and 2018. A review of UG obstetrics and gynecology curricula indicated a lack of gender lens and focus on the reproductive rights of women. Based on these gaps, a gender-integrated curriculum was developed, implemented, and tested with medical students. Significant positive attitudes were seen among male and female students for themes such as access to safe abortion; understanding reproductive health concerns and their complex relationship with gender roles; violence against women as a health issue; and sexuality and health. These results strengthened the resolve to advocate for such a curriculum to be integrated across all medical colleges in the state.

Ecdysis triggering hormone receptors regulate male courtship behavior via antennal lobe interneurons in Drosophila.

Ecdysis triggering hormone receptors (ETHR) regulate the behavioral sequence necessary for cuticle shedding. Recent reports have documented functions for ETHR signaling in adult Drosophila melanogaster. In this study, we report that ETHR silencing in local interneurons of the antennal lobes and fruitless neurons leads to sharply increased rates of male-male courtship. RNAseq analysis of ETHR knockdown flies reveals differential expression of genes involved in axon guidance, courtship behavior and chemosensory functions. Our findings indicate an important role for ETHR in regulation of Drosophila courtship behavior through chemosensory processing in the antennal lobe.

Mifepristone Reduces Food Palatability and Affects Drosophila Feeding and Lifespan.

The Drosophila GeneSwitch system facilitates the spatial and temporal control of gene expression through dietary supplementation of mifepristone (RU486). Because experimental and control groups differ only by treatment with RU486, confounding results from using flies of different genetic backgrounds are eliminated, making GeneSwitch especially useful in studies of aging. However, the effect of RU486 itself on longevity has not been well characterized, particularly in relation to nutritional states known to affect lifespan. Here, we show that RU486 has dose- and diet-dependent effects on longevity in both sexes. On low nutrient diets, RU486 supplementation reduces total food consumption, perhaps exacerbating undernutrition to shorten life. RU486 also inhibits proboscis extension responses to low nutrient diets, suggesting that RU486 has an aversive taste which leads to decreased food consumption and diminished longevity. RU486 is not detrimental to fly lifespan on high nutrient food, correlating with reduced effects of the drug on palatability and total consumption on rich diets. Our results highlight the critical importance of considering how food palatability and nutrient intake might be altered by dietary or drug manipulations in studies of aging and behavior.

Postprandial sleep mechanics in Drosophila.

Food consumption is thought to induce sleepiness. However, little is known about how postprandial sleep is regulated. Here, we simultaneously measured sleep and food intake of individual flies and found a transient rise in sleep following meals. Depending on the amount consumed, the effect ranged from slightly arousing to strongly sleep inducing. Postprandial sleep was positively correlated with ingested volume, protein, and salt-but not sucrose-revealing meal property-specific regulation. Silencing of leucokinin receptor (Lkr) neurons specifically reduced sleep induced by protein consumption. Thermogenetic stimulation of leucokinin (Lk) neurons decreased whereas Lk downregulation by RNAi increased postprandial sleep, suggestive of an inhibitory connection in the Lk-Lkr circuit. We further identified a subset of non-leucokininergic cells proximal to Lkr neurons that rhythmically increased postprandial sleep when silenced, suggesting that these cells are cyclically gated inhibitory inputs to Lkr neurons. Together, these findings reveal the dynamic nature of postprandial sleep.

Acidic Food pH Increases Palatability and Consumption and Extends Drosophila Lifespan.

