Searching for Synthetic Opioid Rescue Agents: Identification of a Potent Opioid Agonist with Reduced Respiratory Depression.

While in the process of designing more effective synthetic opioid rescue agents, we serendipitously identified a new chemotype of potent synthetic opioid. Here, we report that conformational constraint of a piperazine ring converts a mu opioid receptor (MOR) antagonist into a potent MOR agonist. The prototype of the series, which we have termed atoxifent (2), possesses potent in vitro agonist activity. In mice, atoxifent displayed long-lasting antinociception that was reversible with naltrexone. Repeated dosing of atoxifent produced antinociceptive tolerance and a level of withdrawal like that of fentanyl. In rats, while atoxifent produced complete loss of locomotor activity like fentanyl, it failed to produce deep respiratory depression associated with fentanyl-induced lethality. Assessment of brain biodistribution demonstrated ample distribution of atoxifent into the brain with a Tmax of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.

Aromatic hydrazides: A potential solution for Acinetobacter baumannii infections.

The emergence of multidrug-resistant bacteria and the poor efficacy of available antibiotics against these infections have led to the urgent need for novel antibiotics. Acinetobacter baumannii is one of high-priority pathogens due to its ability to mount resistance to different classes of antibiotics. In an effort to provide novel agents in the fight against infections caused by A. baumannii, we synthesized a series of 46 aromatic hydrazides as potential treatments. In this series, 34 compounds were found to be low- to sub-µM inhibitors of A. baumannii growth, with MIC values in the range of 8 µg/mL to ≤0.125 µg/mL against a broad set of multidrug-resistant clinical isolates. These compounds were not highly active against other bacteria. We showed that one of the most potent compounds, 3e, was bacteriostatic and inhibitory to biofilm formation, although it did not disrupt the preformed biofilm. Additionally, we found that these compounds lacked mammalian cytotoxicity. The high antibacterial potency and the lack of mammalian cytotoxicity make these compounds a promising lead series for development of a novel selective anti-A. baumannii antibiotic.

Undergraduate Medical Education Reform in Viet Nam for a Primary Health Care Workforce.

Strong primary health care (PHC) systems require a robust PHC workforce. Traditionally, medical education takes place in academic medical centres that favour subspecialty care rather than PHC settings. This may undervalue primary care as a career and contribute to a shortage of PHC workers. However, designing undergraduate medical education curricula that incorporate early experiences in clinical care delivery at PHC sites remains a challenge, including in many low- and middle-income countries (LMICs). This paper describes how a collaboration between Harvard Medical School and five medical schools in Vietnam, and in-country collaborations among the Vietnamese medical schools, facilitated curricular innovation and co-creation of coursework relevant to PHC through the development of a Practice of Medicine (POM) course. The collaboration implemented a technical assistance strategy consisting of in-person workshops, focused virtual consultations, on-site 'office hours', site visits and observations to each of the five medical universities, and immersion trips to support the creation and implementation of the POM course. A pilot program was started at a single site and then scaled nationally using local customisation, experience, and expertise utilising a train-the-trainers approach. As a result, five new POM courses have been developed by five Vietnamese institutions. Fifty Vietnamese faculty received training to lead the POM course development, and 228 community-based preceptors have been trained to teach students at PHC sites. A total of 52 new PHC and community-based clinical training sites have been added, and 3,615 students have completed or are currently going through a POM course. This experience can serve as a model for future academic collaborations to support the development of a robust PHC workforce for the 21st century.

Exposure to conflict and child health outcomes: evidence from a large multi-country study.

BACKGROUND: Previous research has consistently found evidence of poor health outcomes among children living in conflict areas. However, the methodological focus of these studies has largely been on case studies, chart or registry reviews, qualitative studies, and single country studies. This reflects the need for a comprehensive multi-country analysis of the associations between conflicts and child health over a longer period. This study analyses the adverse impact of exposure to different types of conflicts  from in utero to five years of age, on several child health measures across a large group of countries. Our analysis pools data from multiple countries and time-points, to provide robust evidence on the relationship between conflict and child health. METHODS: Geo-referenced data on various forms of conflict are combined with the Demographic Health Survey dataset, to construct a large unique database of 590,488 pre-school age children across 52 developing countries over the period 1997 to 2018. Our analysis exploits the within-country differences in children's exposure to conflict from in utero to age five, to estimate its association with health outcomes. Our multivariate regression models estimate the links between conflict exposure and child health outcomes, measured using child nutrition outcomes (height-for-age and weight-for-age z-scores) and immunization status. RESULTS AND CONCLUSIONS: Empirical estimates show that even after controlling for a large array of socio-economic and demographic characteristics and location fixed effects, conflict exposure is negatively associated with child nutrition and immunization, across all our measures of conflict. These findings are robust across a range of specifications, alternative measures of conflict and sub-samples.

