Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria.

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

Design and implementation of a mobile health electronic data capture platform that functions in fully-disconnected settings: a pilot study in rural Liberia.

BACKGROUND: Mobile phones and personal digital assistants have been used for data collection in developing world settings for over three decades, and have become increasingly common. However, the use of electronic data capture (EDC) through mobile phones is limited in many areas by inconsistent network connectivity and poor access to electricity, which thwart data transmission and device usage. This is the case in rural Liberia, where many health workers live and work in areas without any access to cellular connectivity or reliable power. Many existing EDC mobile software tools are built for occasionally-disconnected settings, allowing a user to collect data while out of range of a cell tower and transmit data to a central server when he/she regains a network connection. However, few tools exist that can be used indefinitely in fully-disconnected settings, where a user will never have access to the internet or a cell network. This led us to create and implement an EDC software tool that allows for completely offline data transfer and application updating. RESULTS: We designed, pilot-tested, and scaled an open-source fork of Open Data Kit Collect (an Android application that can be used to create EDC systems) that allows for offline Bluetooth-based bidirectional data transfer, enabling a system in which permanently-offline users can collect data and receive application updates. We implemented this platform among a cohort of 317 community health workers and 28 supervisors in a remote area of rural Liberia with incomplete cellular connectivity and low access to power sources. CONCLUSIONS: Running a fully-offline EDC program that completely bypasses the cellular network was found to be feasible; the system is still running, over 4 years after the initial pilot program. The users of this program can theoretically collect data offline for months or years, assuming they receive hardware support when needed. Fully-offline EDC has applications in settings where cellular network coverage is poor, as well as in disaster relief settings in which portions of the communications infrastructure may be temporarily nonfunctional.

A Community Health Worker Intervention to Increase Childhood Disease Treatment Coverage in Rural Liberia: A Controlled Before-and-After Evaluation.

OBJECTIVES: To assess a community health worker (CHW) program's impact on childhood illness treatment in rural Liberia. METHODS: We deployed CHWs in half of Rivercess County in August 2015 with the other half constituting a comparison group until July 2016. All CHWs were provided cash incentives, supply chain support, and monthly clinical supervision. We conducted stratified cluster-sample population-based surveys at baseline (March-April 2015) and follow-up (April-June 2016) and performed a difference-in-differences analysis, adjusted by inverse probability of treatment weighting, to assess changes in treatment of fever, diarrhea, and acute respiratory infection by a qualified provider. RESULTS: We estimated a childhood treatment difference-in-differences of 56.4 percentage points (95% confidence interval [CI] = 36.4, 76.3). At follow-up, CHWs provided 57.6% (95% CI = 42.8, 71.2) of treatment in the intervention group. The difference-in-differences diarrhea oral rehydration therapy was 22.4 percentage points (95% CI = -0.7, 45.5). CONCLUSIONS: Implementation of a CHW program in Rivercess County, Liberia, was associated with large, statistically significant improvements treatment by a qualified provider; however, improvements in correct diarrhea treatment were lower than improvements in coverage. Findings from this study offer support for expansion of Liberia's new National Community Health Assistant Program.

Measuring personal beliefs and perceived norms about intimate partner violence: Population-based survey experiment in rural Uganda.

