Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Intimate partner violence, food insecurity and COVID-19 among newly married women in Nawalparasi district of Nepal: a longitudinal study.

This paper examines factors associated with intimate partner violence (IPV) among newly married women in Nepal, and how IPV was affected by food insecurity and COVID-19. Given evidence that food insecurity is associated with IPV and COVID-19, we explored whether increased food insecurity during COVID-19 is associated with changes in IPV. We used data from a cohort study of 200 newly married women aged 18-25 years, interviewed five times over two years at 6-month intervals (02/2018-07/2020), including after COVID-19-associated lockdowns. Bivariate analysis and mixed-effects logistic regression models were used to examine the association between selected risk factors and recent IPV. IPV increased from 24.5% at baseline to 49.2% before COVID-19 and to 80.4% after COVID-19. After adjusting for covariates, we find that both COVID-19 (OR = 2.93, 95% CI 1.07-8.02) and food insecurity (OR = 7.12, 95% CI 4.04-12.56) are associated with increased odds of IPV, and IPV increased more for food-insecure women post COVID-19 (compared to non-food insecure), but this was not statistically significant (confidence interval 0.76-8.69, p-value = 0.131). Young, newly married women experience high rates of IPV that increase with time in marriage, and COVID-19 has exacerbated this, especially for food-insecure women in the present sample. Along with enforcement of laws against IPV, our results suggest that special attention needs to be paid to women during a crisis time like the current COVID-19 pandemic, especially those who experience other household stressors.

Denial of legal abortion in Nepal.

INTRODUCTION: In Nepal, abortion is legal on request through 12 weeks of pregnancy and up to 28 weeks for health and other reasons. Abortion is available at public facilities at no cost and by trained private providers. Yet, over half of abortions are provided outside this legal system. We sought to investigate the extent to which patients are denied an abortion at clinics legally able to provide services and factors associated with presenting late for care, being denied, and receiving an abortion after being denied. METHODS: We used data from a prospective longitudinal study with 1835 women aged 15-45. Between April 2019 and December 2020, we recruited 1,835 women seeking abortions at 22 sites across Nepal, including those seeking care at any gestational age (n = 537) and then only those seeking care at or after 10 weeks of gestation or do not know their gestational age (n = 1,298). We conducted interviewer-led surveys with these women at the time they were seeking abortion service (n = 1,835), at six weeks after abortion-seeking (n = 1523) and six-month intervals for three years. Using descriptive and multivariable logistic regression models, we examined factors associated with presenting for abortion before versus after 10 weeks gestation, with receiving versus being denied an abortion, and with continuing the pregnancy after being denied care. We also described reasons for the denial of care and how and where participants sought abortion care subsequent to being denied. Mixed-effects models was used to accounting clustering effect at the facility level. RESULTS: Among those recruited when eligibility included seeking abortion at any gestational age, four in ten women sought abortion care beyond 10 weeks or did not know their gestation and just over one in ten was denied care. Of the full sample, 73% were at or beyond 10 weeks gestation, 44% were denied care, and 60% of those denied continued to seek care after denial. Nearly three-quarters of those denied care were legally eligible for abortion, based on their gestation and pre-existing conditions. Women with lower socioeconomic status, including those who were younger, less educated, and less wealthy, were more likely to present later for abortion, more likely to be turned away, and more likely to continue the pregnancy after denial of care. CONCLUSION: Denial of legal abortion care in Nepal is common, particularly among those with fewer resources. The majority of those denied in the sample should have been able to obtain care according to Nepal's abortion law. Abortion denial could have significant potential implications for the health and well-being of women and their families in Nepal.

Adaptation on xylose improves glucose-xylose co-utilization and ethanol production in a carbon catabolite repression (CCR) compromised ethanologenic strain.

