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Murray, M.

Publications and source records attributed to Murray, M..

33 records · Page 2Linked to original sources

Proteasomal subunit depletions differentially affect germline integrity in C. elegans

The 26S proteasome is a multi-subunit protein complex that is canonically known for its ability to degrade proteins in cells and maintain protein homeostasis. Non-canonical or non-proteolytic roles of proteasomal subunits exist, but remain less well studied. We provide characterization of germline-specific functions of different 19S RP proteasome subunits in C. elegans using RNAi specifically from the L4 stage and through generation of endogenously tagged 19S RP lid subunit strains. We show functions for the 19S RP in regulation of proliferation and maintenance of integrity of mitotic zone nuclei, in polymerization of the synaptonemal complex (SC) onto meiotic chromosomes and in the timing of SC subunit redistribution to the short arm of the bivalent, and in turnover of XND-1 proteins at late pachytene. Furthermore, we report that certain 19S RP subunits are required for proper germ line localization of WEE-1.3, a major meiotic kinase. Additionally, endogenous fluorescent labeling revealed that the two isoforms of the essential 19S RP proteasome subunit RPN-6.1 are expressed in a tissue-specific manner in the hermaphrodite. Also, we demonstrate that the 19S RP subunits RPN-6.1 and RPN-7 are crucial for the nuclear localization of the lid subunits RPN-8 and RPN-9 in oocytes, potentially introducing C. elegans germ line as model to study proteasome assembly real-time. Collectively, our data support the premise that certain 19S RP proteasome subunits are playing tissue-specific roles, especially in the germ line. We propose C. elegans as a versatile multicellular model to study the diverse proteolytic and non-proteolytic roles that proteasome subunits play in vivo.

developmental biology↗

Ferroptosis Regulation by the NGLY1/NFE2L1 Pathway

Ferroptosis is an oxidative form of non-apoptotic cell death whose transcriptional regulation is poorly understood. Capncollar (CNC) transcription factors including Nuclear Factor Erythroid-2 Related Factor 1 (NFE2L1/NRF1) and NFE2L2 (NRF2) are important regulators of oxidative stress responses. Here, we report that NFE2L1 expression inhibits ferroptosis, independent of NFE2L2. NFE2L1 inhibits ferroptosis by promoting expression of the key anti-ferroptotic lipid hydroperoxidase glutathione peroxidase 4 (GPX4). NFE2L1 abundance and function are regulated post-translationally by N-glycosylation. Functional maturation of NFE2L1 requires deglycosylation by cytosolic peptide:N-glycanase 1 (NGLY1). We find that loss of NGLY1 or NFE2L1 enhances ferroptosis sensitivity. Expression of wild-type NGLY1 but not a disease-associated NGLY1 mutant inhibits ferroptosis, and this effect is dependent on the presence of NFE2L1. Enhanced ferroptosis sensitivity in NFE2L1 and NFE2L2 knockout cells can be potently reverted by expression of an NFE2L1 mutant containing eight asparagine-to-aspartate protein sequence substitutions, which mimic NGLY1-catalyzed sequence editing. Enhanced ferroptosis sensitivity in NGLY1/NFE2L1 pathway mutants could also be reversed by overexpression of NFE2L2. These results suggest that ferroptosis sensitivity is regulated by NGLY1-catalyzed NFE2L1 deglycosylation, and highlight a broad role for CNC transcription factors in ferroptosis regulation. Significance StatementFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. NFE2L1/NRF1 is a capncollar (CNC) transcription factor whose role in ferroptosis regulation is unclear. Unlike the CNC family member NFE2L2/NRF2, NFE2L1 is an N-glycoprotein whose abundance is regulated by post-translational deglycosylation catalyzed by the enzyme peptide:N-glycanase 1 (NGLY1). Our results indicate that NGLY1-mediated NFE2L1 deglycosylation, resulting in editing of the NFE2L1 amino acid sequence, is necessary for NFE2L1 to inhibit ferroptosis. Mechanistically, NFE2L1 inhibits ferroptosis by via the anti-ferroptotic protein GPX4. This work demonstrates that CNC transcription factors beyond NFE2L2 can regulate ferroptosis. This work may suggest a role of misregulation of ferroptosis in NGLY1 deficiency, an ultrarare genetic disorder.

cell biology↗

The omega-3-fatty acid docosahexaenoic acid modulates intracellular myo-inositol and its biosynthetic genes.