BACKGROUND: Demographic and Health Surveys (DHS) conducted throughout sub-Saharan Africa indicate there is widespread acceptance of intimate partner violence, contributing to an adverse health risk environment for women. While qualitative studies suggest important limitations in the accuracy of the DHS methods used to elicit attitudes toward intimate partner violence, to date there has been little experimental evidence from sub-Saharan Africa that can be brought to bear on this issue. METHODS AND FINDINGS: We embedded a randomized survey experiment in a population-based survey of 1,334 adult men and women living in Nyakabare Parish, Mbarara, Uganda. The primary outcomes were participants' personal beliefs about the acceptability of intimate partner violence and perceived norms about intimate partner violence in the community. To elicit participants' personal beliefs and perceived norms, we asked about the acceptability of intimate partner violence in five different vignettes. Study participants were randomly assigned to one of three survey instruments, each of which contained varying levels of detail about the extent to which the wife depicted in the vignette intentionally or unintentionally violated gendered standards of behavior. For the questions about personal beliefs, the mean (standard deviation) number of items where intimate partner violence was endorsed as acceptable was 1.26 (1.58) among participants assigned to the DHS-style survey variant (which contained little contextual detail about the wife's intentions), 2.74 (1.81) among participants assigned to the survey variant depicting the wife as intentionally violating gendered standards of behavior, and 0.77 (1.19) among participants assigned to the survey variant depicting the wife as unintentionally violating these standards. In a partial proportional odds regression model adjusting for sex and village of residence, with participants assigned to the DHS-style survey variant as the referent group, participants assigned the survey variant that depicted the wife as intentionally violating gendered standards of behavior were more likely to condone intimate partner violence in a greater number of vignettes (adjusted odds ratios [AORs] ranged from 3.87 to 5.74, with all p < 0.001), while participants assigned the survey variant that depicted the wife as unintentionally violating these standards were less likely to condone intimate partner violence (AORs ranged from 0.29 to 0.70, with p-values ranging from <0.001 to 0.07). The analysis of perceived norms displayed similar patterns, but the effects were slightly smaller in magnitude: participants assigned to the "intentional" survey variant were more likely to perceive intimate partner violence as normative (AORs ranged from 2.05 to 3.51, with all p < 0.001), while participants assigned to the "unintentional" survey variant were less likely to perceive intimate partner violence as normative (AORs ranged from 0.49 to 0.65, with p-values ranging from <0.001 to 0.14). The primary limitations of this study are that our assessments of personal beliefs and perceived norms could have been measured with error and that our findings may not generalize beyond rural Uganda. CONCLUSIONS: Contextual information about the circumstances under which women in hypothetical vignettes were perceived to violate gendered standards of behavior had a significant influence on the extent to which study participants endorsed the acceptability of intimate partner violence. Researchers aiming to assess personal beliefs or perceived norms about intimate partner violence should attempt to eliminate, as much as possible, ambiguities in vignettes and questions administered to study participants. TRIAL REGISTRATION: ClinicalTrials.gov NCT02202824.

IL-33 drives biphasic IL-13 production for noncanonical Type 2 immunity against hookworms.

Parasitic helminths are a major cause of chronic human disease, affecting more than 3 billion people worldwide. Host protection against most parasitic helminths relies upon Type 2 cytokine production, but the mechanisms that regulate interleukin (IL) 4 and 13 production from CD4(+) T helper 2 cells (T(H)2) and innate lymphoid type 2 cells (ILC2s) remain incompletely understood. The epithelial cell-derived cytokines IL-25 and IL-33 promote Type 2 responses, but the extent of functional redundancy between these cytokines is unclear and whether Type 2 memory relies upon either IL-25 or IL-33 is unknown. Herein, we demonstrate a pivotal role for IL-33 in driving primary and anamnestic immunity against the rodent hookworm Nippostrongylus brasiliensis. IL-33-deficient mice have a selective defect in ILC2-derived IL-13 during both primary and secondary challenge infections but generate stronger canonical CD4(+) T helper 2 cells responses (IL-4, IgE, mast cells, and basophils) than WT controls. Lack of IL-13 production in IL-33-deficient mice impairs resistin-like molecule beta (RELMß) expression and eosinophil recruitment, which are two mechanisms that eliminate N. brasiliensis parasites from infected hosts. Thus, IL-33 is requisite for IL-13 but not IL-4-driven Type 2 responses during hookworm infection.

Understanding and predicting social media use among community health center patients: a cross-sectional survey.

BACKGROUND: The use of social media by health care organizations is growing and provides Web-based tools to connect patients, caregivers, and providers. OBJECTIVE: The aim was to determine the use and factors predicting the use of social media for health care-related purposes among medically underserved primary care patients. METHODS: A cross-sectional survey was administered to 444 patients of a federally qualified community health center. RESULTS: Community health center patients preferred that their providers use email, cell phones for texting, and Facebook and cell phone apps for sharing health information. Significantly more Hispanic than white patients believed their providers should use Facebook (P=.001), YouTube (P=.01), and Twitter (P=.04) for sharing health information. Use and intentions to use social media for health-related purposes were significantly higher for those patients with higher subjective norm scores. CONCLUSIONS: Understanding use and factors predicting use can increase adoption and utilization of social media for health care-related purposes among underserved patients in community health centers.

TGF-ß-responsive myeloid cells suppress type 2 immunity and emphysematous pathology after hookworm infection.