BACKGROUND: Sugar hydrolysates from lignocellulosic biomass are majorly composed of glucose and xylose that can be fermented to biofuels. Bacteria, despite having the natural ability to consume xylose are unable to consume it in presence of glucose due to a carbon catabolite repression (CCR) mechanism. This leads to overall reduced productivity as well as incomplete xylose utilization due to ethanol build-up from glucose utilization. In our effort to develop a strain for simultaneous fermentation of glucose and xylose into ethanol, we deleted ptsG in ethanologenic E. coli SSK42 to make it deficient in CCR and performed adaptive laboratory evolution to achieve accelerated growth rate, sugar consumption and ethanol production. Finally, we performed proteomics study to identify changes that might have been responsible for the observed improved phenotype of the evolved strain. RESULTS: The parental strain of SSK42, i.e., wild-type E. coli B, did not co-utilize glucose and xylose as expected. After deleting the ptsG gene encoding the EIIBCGlc subunit of PTS system, glucose consumption is severely affected in wild-type E. coli B. However, the ethanologenic, SSK42 strain, which was evolved in our earlier study on both glucose and xylose, didn't show such a drastic effect of EIIBCGlc deletion, instead consumed glucose first, followed by xylose without delay for switching from one sugar to another. To improve growth on xylose and co-utilization capabilities, the ptsG deleted SSK42 was evolved on xylose. The strain evolved for 78 generations, strain SCD78, displayed significant co-utilization of glucose and xylose sugars. At the bioreactor level, the strain SCD78 produced 3-times the ethanol titer of the parent strain with significant glucose-xylose co-utilization. The rate of glucose and xylose consumption also increased 3.4-fold and 3-fold, respectively. Proteome data indicates significant upregulation of TCA cycle proteins, respiration-related proteins, and some transporters, which may have a role in increasing the total sugar consumption and co-utilization of sugars. CONCLUSION: Through adaptive evolution, we have obtained a strain that has a significant glucose-xylose co-utilization phenotype with 3-fold higher total sugar consumption rate and ethanol production rate compared to the unevolved strain. This study also points out that adaptation on xylose is enough to impart glucose-xylose co-utilization property in CCR compromised ethanologenic strain SSK42.

Salt-stress adaptation of yeast as a simple method to improve high-gravity fermentation in an industrial medium.

While Saccharomyces cerevisiae is a popular organism to produce ethanol, its fermentation performance is affected at high sugar concentrations due to osmotic stress. We hypothesized that adaptation under ionic stress conditions will improve the fermentation performance at high sugar concentrations due to cross-stress adaptation. We, therefore, adapted a high-performance yeast strain, S. cerevisiae CEN.PK 122, to increasing salt concentrations in an industrial medium. Control cells were adapted in the medium without added salt. The cells adapted to 3.5% (w/v) salt concentration demonstrated a superior performance when fermenting 10-30% (w/v) glucose. When fermenting 30% (w/v) glucose, the ethanol yields of the adapted cells (0.49 ± 0.01 g g-1) were about 30% higher than the control cells (0.37 ± 0.01 g g-1) and are comparable with the best reported to date for any medium employed. Similar improvements were also observed when fermenting 10% (w/v) sucrose. However, little improvement in fermentation was observed at the higher temperature tested (40 °C), even though the growth of the adapted cells was greater when tested in YPD medium. The improvements in fermentation at 30 °C were primarily related to the faster growth of the adapted cells and not to an increase in specific intake rates. Additionally, a significantly reduced lag phase was also observed when fermenting 30% (w/v) glucose. Thus, our work shows the application of a simple strategy to significantly improve high-gravity fermentation (HGF) performance through adaptation. KEY POINTS: • Cell adapted on 3.5% NaCl made 28% more ethanol when fermenting 30% glucose. • The adapted cells had reduced lag phase, grew faster, and produced less glycerol. • The improvements were not related to increased specific rates of production.

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture.

BACKGROUND: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together. RESULTS: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96 and 120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144 and 168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration. CONCLUSIONS: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

Determinants of imbalanced sex ratio at birth in Nepal: evidence from secondary analysis of a large hospital-based study and nationally-representative survey data.