Bipolar disorder is a debilitating mood disorder characterized by recurring episodes of mania and depression. It affects 2.6% of adults and has a lifetime prevalence among adults of 3.9%. Current mood stabilizers are not always effective and/or are not well tolerated by many patients; thus, there is a need to develop or identify more effective and less harmful treatments. Omega-3-fatty acids have been shown to be effective in the treatment of bipolar disorder; however, their mechanism of action is unknown. Myo-inositol depletion has been hypothesized as the mechanism by which mood stabilizers exert their therapeutic effect. Using an enzymatic assay, we determined intracellular myo-inositol levels increased more than 2-fold when cells were grown in the presence of the omega-3 fatty acid docosahexaenoic acid (DHA). RT-qPCR was used to characterize the effects of DHA on genes in the myo-inositol biosynthetic pathway. We show DHA increases relative expression and has a concentration-dependent impact on INO1 and INM1, which encode myo-inositol-1-phosphate synthase and myo-inositol monophosphate 1-phosphatase, respectively. We therefore conclude that the omega-3-fatty acid DHA exerts its therapeutic effect on bipolar disorder by increasing intracellular myo-inositol, which may be accomplished by upregulating its biosynthetic genes.

molecular biology↗

A sex-specific evolutionary interaction between ADCY9 and CETP

Pharmacogenomic studies have revealed associations between rs1967309 in the adenylyl cyclase type 9 (ADCY9) gene and clinical responses to the cholesteryl ester transfer protein (CETP) modulator dalcetrapib, however, the mechanism behind this interaction is still unknown. Here, we characterized selective signals at the locus associated with the pharmacogenomic response in human populations and we show that rs1967309 region exhibits signatures of positive selection in several human populations. Furthermore, we identified a variant in CETP, rs158477, which is in long-range linkage disequilibrium with rs1967309 in the Peruvian population. The signal is mainly seen in males, a sex-specific result that is replicated in the LIMAA cohort of over 3,400 Peruvians. Analyses of RNA-seq data further suggest an epistatic interaction on CETP expression levels between the two SNPs in multiple tissues, which also differs between males and females. We also detected interaction effects of the two SNPs with sex on cardiovascular phenotypes in the UK Biobank, in line with the sex-specific genotype associations found in Peruvians at these loci. We propose that ADCY9 and CETP coevolved during recent human evolution due to sex-specific selection, which points towards a biological link between dalcetrapibs pharmacogene ADCY9 and its therapeutic target CETP.

genetics↗

Genome-wide association study reveals an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels

Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. Additionally, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels.

genetics↗

Programmatic goals and spatial epidemiology influence the merit of targeted versus of population-wide interventions for yaws eradication

Infectious disease eradication programs often pursue spatially targeted interventions, but how well they perform might depend on the underlying spatial epidemiology and the specific goal of the program. We use a stochastic compartmental metapopulation model of yaws transmission to investigate how total targeted treatment (TTT) performs compared to mass drug administration (MDA) in different settings. While TTT can efficiently control the prevalence of active yaws disease, we consistently found that multiple rounds of TTT are required to match the impact of 1 round of MDA on the prevalence of latent yaws infection. When complete eradication of yaws is the goal, MDA can achieve the same result as TTT more quickly and probably at lower cost. We found that the performance of TTT is improved when there is little mixing between subpopulations and when there is spatial heterogeneity in transmissibility, but even in these settings, our model suggests that MDA will still outperform TTT.\n\nSignificance StatementYaws is a neglected tropical disease that causes skin lesions. Eradicating yaws is challenging because people can be infected but not show any signs or symptoms for years. Using simulations, we found that targeting antibiotics to people with active yaws and to their neighbors is a good way to combat active yaws, but treating entire populations is a better way to get rid of all infections, including the hidden ones. Also, targeted treatment works better in populations in which people do not move around much and in which how easily the disease is transmitted varies from place to place. Overall, a targeted treatment strategy uses fewer antibiotics but takes longer than mass treatment to achieve results.

epidemiology↗

Complete blood count with differential: An effective diagnostic for IBS subtype in the context of BMI?