Transforming growth factor ß (TGF-ß) regulates inflammation, immunosuppression, and wound-healing cascades, but it remains unclear whether any of these functions involve regulation of myeloid cell function. The present study demonstrates that selective deletion of TGF-ßRII expression in myeloid phagocytes i) impairs macrophage-mediated suppressor activity, ii) increases baseline mRNA expression of proinflammatory chemokines/cytokines in the lung, and iii) enhances type 2 immunity against the hookworm parasite Nippostrongylus brasiliensis. Strikingly, TGF-ß-responsive myeloid cells promote repair of hookworm-damaged lung tissue, because LysM(Cre)TGF-ßRII(flox/flox) mice develop emphysema more rapidly than wild-type littermate controls. Emphysematous pathology in LysM(Cre)TGF-ßRII(flox/flox) mice is characterized by excessive matrix metalloprotease (MMP) activity, reduced lung elasticity, increased total lung capacity, and dysregulated respiration. Thus, TGF-ß effects on myeloid cells suppress helminth immunity as a consequence of restoring lung function after infection.

Black-White disparities in maternal vulnerability and adverse pregnancy outcomes: an ecological population study in the United States, 2014-2018.

Background: Systematic information on the association between community-level determinants and maternal health outcomes and disparities is needed. We aimed to investigate multi-dimensional place-based contributions to Black-White maternal health disparities in the United States. Methods: We constructed the Maternal Vulnerability Index, a geospatial measure of vulnerability to poor maternal health. The index was linked to 13m live births and maternal deaths to mothers aged 10-44 for 2014-2018 in the United States. We quantified racial disparities in exposure to higher risk environments, and used logistic regression to estimate associations between race, vulnerability, and maternal death (n = 3633), low birthweight (n = 1.1m), and preterm birth (n = 1.3m). Findings: Black mothers lived in disproportionately higher maternal vulnerability counties, when compared to White mothers (median of 55 vs 36/100 points). Giving birth in the highest-quartile MVI counties was associated with an increase in the odds of poor outcomes when compared to the lowest-quartile (aOR 1.43 [95% CI 1.20-1.71] for mortality, 1.39 [1.37-1.41] for low birthweight and 1.41 [1.39-1.43] for preterm birth, adjusted for age, educational attainment level and race/ethnicity). Racial disparities exist in low- and high-vulnerability counties: Black mothers in the least vulnerable counties remain at higher risk of maternal mortality, preterm birth, and low birthweight as White mothers in the most vulnerable. Interpretation: Exposure to community maternal vulnerability is associated with increased odds of adverse outcomes, but the Black-White gap in outcomes remained under all vulnerability levels. Our findings suggest that locally-informed precision health interventions and further research into racism are needed to achieve maternal health equity. Funding: Bill & Melinda Gates Foundation (grant number INV-024583).

Normative vs personal attitudes toward persons with HIV, and the mediating role of perceived HIV stigma in rural Uganda.

BACKGROUND: HIV stigma has well-documented negative impacts on HIV testing, transmission risk behavior, initiation of and adherence to antiretroviral therapy, and retention in care. We sought to assess the extent to which anticipated HIV stigma is based on misperceptions of normative attitudes toward persons with HIV, and to determine whether persons with HIV have stronger misperceptions compared with HIV-negative persons or persons of unknown serostatus. We also sought to estimate the association between normative attitudes about persons with HIV and personal attitudes about persons with HIV, and to determine the extent to which anticipated stigma mediates this association. METHODS: We conducted a whole-population survey of 1776 persons living in 8 rural villages in southwestern Uganda. Negative attitudes toward persons with HIV, and anticipated stigma, were measured using a newly validated 15-item scale measuring multiple dimensions of HIV stigma, including social distance, blaming attitudes, and concerns about reciprocity. We used multivariable regression to estimate the association between normative attitudes about persons with HIV and personal attitudes toward persons with HIV, and to determine the extent to which perceptions of normative attitudes (anticipated stigma) mediated this association. RESULTS: Study participants believed that negative attitudes toward persons with HIV were more pervasive than they actually are. Perceptions of the extent to which these negative attitudes are normative mediated more than one-third of the association between normative attitudes and their personal attitudes. In contrast to what we originally hypothesized, persons with HIV were less likely to misperceive these norms and perceived normative attitudes to be less stigmatizing than did others in the general population. CONCLUSIONS: Interventions designed to accurately describe normative attitudes toward persons with HIV may reduce HIV stigma without directly focusing on the educational components that are typically embedded in anti-stigma interventions.

Measuring Knowledge of Community Health Workers at the Last Mile in Liberia: Feasibility and Results of Clinical Vignette Assessments.

INTRODUCTION: Community health workers (CHWs) can provide lifesaving treatment for children in remote areas, but high-quality care is essential for effective delivery. Measuring the quality of community-based care in remote areas is logistically challenging. Clinical vignettes have been validated in facility settings as a proxy for competency. We assessed feasibility and effectiveness of clinical vignettes to measure CHW knowledge of integrated community case management (iCCM) in Liberia's national CHW program. METHODS: We developed 3 vignettes to measure knowledge of iCCM illnesses (malaria, diarrhea, and pneumonia) in 4 main areas: assessment, diagnosis, treatment, and caregiver instructions. Trained nurse supervisors administered the vignettes to CHWs in 3 counties in rural Liberia as part of routine program supervision between January and May 2019, collected data on CHW knowledge using a standardized checklist tool, and provided feedback and coaching to CHWs in real time after vignette administration. Proportions of vignettes correctly managed, including illness classification, treatment, and referral where necessary, were calculated. We assessed feasibility, defined as the ability of clinical supervisors to administer the vignettes integrated into their routine activities once per year for each CHW, and effectiveness, defined as the ability of the vignettes to measure the primary outcomes of CHW knowledge of diagnosis and treatment including referrals. RESULTS: We were able to integrate this assessment into routine supervision, facilitate real-time coaching, and collect data on iCCM knowledge among 155 CHWs through delivery of 465 vignettes. Diagnosis including severity was correct in 65%-82% of vignettes. CHWs correctly identified danger signs in 44%-50% of vignettes, correctly proposed referral to the facility in 63% of vignettes including danger signs, and chose correct lifesaving treatment in 23%-65% of vignettes. Both diagnosis and lifesaving treatment rates were highest for malaria and lowest for severe pneumonia. CONCLUSION: Administration of vignettes to assess knowledge of correct iCCM case management was feasible and effective in producing results in this setting. Proportions of correct diagnosis and lifesaving treatment varied, with high proportions for uncomplicated disease, but lower for more severe cases, with accurate recognition of danger signs posing a challenge. Future work includes validation of vignettes for use with CHWs through direct observation, strengthening supportive supervision, and program interventions to address identified knowledge gaps.

Prevalence and correlates of chronic obstructive pulmonary disease and chronic respiratory symptoms in rural southwestern Uganda: a cross-sectional, population-based study.

BACKGROUND: The global burden of chronic obstructive pulmonary disease (COPD) disproportionately affects resource-limited settings such as sub-Saharan Africa (SSA), but population-based prevalence estimates in SSA are rare. We aimed to estimate the population prevalence of COPD and chronic respiratory symptoms in rural southwestern Uganda. METHODS: Adults at least 18 years of age who participated in a population-wide census in rural southwestern Uganda completed respiratory questionnaires and lung function testing with bronchodilator challenge at health screening events in June 2015. We defined COPD as post-bronchodilator forced expiratory volume in one second to forced vital capacity ratio less than the lower limit of normal. We fit multivariable linear and log binomial regression models to estimate correlates of abnormal lung function and respiratory symptoms, respectively. We included inverse probability of sampling weights in models to facilitate population-level estimates. RESULTS: Forty-six percent of census participants (843/1814) completed respiratory questionnaires and spirometry, of which 565 (67%) met acceptability standards. COPD and respiratory symptom population prevalence were 2% (95% confidence interval (CI) = 1%-3%) and 30% (95% CI = 25%-36%), respectively. Respiratory symptoms were more prevalent and lung function was lower among women and ever-smokers (P < 0.05). HIV serostatus was associated with neither respiratory symptoms nor lung function. CONCLUSIONS: COPD population prevalence was low despite prevalent respiratory symptoms. This work adds to the growing body of literature depicting lower-than-expected COPD prevalence estimates in SSA and raises questions about whether the high respiratory symptom burden in rural southwestern Uganda represents underlying structural lung disease not identified by screening spirometry.

Satellite images and machine learning can identify remote communities to facilitate access to health services.

OBJECTIVE: Community health systems operating in remote areas require accurate information about where people live to efficiently provide services across large regions. We sought to determine whether a machine learning analyses of satellite imagery can be used to map remote communities to facilitate service delivery and planning. MATERIALS AND METHODS: We developed a method for mapping communities using a deep learning approach that excels at detecting objects within images. We trained an algorithm to detect individual buildings, then examined building clusters to identify groupings suggestive of communities. The approach was validated in southeastern Liberia, by comparing algorithmically generated results with community location data collected manually by enumerators and community health workers. RESULTS: The deep learning approach achieved 86.47% positive predictive value and 79.49% sensitivity with respect to individual building detection. The approach identified 75.67% (n = 451) of communities registered through the community enumeration process, and identified an additional 167 potential communities not previously registered. Several instances of false positives and false negatives were identified. DISCUSSION: Analysis of satellite images is a promising solution for mapping remote communities rapidly, and with relatively low costs. Further research is needed to determine whether the communities identified algorithmically, but not registered in the manual enumeration process, are currently inhabited. CONCLUSIONS: To our knowledge, this study represents the first effort to apply image recognition algorithms to rural healthcare delivery. Results suggest that these methods have the potential to enhance community health worker scale-up efforts in underserved remote communities.

Population Prevalence and Correlates of Prolonged QT Interval: Cross-Sectional, Population-Based Study From Rural Uganda.

OBJECTIVES: We aimed to estimate the prevalence and correlates of QT interval prolongation in rural Uganda. BACKGROUND: Major electrocardiographic abnormalities, including prolonged QT interval, have been shown to be independently predictive of adverse cardiovascular events among Western populations. Cardiovascular diseases are on the rise in sub-Saharan Africa with poorly characterized context-specific risk factors. An important question is whether ECG screening might have value in cardiovascular disease risk stratification in SSA. METHODS: We conducted a cross-sectional survey in a sample of adults participating in an ongoing whole-population cohort in Mbarara, Uganda, in 2015. Of 1,814 subjects enrolled in the parent whole-population cohort, 856 (47%) participated in the study. Participants completed 12-lead electrocardiography and cardiovascular disease risk factors assessment. We summarized sex-specific, heart rate variation-adjusted QT (QTa) defining prolonged QTa as >460 ms in women and >450 ms in men. We fit linear and logistic regression models to estimate correlates of (continuous) QTa interval length and (dichotomous) prolonged QTa. Models included inverse probability of sampling weights to generate population-level estimates accounting for study nonparticipation. RESULTS: We assessed data from 828 participants with electrocardiograms. The weighted population mean age was 38.4 years (95% confidence interval: 36.3-40.4). The weighted population was 50.4% female, 11.5% had elevated blood pressure, and 57.6% had a high-sensitivity C-reactive protein >1 mg/dl. The population mean QTa was 409.1 ms (95% confidence interval: 405.1-413.1), and 10.3% (95% confidence interval: 7.8-13.5) met criteria for prolonged QTa. Women had a higher mean QTa (421.6 ms vs. 396.3 ms; p < 0.001), and a higher proportion of women had a prolonged QTa (14.0% vs. 9.3%; p = 0.122) than did men. In multivariable-adjusted regression models, female sex and hypertension correlated with higher mean QTa and meeting criteria for prolonged QTa, respectively. CONCLUSIONS: QT interval prolongation is highly prevalent in rural Uganda and may be more common than in high-income settings. Female sex, age, and high blood pressure correlated with QT interval prolongation. Future work should assess whether genetic predisposition or environmental factors in sub-Saharan African populations contribute to prolonged QT and clarify consequences.

Strong underwater adhesives made by self-assembling multi-protein nanofibres.

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m(-2), which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

Differential colonization with segmented filamentous bacteria and Lactobacillus murinus do not drive divergent development of diet-induced obesity in C57BL/6 mice.

Alterations in the gut microbiota have been proposed to modify the development and maintenance of obesity and its sequelae. Definition of underlying mechanisms has lagged, although the ability of commensal gut microbes to drive pathways involved in inflammation and metabolism has generated compelling, testable hypotheses. We studied C57BL/6 mice from two vendors that differ in their obesogenic response and in their colonization by specific members of the gut microbiota having well-described roles in regulating gut immune responses. We confirmed the presence of robust differences in weight gain in mice from these different vendors during high fat diet stress. However, neither specific, highly divergent members of the gut microbiota (Lactobacillus murinus, segmented filamentous bacteria) nor the horizontally transmissible gut microbiota were found to be responsible. Constitutive differences in locomotor activity were observed, however. These data underscore the importance of selecting appropriate controls in this widely used model of human obesity.