BACKGROUND: Despite the prevalent use of Drosophila as a model in studies of nutrition, the effects of fundamental food properties, such as pH, on animal health and behavior are not well known. OBJECTIVES: We examined the effect of food pH on adult Drosophila lifespan, feeding behavior, and microbiota composition and tested the hypothesis that pH-mediated changes in palatability and total consumption are required for modulating longevity. METHODS: We measured the effect of buffered food (pH 5, 7, or 9) on male gustatory responses (proboscis extension), total food intake, and male and female lifespan. The effect of food pH on germfree male lifespan was also assessed. Changes in fly-associated microbial composition as a result of food pH were determined by 16S ribosomal RNA gene sequencing. Male gustatory responses, total consumption, and male and female longevity were additionally measured in the taste-defective Pox neuro (Poxn) mutant and its transgenic rescue control. RESULTS: An acidic diet increased Drosophila gustatory responses (40-230%) and food intake (5-50%) and extended survival (10-160% longer median lifespan) compared with flies on either neutral or alkaline pH food. Alkaline food pH shifted the composition of fly-associated bacteria and resulted in greater lifespan extension (260% longer median survival) after microbes were eliminated compared with flies on an acidic (50%) or neutral (130%) diet. However, germfree flies lived longer on an acidic diet (5-20% longer median lifespan) compared with those on either neutral or alkaline pH food. Gustatory responses, total consumption, and longevity were unaffected by food pH in Poxn mutant flies. CONCLUSIONS: Food pH can directly influence palatability and feeding behavior and affect parameters such as microbial growth to ultimately affect Drosophila lifespan. Fundamental food properties altered by dietary or drug interventions may therefore contribute to changes in animal physiology, metabolism, and survival.

Safety and Efficacy of Oral Mifepristone in Pre-induction Cervical Ripening and Induction of Labour in Prolonged Pregnancy.

OBJECTIVES: To study the safety and efficacy of oral mifepristone in pre-induction cervical ripening and induction of labour in prolonged pregnancy. METHODS: This is a single blind randomized control trial. 100 women with prolonged pregnancy beyond 40 weeks and Bishop score <6 were recruited, and randomly allocated into two groups. Women who received Tab. Mifepristone 200 mg orally were assigned in Study Group (n = 50) and who received placebo orally were assigned in Control Group (n = 50) At the end of 24 h, change in the Bishop's score was assessed and Tab. Misoprostol 25 µg was administered intravaginally every 4 h, maximum 6 doses for induction/augmentation of labour. Analysis regarding safety and efficacy of the drug was done with regards to maternal and perinatal outcome. RESULTS: Among 100 subjects, 50 received mifepristone and 50 received placebo. Mean induction to delivery interval was 1,907 ± 368.4 min for Study Group versus 2,079 ± 231.6 min for Control Group. The improvement in mean Bishop score was 5.0408 ± 1.90 for Study Group compared with 3.26 ± 1.15 was for Control Group after 24 h. Mean dose of misoprostol in Study Group was 40 ± 27.2, while the same in Control Group was 52 ± 19.46. Eight (16 %) women in Study Group and two (4 %) women in Control Group delivered vaginally within 24 h without any need of augmentation. There were 6 (12 %) cesareans and 2 (4 %) instrumental deliveries in Study Group and 8 (16 %) cesareans and 5 (10 %) instrumental deliveries in the Control Group. There was no statistically significant difference in perinatal outcomes between two groups. CONCLUSIONS: Mifepristone had a modest effect on cervical ripening when given 24 h prior to labour induction and appearing to reduce need for misoprostol compared with placebo.

Microbes Promote Amino Acid Harvest to Rescue Undernutrition in Drosophila.

Microbes play an important role in the pathogenesis of nutritional disorders such as protein-specific malnutrition. However, the precise contribution of microbes to host energy balance during undernutrition is unclear. Here, we show that Issatchenkia orientalis, a fungal microbe isolated from field-caught Drosophila melanogaster, promotes amino acid harvest to rescue the lifespan of undernourished flies. Using radioisotope-labeled dietary components (amino acids, nucleotides, and sucrose) to quantify nutrient transfer from food to microbe to fly, we demonstrate that I. orientalis extracts amino acids directly from nutrient-poor diets and increases protein flux to the fly. This microbial association restores body mass, protein, glycerol, and ATP levels and phenocopies the metabolic profile of adequately fed flies. Our study uncovers amino acid harvest as a fundamental mechanism linking microbial and host metabolism, and highlights Drosophila as a platform for quantitative studies of host-microbe relationships.