Conflicts and son preference: Micro-level evidence from 58 countries.

Research on the association between armed conflict and son preference has largely been based on single-country studies, often presenting descriptive patterns. This paper empirically analyzes the association between conflict and son preference using a sample of more than 1.1 million individuals from 58 countries over the period 2003-2018. We empirically show that both the incidence and intensity of conflict exposure are associated with greater son preference. Moreover, conflict-exposed individuals are likely to realise their preference for sons, as reflected in the systematically higher prevalence of sons over daughters among these individuals. To explore the aggregate effects of these findings, we conduct a cross-country analysis of sex ratios and show that history of conflict exposure plays an important role in explaining the cross-country differences in sex ratios.

Is There an Informal Employment Penalty in Food Security? Evidence from Rural Vietnam.

In this paper, we investigate the association between informal non-farm wage employment and household food security in rural Vietnam. The data for our analyses come from a nationally representative panel dataset of 1390 rural households from Vietnam Household Living Standards Survey. We conduct multivariate regression analysis using robust quantitative tools to show that informal employment is associated with a reduction in the consumption of nutritious foods. Our analysis also shows that informal employment reduces consumption of vegetables and fruits, using both the calorie- and expenditure-based shares of food groups.

Dans cet article, nous étudions l'association entre l'emploi salarié informel non agricole et la sécurité alimentaire des ménages dans les zones rurales du Vietnam. Les données utilisées pour nos analyses proviennent d'un ensemble de données de panel, représentatif au niveau national, de 1 390 ménages ruraux ayant participé à l'enquête sur le niveau de vie des ménages au Vietnam. Nous conduisons une analyse de régression multivariée à l'aide de solides outils quantitatifs pour démontrer que l'emploi informel est associé à une réduction de la consommation d'aliments nutritifs. Notre analyse montre également que l'emploi informel est associé à une consommation moindre de légumes et de fruits, que ce soit en termes de calories consommées et de dépenses financières pour chaque groupe d'aliments.

Exposure to conflicts and the continuum of maternal healthcare: Analyses of pooled cross-sectional data for 452,192 women across 49 countries and 82 surveys.

BACKGROUND: Violent conflicts are observed in many parts of the world and have profound impacts on the lives of exposed individuals. The limited evidence available from specific country or region contexts suggest that conflict exposure may reduce health service utilization and have adverse affects on health. This study focused on identifying the association between conflict exposure and continuum of care (CoC) services that are crucial for achieving improvements in reproductive, maternal, newborn, and child health and nutrition (RMNCHN). METHODS AND FINDINGS: We combined data from 2 sources, the Demographic Health Surveys (DHS) and the Uppsala Conflict Data Program's (UCDP) Georeferenced Event Dataset, for a sample of 452,192 women across 49 countries observed over the period 1997 to 2018. We utilized 2 consistent measures of conflict-incidence and intensity-and analyzed their association with maternal CoC in 4 key components: (i) at least 1 antenatal care (ANC) visit; (ii) 4 or more ANC visits; (iii) 4 or more ANC visits and institutional delivery; and (iv) 4 or more ANC visits, institutional delivery, and receipt of postnatal care (PNC) either for the mother or the child within 48 hours after birth. To identify the association between conflict exposure and components of CoC, we estimated binary logistic regressions, controlling for a large set of individual and household-level characteristics and year-of-survey and country/province fixed-effects. This empirical setup allows us to draw comparisons among observationally similar women residing in the same locality, thereby mitigating the concerns over unobserved heterogeneity. Around 39.6% (95% CI: 39.5% to 39.7%) of the sample was exposed to some form of violent conflict at the time of their pregnancy during the study period (2003 to 2018). Although access to services decreased for each additional component of CoC in maternal healthcare for all women, the dropout rate was significantly higher among women who have been exposed to conflict, relative to those who have not had such exposure. From logistic regression estimates, we observed that relative to those without exposure to conflict, the odds of utilization of each of the components of CoC was lower among those women who were exposed to at least 1 violent conflict. We estimated odds ratios of 0.86 (95% CI: 0.82 to 0.91, p < 0.001) for at least 1 ANC; 0.95 (95% CI: 0.91 to 0.98, p < 0.005) for 4 or more ANC; and 0.92 (95% CI: 0.89 to 0.96, p < 0.001) for 4 or more ANC and institutional delivery. We showed that both the incidence of exposure to conflict as well as its intensity have profound negative implications for CoC. Study limitations include the following: (1) We could not extend the CoC scale beyond PNC due to inconsistent definitions and the lack of availability of data for all 49 countries across time. (2) The measure of conflict intensity used in this study is based on the number of deaths due to the absence of information on other types of conflict-related harms. CONCLUSIONS: This study showed that conflict exposure is statistically significantly and negatively associated with utilization of maternal CoC services, in each component of the CoC scale. These findings have highlighted the challenges in achieving the Sustainable Development Goal 3 in conflict settings, and the need for more concerted efforts in ensuring CoC, to mitigate its negative implications on maternal and child health.