The Complete Blood Count with Differential hematological assay is a mainstay diagnostic for point-of-care clinical diagnoses for a spectrum of diseases including infection, inflammation, anemia, and leukemia, and CBC-D profiles are under investigation as early prognostic biomarkers for leukemias and other diseases. Chronic abdominal pain (CAP) and irritable bowel syndrome (IBS) are prevalent gastrointestinal disorders in the United States, with obesity among the most common comorbidities. Often, IBS-like gastrointestinal (GI) symptoms persist after resolution of known inflammation and/or enteropathogenic infection, and current literature contains significant discussion of the extent to which IBS is within the biological spectrum of inflammatory disease. Obesity is also associated with generalized signatures of inflammation and may confound accurate diagnoses. We performed ANOVA, multiple means comparisons, statistical analyses of CBC data from our \"Brain-Gut Natural History\" (BGNH) clinical cohort, with additional ELISA assays for lipopolysaccharide binding protein (LBP), IL-10, cortisol, and ACTH, signatures of immune-inflammatory response and Hypothalamic-Pituitary-Adrenal (HPA) axis activity, respectively. BGNH cohort includes healthy and overweight individuals diagnosed with IBS diarrhea-(IBS-D) and constipation-predominant (IBS-C) subtypes. We identified several potentially significant markers for IBS-D and IBS-C, notably IL-10, mean platelet volume (MPV), with LBP and monocyte percent also showing some statistical significance. Weight also showed significant results for overweight vs. normal weight, regardless of IBS subtype, particularly for Cortisol. CBC-D predictive profiles for IBS subtype and weight were identified using discriminant functions analysis and show that predictivity of marker profiles have poor performance relative to their normal weight subsets. Further refinement of this analysis will be performed utilizing increased sample size, additional molecular profiles, and enhanced statistical analysis.

immunology↗

OVOL2 induces mesenchymal-to-epithelial transition in fibroblasts and enhances reprogramming to epithelial lineages

Mesenchymal-to-epithelial transition (MET) is an important step in cell reprogramming from fibroblasts (a cell type frequently used for this purpose) to various epithelial cell types. However, the mechanism underlying MET induction in fibroblasts remains to be understood. The present study aimed to identify the transcription factors (TFs) that efficiently induce MET in dermal fibroblasts. OVOL2 was identified as a potent inducer of key epithelial genes, and OVOL2 cooperatively enhanced MET induced by HNF1A, TP63, and KLF4, which are known reprogramming TFs to epithelial lineages. In TP63/KLF4-induced keratinocyte-like cell-state reprogramming, OVOL2 greatly facilitated the activation of epithelial and keratinocyte-specific genes. This was accompanied by enhanced changes in chromatin accessibility across the genome. Mechanistically, motif enrichment analysis revealed that the target loci of KLF4 and TP63 become accessible upon induction of TFs, whereas the OVOL2 target loci become inaccessible. This indicates that KLF4 and TP63 positively regulate keratinocyte-associated genes whereas OVOL2 suppresses fibroblast-associated genes. The exogenous expression of OVOL2 therefore disrupts fibroblast lineage identity and facilitates fibroblast cell reprogramming into epithelial lineages cooperatively with tissue-specific reprogramming factors. Identification of OVOL2 as a MET inducer and an epithelial reprogramming enhancer in fibroblasts provides new insights into cellular reprogramming improvement for future applications.

cell biology↗

Isoniazid preventive therapy protects against tuberculosis among household contacts of isoniazid-resistant patients.

BackgroundThe World Health Organization recommends the use of isoniazid (INH) alone or combination INH and rifapentine therapy to treat latent tuberculosis infection (LTBI) in groups at high risk of tuberculosis (TB) progression. The recent rise of INH- and multi-drug resistant (MDR) TB has complicated the choice of LTBI treatment regimen. We examine the risk of TB disease among household contacts (HHCs) who received INH after being exposed to patients with drug-sensitive, INH-resistant, or MDR tuberculosis. MethodsIn this prospective cohort study conducted Between September 2009 and August 2012 in Lima, Peru, we identified 4,500 index TB patients and measured incident TB disease in their 14,044 HHCs over a one-year follow-up period. HHCs under 19 years of age were offered INH preventive therapy (IPT). We used a Cox frailty proportional hazards model to evaluate whether the effect of IPT on incident TB disease varied by the resistance profile of the index case. We repeated the analyses in a second independent dataset. FindingsAmong 4,216 HHCs under 19 years of age, 2,106 (50%) initiated IPT at enrolment. The protective effect of INH was more extreme in HHCs exposed to drug-sensitive (Hazard Ratio [95% confidence interval]=0{middle dot}2[0{middle dot}20-0{middle dot}50]) and to MDR-TB (0{middle dot}26[0{middle dot}08-0{middle dot}77]) compared to those exposed to mono-INH-resistant (0{middle dot}80[0{middle dot}23 to 2{middle dot}79]). Among those who received at least three months of INH, effectiveness increased across all three groups (INH-sensitive:0{middle dot}20 [0{middle dot}10 to 040]; MDR:0{middle dot}16 [0 02-127]; mono-INH-resistant:0{middle dot}72 [0{middle dot}16-3{middle dot}16]). In the second independent study, TB occurred in none of the 76 HHCs who received IPT compared to 3% (8/273) of those who did not. InterpretationWe found that IPT use is associated with reduced incidence of TB disease among contacts of MDR-TB patients. This finding suggests that INH may have role in the management of MDR-LTBI. FundingNational Institutes of Health and the National Institute of Allergy and Infectious Diseases CETR (U19AI109755) and TBRU (U19AI111224)

epidemiology↗

RESISTANCE AT NO COST: THE TRANSMISSIBILITY AND POTENTIAL FOR DISEASE PROGRESSION OF DRUG-RESISTANT M. TUBERCULOSIS

BackgroundThe future trajectory of drug resistant tuberculosis strongly depends on the fitness costs of drug resistance mutations. Here, we measured the association of phenotypic drug resistance and the risk of TB infection and disease among household contacts (HHCs) of patients with pulmonary TB.\n\nMethodsWe evaluated 12767 HHCs of patients with drug sensitive and resistant pulmonary TB at baseline, two, six, and 12 months to ascertain infection status and to determine whether they developed tuberculosis disease. We also assessed the impact of drug resistance phenotype on the likelihood that a TB strain shared a genetic fingerprint with at least one other TB patient in the cohort.\n\nFindingsAmong 3339 TB patients for whom were DST available, 1274 (38%) had TB that was resistant to at least one drug and 478 (14*3%) had multi-drug resistant (MDR) TB, i.e. TB resistant to both INH and rifampicin. Compared to HHCs of drug sensitive TB patients, those exposed to a patient with MDR-TB had an 8% (95% CI: 4-13%) higher risk of infection by the end of follow up. We found no statistically significant difference in the relative hazard of incident TB disease among HHCs exposed to MDR-TB compared to DS-TB (Adjusted HR 1*28 [(95% CI: *9-1*83]). Patients with MDR-TB were more likely to be part of a genetic cluster than were DS-TB patients.\n\nInterpretationClinical strains of MDR M. tuberculosis are neither less transmissible than drug sensitive strains nor less likely to cause disease. (ClinicalTrials.gov number, NCT00676754)\n\nFundingNational Institutes of Health: NIH/NIAID CETR U19AI109755\n\nStatementAll authors have seen the manuscript and approved the manuscript.

epidemiology↗

Genome wide association with quantitative resistance phenotypes in Mycobacterium tuberculosis reveals novel resistance genes and regulatory regions

Drug resistance is threatening attempts at tuberculosis epidemic control. Molecular diagnostics for drug resistance that rely on the detection of resistance-related mutations could expedite patient care and accelerate progress in TB eradication. We performed minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We then used a linear mixed model to evaluate genome wide associations between mutations in MTB genes or noncoding regions and drug resistance, followed by validation of our findings in an independent dataset of 792 patient isolates. Novel associations at 13 genomic loci were confirmed in the validation set, with 2 involving noncoding regions. We found promoter mutations to have smaller average effects on resistance levels than gene body mutations in genes where both can contribute to resistance. Enabled by a quantitative measure of resistance, we estimated the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. We also report the proportion of variation in resistance levels explained by the novel loci identified here. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causative or compensatory loci, and emphasizes the contribution of the noncoding portion of the genome.

evolutionary biology↗

Rifampicin and rifabutin resistance in 1000 Mycobacterium tuberculosis clinical isolates

SynopsisDrug resistant tuberculosis (TB) remains a public health challenge with limited treatment options and high associated mortality. Rifamycins are among the most potent anti-TB drugs, and the loss of susceptibility to these agents, a hallmark of MDR TB, is considered a substantial therapeutic challenge. Rifamycins are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross resistance between the two rifamycin drugs rifampicin (RIF) and rifabutin (RFB). We performed whole genome sequencing of 1005 MTB clinical isolates and measured minimum inhibitory concentration (MIC) to both agents on 7H10 agar using the indirect proportion method. Of the 1005 isolates, 767 were RIF resistant, and of these, 211 (27%) were sensitive to RFB at the critical concentration of 0.5ug/ml; 101/211 isolates had the rpoB mutation D435V (E.coli D516V). Isolates with discrepant resistance (RIF R and RFB S) 16.9 times more likely to harbor a D435V mutation as those resistant to both agents (OR 95% CI 10.5-27.9, P-value <10-40). To further understand this discrepancy, we generated both D435V and S450L (E.coli S531L) rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamar blue reduction assay. Compared with wildtype, D435V increased the 50% inhibitory concentration (IC50) to both RIF and RFB, however in both cases to a lesser degree than the S450L mutation. The observation that the rpoB D435V mutation produces an increase in the IC50 for both drugs contrasts with findings from previous smaller studies that suggested that isolates with D435V mutation remain RFB susceptible despite being RIF resistant. Our finding thus suggests that the recommended critical testing concentration for RFB should be revised.

evolutionary biology↗

Spatial-temporal clustering analysis of yaws on Lihir Island, Papua New Guinea to enhance planning and implementation of eradication programs

BackgroundIn the global program for the eradication of yaws, assessments of the prevalence of the disease are used to decide where to initiate mass treatment. However, the smallest administrative unit which should be used as the basis for making decisions is not clear. We investigated spatial and temporal clustering of yaws to help inform the choice of implementation unit.\n\nMethodology/Principal findingsWe analyzed 11 years of passive surveillance data on incident yaws cases (n = 1448) from Lihir Island, Papua New Guinea. After adjusting for age, sex, and trends in health-seeking, we detected three non-overlapping spatiotemporal clusters (p < 1 x 10-17, p = 1.4 x 10-14, p = 1.4 x 10-8). These lasted from 28 to 47 months in duration and each encompassed between 4 and 6 villages. We also assessed spatial clustering of prevalent yaws cases (n = 532) that had been detected in 7 biannual active case finding surveys beginning in 2013. We identified 1 statistically significant cluster in each survey. We considered the possibility that schools that serve multiple villages might be loci of transmission, but we found no evidence that incident cases of yaws among 8- to 14-year-olds clustered within primary school attendance areas (p = 0.684).\n\nConclusions/SignificanceThese clusters likely reflect transmission of yaws across village boundaries; villages may be epidemiologically linked to a degree such that mass drug administration may be more effectively implemented at a spatial scale larger than the individual village.\n\nAuthor SummaryThe World Health Organization aims to eradicate yaws using mass drug administration (MDA), which consists of treating everyone in an administrative unit with antibiotics. The administrative unit in a country which is used as the basis for making decisions about implementing MDA is called the implementation unit. Prevalence assessments are used to identify endemic communities for mass treatment programs, but the spatial scale (e.g. village, sub-district, district, or province) at which mass treatment should be implemented is currently unclear. The choice of implementation unit depends on many factors; one of these is the underlying transmission patterns of the disease. Using data from Lihir Island, Papua New Guinea, we found that geographic clusters of yaws often spanned multiple villages. These clusters likely reflect transmission of the infectious disease across village boundaries and suggest that it may be best to implement MDA at a spatial scale larger than the individual village, for example at sub-district level.

epidemiology↗

Downregulation of the central noradrenergic system by Toxoplasma gondii infection

The parasitic protozoan Toxoplasma gondii becomes encysted in brain and muscle tissue during chronic infection, a stage that was previously thought to be dormant but has been found to be active and associated with physiological effects in the host. Dysregulation of catecholamines in the CNS has previously been observed in chronically-infected animals. In the study described here, the noradrenergic system was suppressed with decreased levels of norepinephrine in brains of infected animals and in infected neuronal cells in vitro. Expression of dopamine {beta}-hydroxylase (DBH), essential for synthesis of norepinephrine from dopamine, was the most differentially-expressed gene in infections in vitro and was down-regulated in infected brain tissue, particularly in the prefrontal cortex and dorsal locus coeruleus/pons region. The down-regulated DBH expression in infected rat catecholaminergic and human neuronal cells corresponded with decreased norepinephrine and increased dopamine. As the DBH suppression was observed in vitro, this effect is not caused by neuroinflammation. Silencing of DBH expression was specific for T. gondii infection and was not observed with CMV infection. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically-infected animals, with a high correlation between DBH expression and infection intensity. These findings together provide a plausible mechanism to explain prior discrepancies in changes to CNS neurotransmitters levels with infection. The suppression of norepinephrine synthesis observed here may, in part, explain behavioural effects of infection, associations with mental illness, and neurological consequences of infection such as the loss of coordination and motor impairments associated with human toxoplasmosis.

microbiology↗

Catching up to a fast-moving target: Evaluation of a health system strengthening intervention in rural Rwanda 2005-2010 using data from repeated cross-sectional surveys

IntroductionAlthough Rwandas health system underwent major reforms and improvements after the 1994 Genocide, the health system and population health in the southeast lagged behind other areas. In 2005 Partners In Health and the Rwandan Ministry of Health began a health system strengthening intervention in this region.\n\nMethodsCombining results from the 2005 and 2010 Demographic and Health Surveys with those from a supplemental 2010 survey, we compared changes in health system output indicators and population health outcomes between 2005 and 2010 as reported by 21,338 women living in the intervention area and similar rural areas, controlling for potential confounding by economic and demographic variables.\n\nResultsOverall health system coverage improved similarly in both regions between 2005 and 2010, with an indicator of composite coverage of child health interventions increasing from 57.9% to 75.0% in the intervention area and from 58.7% to 73.8% in other rural areas. Despite experiencing poorer health outcomes in 2005, the intervention area caught up to or exceeded other rural areas on 23 of 25 indicators. Most notably, under-five mortality declined by an annual rate of 12.8% in the intervention area, from 229.8 to 83.2 deaths per 1000 live births, and by 8.9% in other rural areas, from 157.7 to 75.8 deaths per 1000 live births. Improvements were most dramatic among the poorest households.\n\nConclusionWe observed dramatic improvements in population health outcomes including under-five mortality between 2005 and 2010 in rural Rwanda generally, and in the intervention area specifically.\n\nSUMMARY BOXO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LIMuch of the evidence that health system strengthening in rural Africa has improved health outcomes comes from studies of targeted regional interventions such as performance based financing or community health worker programs, rather than integrated interventions that encompass multiple components including infrastructure and supply chain investments, health management information system, workforce training and incentives at all levels, community health workers, and free services for poor patients.\nC_LIO_LIIn addition to these experimental or quasi-experimental studies, a series of case studies have documented individual nations, pathways to achieving millennium development goal 4 target, the reduction of under-five mortality by two thirds between 1990 and 2015.\nC_LIO_LIThese reports suggest that improvements in coverage of reproductive, maternal and child health indicators explain some, but not all, of the decline in child mortality and that these successes occurred in the context of national gains in health, nutrition and food security, sanitation, poverty reduction, and access to clean water.\nC_LI\n\nWhat are the new findings?O_LICoverage of most maternal and child health care interventions improved at a similar pace in our rural intervention area and other rural areas.\nC_LIO_LIDespite experiencing poorer health outcomes in 2005, our rural intervention area caught up to or exceeded other rural areas on 23 of 25 population health indicators by 2010.\nC_LIO_LIInfant and under-5 mortality declined in our rural intervention area even more precipitously than in other rural areas of Rwanda between 2005 and 2010.\nC_LI\n\nHow might this influence practice?O_LIThe process of strengthening national health systems often involves trade-offs between a focus on first testing individual programs that distributed widely, as is often practiced by pilot programs with multilateral institutions, or implementing multiple simultaneous programs locally. Our results show that integrated health system strengthening interventions can be locally adapted to enable the rapid expansion of health care coverage as well as dramatic improvements in population health outcomes.\nC_LIO_LIIntegrated multi-level interventions can also help narrow the health care coverage and outcome gap between richer and poorer members of a society.\nC_LIO_LINational governments can leverage nongovernmental partners to achieve the health related sustainable development goals through joint implementation of national health policy.\nC_LI

epidemiology↗