Quantifying Drosophila food intake: comparative analysis of current methodology.

Food intake is a fundamental parameter in animal studies. Despite the prevalent use of Drosophila in laboratory research, precise measurements of food intake remain challenging in this model organism. Here, we compare several common Drosophila feeding assays: the capillary feeder (CAFE), food labeling with a radioactive tracer or colorimetric dye and observations of proboscis extension (PE). We show that the CAFE and radioisotope labeling provide the most consistent results, have the highest sensitivity and can resolve differences in feeding that dye labeling and PE fail to distinguish. We conclude that performing the radiolabeling and CAFE assays in parallel is currently the best approach for quantifying Drosophila food intake. Understanding the strengths and limitations of methods for measuring food intake will greatly advance Drosophila studies of nutrition, behavior and disease.

Clinical Study of Bacillus coagulans Unique IS-2 (ATCC PTA-11748) in the Treatment of Patients with Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most prevalent vaginal infection worldwide and is characterized by reduction of native lactobacilli. Antimicrobial therapy used to cure the disease is often found to be ineffective. We postulate that Bacillus coagulans Unique IS-2 (Unique Biotech Limited, India) might provide an appendage to antimicrobial treatment and improve curing rate. In the present study 40 Indian women diagnosed with BV by the presence of symptoms including white discharge, pH greater than 4.7, burning micturation, itching, soreness and redness at vulva. The subjects were divided in 2 groups probiotic (n = 20) and control (n = 20) based on age (control group, 33 ± 3 years and probiotic group, 32.5 ± 3 years), history of previous vaginosis (control group, 75% or 15/20 and probiotic group, 75% or 15/20) and severity of current vaginosis infection (burning micturation and itching, 35% in each group). Probiotic group subjects were assigned to receive a dose of antibiotic therapy [Ofloxacin-Ornidazole with strength of 200-500 mg per capsule/day for 5 days along with vaginal peccaries (co-kimaxazol) for 3 days] simultaneously with two probiotic capsules (10(9) CFUs of Bacillus coagulans Unique IS-2 per capsule). The control group received only antibiotic therapy. At the end of the treatment the 80% of probiotic group subjects showed significant positive response as revealed by reduction of vaginosis symptoms compared to the control group which exhibited reduction in 45% subjects only. Thus, the results of present study indicate that strain Bacillus coagulans Unique IS-2 can provide benefits to women being treated with antibiotics to cure an infectious condition.

Kinetic evaluation of H2S and NH3 biofiltration for two media used for wastewater lift station emissions.

In this study, biofiltration using a natural wood chip medium and a commercial biofiltration medium was evaluated for the removal of moderate concentrations of hydrogen sulfide (H2S) (up to 100 parts per million by volume [ppmv]) in the presence of significant concentrations of ammonia (NH3). These levels were chosen as representative of wastewater lift station emissions in the Brownsville, TX, area. NH3-removing portions of the biofilms may compete with H2S-removing portions and inhibit H2S removal. H2S process removal efficiencies for the commercial and natural media ranged from 90 to 96% depending on inlet loading and media type and bed height. Kinetic analysis of the H2S removal process followed apparent first-order reaction behavior. The average first-order reaction rates were 0.03 sec(-1) for the commercial medium and 0.09 sec(-1) for the natural medium. Pressure drops across the columns ranged from 0.41 in. H2O/ft for the commercial medium to 1.41 in. H2O/ft for the natural medium. NH3 gas levels of up to 80 ppmv did not affect the H2S removal process efficiency, and calculated kinetic rate constants for H2S removal remained almost the same. The NH3 gas also was removed simultaneously with the H2S up to 98% removal efficiency by the commercial medium.