OBJECTIVES: To quantify sex ratios at births (SRBs) in hospital deliveries in Nepal, and understand the socio-demographic correlates of skewed SRB. Skewed SRBs in hospitals could be explained by sex selective abortion, and/or by decision to have a son delivered in a hospital-increased in -utero investments for male fetus. We use data on ultrasound use to quantify links between prenatal knowledge of sex, parity and skewed SRBs. DESIGN: Secondary analysis of: (1) de-identified data from a randomizedrandomised controlled trial, and (2) 2011 Nepal Demographic and Health Survey (NDHS). SETTING: Nepal. PARTICIPANTS: (1) 75 428 women who gave birth in study hospitals, (2) NDHS: 12 674 women aged 15-49 years. OUTCOME MEASURES: SRB, and conditional SRB of a second child given first born male or female were calculated. RESULTS: Using data from 75 428 women who gave birth in six tertiary hospitals in Nepal between September 2015 and March 2017, we report skewed SRBs in these hospitals, with some hospitals registering deliveries of 121 male births per 100 female births. We find that a nationally representative survey (2011 NDHS) reveals no difference in the number of hospital delivery of male and female babies. Additionally, we find that: (1) estimated SRB of second-order births conditional on the first being a girl is significantly higher than the biological SRB in our study and (2) multiparous women are more likely to have prenatal knowledge of the sex of their fetus and to have male births than primiparous women with the differences increasing with increasing levels of education. CONCLUSIONS: Our analysis supports sex-selective abortion as the dominant cause of skewed SRBs in study hospitals. Comprehensive national policies that not only plan and enforce regulations against gender-biased abortions and, but also ameliorate the marginalizedmarginalised status of women in Nepal are urgently required to change this alarming manifestation of son preference. TRIAL REGISTRATION NUMBER: NCT02718222.

Providers' perspectives on denial of abortion care in Nepal: a cross sectional study.

BACKGROUND: Despite legalization of abortion in Nepal in 2002, many women are still unable to access legal services. This paper examines providers' views, experiences with abortion denial, and knowledge related to abortion provision, and identifies areas for improvement in quality of care. METHODS: We conducted a structured survey with 106 abortion care providers at 55 government-approved safe abortion facilities across five districts of Nepal in 2017. We assessed reasons for denial of abortion care, knowledge about laws, barriers to provision and attitudes towards abortion. RESULTS: Almost all providers (96%) reported that they have ever refused clients for abortion services. Common reasons included beyond 12 weeks gestation (93%), sex selective abortion (86%), and medical contraindications (85%). One in four providers denied abortion for lack of drugs or trained personnel, and one third denied services when they perceived that the woman's reasons for abortion were insufficient. Only a third of providers knew all three legal indications for abortion -- less than or equal to 12 weeks of pregnancy on request, up to 18 weeks for rape or incest, and any time for maternal or fetal health risk. Overall, providers were in favor of legal abortion but a substantial proportion had mixed or negative attitudes about the service. CONCLUSIONS: Improvements in training to address providers' inadequate knowledge about the abortion law may reduce inappropriate denial of abortion. Establishing referral networks in the case of abortion denial and ensuring regular supply of medical abortion drugs would help more women access abortion care in Nepal.

Improvement in ethanol productivity of engineered E. coli strain SSY13 in defined medium via adaptive evolution.

E. coli has the ability to ferment both C5 and C6 sugars and produce mixture of acids along with small amount of ethanol. In our previous study, we reported the construction of an ethanologenic E. coli strain by modulating flux through the endogenous pathways. In the current study, we made further changes in the strain to make the overall process industry friendly; the changes being (1) removal of plasmid, (2) use of low-cost defined medium, and (3) improvement in consumption rate of both C5 and C6 sugars. We first constructed a plasmid-free strain SSY13 and passaged it on AM1-xylose minimal medium plate for 150 days. Further passaging was done for 56 days in liquid AM1 medium containing either glucose or xylose on alternate days. We observed an increase in specific growth rate and carbon utilization rate with increase in passage numbers until 42 days for both glucose and xylose. The 42nd day passaged strain SSK42 fermented 113 g/L xylose in AM1 minimal medium and produced 51.1 g/L ethanol in 72 h at 89% of maximum theoretical yield with ethanol productivity of 1.4 g/L/h during 24-48 h of fermentation. The ethanol titer, yield and productivity were 49, 40 and 36% higher, respectively, for SSK42 as compared to unevolved SSY13 strain.