The Internalized Homophobia Scale for Vietnamese Sexual Minority Women: Conceptualization, Factor Structure, Reliability, and Associations With Hypothesized Correlates.

We developed the first Vietnamese Internalized Homophobia (IH) scale for use with Vietnamese sexual minority women (SMW). Drawing from existing IH scales in the international literature and based on prior qualitative research about SMW in the Viet Nam context, the scale covers two domains: self-stigma (negative attitudes toward oneself as a sexual minority person) and sexual prejudice (negative attitudes toward homosexuality/same-sex relations in general). Scale items, including items borrowed from existing scales and items based on local expressions, were reviewed and confirmed by members of the target population. Quantitative evaluation used data from an anonymous web-based survey of Vietnamese SMW, including those who identified as lesbian (n = 1187), or as bisexual (n = 641) and those who were unsure about their sexual identity (n = 353). The scale was found to consist of two highly correlated factors reflecting self-stigma (not normal/wholesome and self-reproach and wishing away same-sex sexuality) and one factor reflecting sexual prejudice, and to have excellent internal consistency. Construct validity was evidenced by subscale associations with a wide range of hypothesized correlates, including perceived sexual stigma, outness, social support, connection to other SMW, relationship quality, psychological well-being, anticipation of heterosexual marriage, and endorsement of same-sex marriage legalization. Self-stigma was more strongly associated with psychosocial correlates, and sexual prejudice was more associated with endorsement of legal same-sex marriage. The variations in these associations across the hypothesized correlates and across sexual identity groups were consistent with the minority stress model and the IH literature, and exhibited context-specific features, which are discussed.

Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

Expression of rRNA genes and nucleolus formation at ectopic chromosomal sites in the yeast Saccharomyces cerevisiae.

We constructed yeast strains in which rRNA gene repeats are integrated at ectopic sites in the presence or absence of the native nucleolus. At all three ectopic sites analyzed, near centromere CEN5, near the telomere of chromosome VI-R, and in middle of chromosome V-R (mid-V-R), a functional nucleolus was formed, and no difference in the expression of rRNA genes was observed. When two ribosomal DNA (rDNA) arrays are present, one native and the other ectopic, there is codominance in polymerase I (Pol I) transcription. We also examined the expression of a single rDNA repeat integrated into ectopic loci in strains with or without the native RDN1 locus. In a strain with reduced rRNA gene copies at RDN1 (approximately 40 copies), the expression of a single rRNA gene copy near the telomere was significantly reduced relative to the other ectopic sites, suggesting a less-efficient recruitment of the Pol I machinery from the RDN1 locus. In addition, we found a single rRNA gene at mid-V-R was as active as that within the 40-copy RDN1. Combined with the results of activity analysis of a single versus two tandem copies at CEN5, we conclude that tandem repetition is not required for efficient rRNA gene transcription.

Role of histone deacetylase Rpd3 in regulating rRNA gene transcription and nucleolar structure in yeast.

The 35S rRNA genes at the RDN1 locus in Saccharomyces cerevisiae can be transcribed by RNA polymerase (Pol) II in addition to Pol I, but Pol II transcription is usually silenced. The deletion of RRN9 encoding an essential subunit of the Pol I transcription factor, upstream activation factor, is known to abolish Pol I transcription and derepress Pol II transcription of rRNA genes, giving rise to polymerase switched (PSW) variants. We found that deletion of histone deacetylase gene RPD3 inhibits the appearance of PSW variants in rrn9 deletion mutants. This inhibition can be explained by the observed specific inhibition of Pol II transcription of rRNA genes by the rpd3Delta mutation. We propose that Rpd3 plays a role in the maintenance of an rRNA gene chromatin structure(s) that allows Pol II transcription of rRNA genes, which may explain the apparently paradoxical previous observation that rpd3 mutations increase, rather than decrease, silencing of reporter Pol II genes inserted in rRNA genes. We have additionally demonstrated that Rpd3 is not required for inhibition of Pol I transcription by rapamycin, supporting the model that Tor-dependent repression of the active form of rRNA genes during entry into stationary phase is Rpd3 independent.

Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast.

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes.

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

Silencing in yeast rDNA chromatin: reciprocal relationship in gene expression between RNA polymerase I and II.

About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae. Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes. Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active. By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats. Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA.