Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Protective efficacy of an attenuated Mtb ΔLprG vaccine in mice.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Rhizarian 'Novel Clade 10' Revealed as Abundant and Diverse Planktonic and Terrestrial Flagellates, including Aquavolon n. gen.

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria.

Oxygen and pulse oximetry in childhood pneumonia: surveys of clinicians and student clinicians in Cambodia.

OBJECTIVE: To better understand the availability of oxygen and pulse oximetry, barriers to use, clinician perceptions and practices regarding their role in the management of childhood pneumonia, and the formal education and training regarding these technologies received by student clinicians in Cambodia. METHODS: In the clinician survey, we surveyed 81 clinicians practising at all national paediatric, provincial and district referral hospitals throughout Cambodia. Respondents were primarily physicians whose scope of practice included paediatrics, and most reported the presence of oxygen (93% (95% confidence interval (CI) [87, 98])) but less availability of pulse oximetry (51% (95% CI [39, 61])). RESULTS: Common barriers to use included a lack of policies and guidelines, as well as a lack of training. In the student clinician survey, 332 graduating medical and nursing students were surveyed, and most reported learning about oxygen (96% (95% CI [94, 98])) and pulse oximetry (72% (95% CI [67, 77])) during their training. CONCLUSIONS: Data from both surveys indicate that despite their utility, oxygen and pulse oximetry may be underused in Cambodia. The reported barriers and perceptions of the tools indicate a clear role for improved training for clinicians and students on the use of oxygen and pulse oximetry, the value of oxygen and pulse oximetry for managing childhood pneumonia, and the need for improved policies and guidelines governing their use.

Variation in metabolic demand following severe pediatric traumatic brain injury: A case review.

A traumatic brain injury (TBI) is one of the most common pediatric traumas among children in the United States. Appropriate nutrition support, including the initiation of early enteral nutrition, within the first 48 h after injury is crucial for children with a TBI. It is important that clinicians avoid both underfeeding and overfeeding, as both can lead to poor outcomes. However, the variable metabolic response to a TBI can make determining appropriate nutrition support difficult. Because of the dynamic metabolic demand, indirect calorimetry (IC) is recommended, instead of predictive equations, to measure energy requirements. Although IC is suggested and ideal, few hospitals have the technology available. This case review discusses the variable metabolic response, identified using IC, in a child with a severe TBI. The case report highlights the ability of the team to meet measured energy requirements early, even in the setting of fluid overload. It also highlights the presumed positive impact of early and appropriate nutrition provision on the patient's clinical and functional recovery. Further research is needed to investigate the metabolic response to TBIs in children and the impact optimal feedings based on the measured resting energy expenditure have on clinical, functional, and rehabilitation outcomes.

Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21.

We performed a genome-wide association study (GWAS) in 1705 Parkinson's disease (PD) UK patients and 5175 UK controls, the largest sample size so far for a PD GWAS. Replication was attempted in an additional cohort of 1039 French PD cases and 1984 controls for the 27 regions showing the strongest evidence of association (P< 10(-4)). We replicated published associations in the 4q22/SNCA and 17q21/MAPT chromosome regions (P< 10(-10)) and found evidence for an additional independent association in 4q22/SNCA. A detailed analysis of the haplotype structure at 17q21 showed that there are three separate risk groups within this region. We found weak but consistent evidence of association for common variants located in three previously published associated regions (4p15/BST1, 4p16/GAK and 1q32/PARK16). We found no support for the previously reported SNP association in 12q12/LRRK2. We also found an association of the two SNPs in 4q22/SNCA with the age of onset of the disease.

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model.

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10 -10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

A functional calcium-binding site in the metalloprotease domain of ADAMTS13.

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca(2+) ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca(2+) concentrations, a high-affinity functional ADAMTS13 Ca(2+)-binding site was suggested with K(D(app)) of 80 muM (+/- 15 muM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca(2+)-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca(2+) dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca(2+)-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K(D(app)) for Ca(2+) of ADAMTS13, and for D187A and E212A also reduced the V(max) to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca(2+) revealed an approximately 13-fold reduction in specificity constant, k(cat)/K(m), contributed by changes in both K(m) and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca(2+) upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

Mechanistic Understanding of High Flow Nasal Cannula Therapy and Pressure Support with an In Vitro Infant Model.

Despite the increased use of high flow nasal cannula therapy, little has been done to predict airway pressures for a full breath cycle. A 3-month-old infant in vitro model was developed, which included the entire upper airway and the first three bifurcations of the lungs. A breathing simulator was used to create a realistic breath pattern, and high flow was provided using a Vapotherm unit. Four cannulas of varying sizes were used to assess the effects of the inner diameter and nasal occlusion of the cannulas on airway pressures. At 8 L min-1, end expiratory pressures of 0.821-1.306 cm H2O and 0.828-1.133 cm H2O were produced in the nasopharynx and trachea, respectively. Correlations were developed to predict full breath cycle airway pressures, based on the gas flow rate delivered, cannula dimensions, as well as the breathing flow rate, for the nasopharynx and trachea. Pearson correlation coefficients for the nasopharynx and trachea correlations were 0.991 and 0.992, respectively. The developed correlations could be used to determine the flow rate necessary for a cannula to produce pressures similar to CPAP settings. The proposed correlations accurately predict the regional airway pressure up to and including 7 cm H2O of support for the entire breath cycle.

Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD.

BACKGROUND: It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP) pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8. RESULTS: Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively). Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia) and the Surui (America). Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. CONCLUSION: The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in genetic epidemiology.

SNP analysis to results (SNPator): a web-based environment oriented to statistical genomics analyses upon SNP data.

UNLABELLED: Single nucleotide polymorphisms (SNPs) are the most widely used marker in studies to assess associations between genetic variants and complex traits or diseases. They are also becoming increasingly important in the study of the evolution and history of humans and other species. The analysis and processing of SNPs obtained thanks to high-throughput technologies imply the time consuming and costly use of different, complex and usually format-incompatible software. SNPator is a user-friendly web-based SNP data analysis suite that integrates, among many other algorithms, the most common steps of a SNP association study. It frees the user from the need to have large computer facilities and an in depth knowledge of genetic software installation and management. Genotype data is directly read from the output files of the usual genotyping platforms. Phenotypic data on the samples can also be easily uploaded. Many different quality control and analysis procedures can be performed either by using built-in SNPator algorithms or by calling standard genetic software. AVAILABILITY: Access is granted from the SNPator webpage http://www.snpator.org.

Novel Pyrazole-Containing Compounds Active against Mycobacterium tuberculosis.

In this study, a series of 49 five-membered heterocyclic compounds containing either a pyridine- or a pyrrole-type nitrogen were synthesized and tested against Mycobacterium tuberculosis. Among them, only the 1,3,5-trisubstituted pyrazoles 5-49 exhibited minimum inhibitory concentration values in the low micromolar range, and some also exhibited an improved physicochemical profile without cytotoxic effects. Three pyrazoles were subjected to an animal tuberculosis efficacy model, and compound 6 induced a statistically significant difference in lung bacterial counts compared with untreated mice. Moreover, to determine the target of this series, resistors were generated, and whole genome sequencing revealed mutations in the mmpL3 gene.

Worldwide genetic variation in dopamine and serotonin pathway genes: implications for association studies.

The dopamine and serotonin systems are two of the most important neurotransmitter pathways in the human nervous system and their roles in controlling behavior and mental status are well accepted. Genes from both systems have been widely implicated in psychiatric and behavioral disorders, with numerous reports of associations and almost equally as numerous reports of the failure to replicate a previous finding of association. We investigate a set of 21 dopamine and serotonin genes commonly tested for association with psychiatric disease in a set of 39 worldwide populations representing global genetic diversity to see whether the failure to replicate findings of association may be explained by population based differences in allele frequencies and linkage disequilibrium (LD) in this gene set. We present results demonstrating a surprising homogeneity of the allele frequencies across worldwide populations in these genes. LD both for populations within continent groupings and across continental regions also showed a remarkable similarity. These findings taken together suggest that ethnic differences in these parameters are not major generators of artifacts in genetic association studies of psychiatric disorders with genes from this set. Therefore, factors other than ethnic differences in genetic variation may explain the discrepancies reported among genetic association studies with this set of genes to date. The transferability of tagSNPs defined in the HapMap populations to other worldwide populations was also investigated and found to be high. A list of tagSNPs per gene and continental region is proposed providing a guide for future association studies with these genes.

Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation.

Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of Mycobacterium tuberculosis in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.

An Examination of the Sociodemographic and Health Determinants of Major Depressive Disorder Among Black Women.

OBJECTIVES: Black women disproportionately share the distribution of risk factors for physical and mental illnesses. The goal of this study was to examine the sociodemographic and health correlates of major depressive disorder (MDD) symptoms among black women. METHODS: Pooled data from the 2005-2010 National Health and Nutrition Examination Survey (NHANES) were used to assess the sociodemographic and health correlates of MDD symptoms among black women (n = 227). Multivariate logistic regression techniques assessed the association between MDD symptoms and age, socioeconomic status, health status, and health behaviors. RESULTS: Poverty income ratio and smoking status were significantly associated with the likelihood of having MDD symptoms. Black women who were smokers were also more likely to have MDD symptoms compared to non-smokers [OR = 8.05, 95% CI = (4.56, 14.23)]. After controlling for all other socioeconomic and health variables, this association remained statistically significant. In addition, after controlling for all other variables, the multivariate analyses showed that black women below 299% federal poverty level (FPL) were nearly three times more likely to have MDD symptoms compared to women above 300% FPL [OR = 2.82, 95% CI = (1.02, 7.96)]. CONCLUSIONS: These analyses suggest that poverty and smoking status are associated with MDD symptoms among black women. A deeper understanding of the underlying mechanisms and key factors which influence MDD symptoms are needed in order to develop and create mental health programs targeting women of color.

High-throughput sequencing of microbial eukaryotes in Lake Baikal reveals ecologically differentiated communities and novel evolutionary radiations.

We performed high-throughput 18S rDNA V9 region sequencing analyses of microeukaryote (protist) communities at seven sites with depths ranging from 0 to 1450 m in the southern part of Lake Baikal. We show that microeukaryotic diversity differed according to water column depth and sediment depth. Chrysophytes and perkinsids were diverse in subsurface samples, novel radiations of petalomonads and Ichthyobodo relatives were found in benthic samples, and a broad range of divergent OTUs were detected in deep subbenthic samples. Members of clades usually associated with marine habitats were also detected, including syndineans for the first time in freshwater systems. Fungal- and cercozoan-specific c. 1200 bp amplicon clone libraries also revealed many novel lineages in both planktonic and sediment samples at all depths, a novel radiation of aphelids in shallower benthic samples, and partitioning of sarcomonad lineages in shallow vs deep benthic samples. Putative parasitic lineages accounted for 12.4% of overall reads, including a novel radiation of Ichthyobodo (fish parasite) relatives. Micrometazoans were also analysed, including crustaceans, rotifers and nematodes. The deepest (>1000 m) subsurface sediment samples harboured some highly divergent sequence types, including heterotrophic flagellates, parasites, putative metazoans and sequences likely representing organisms originating from higher up in the water column.

Progresses and challenges of utilizing traditional birth attendants in maternal and child health in Nigeria.

BACKGROUND AND OBJECTIVES: Despite advances in modern healthcare, Traditional Birth Attendants (TBA) have continued to be heavily utilized in rural communities in Nigeria. Major disparities in maternal health care in Nigeria remain present despite the goal of the United Nations Millennium Development Goal to reduce maternal mortality by 2015. The objective of this study is to review the contribution of TBAs in the birthing process in Nigeria, and to examine barriers and opportunities for utilizing TBAs in improving maternal and child health outcomes in Nigeria. METHODS: A literature review of two major electronic databases was conducted using the PRISMA framework to identify English language studies conducted between 2006 and 2016. Inclusion criteria included articles that examined the role of traditional birth attendants as a factor influencing maternal health in Nigeria. RESULTS: The value of TBAs has not been fully examined as few studies have aimed to examine its potential role in reducing maternal mortality with proper training. Eight manuscripts that were examined highlighted the role of TBAs in maternal health including outcomes of utilizing trained versus non-trained TBAs. CONCLUSION AND GLOBAL HEALTH IMPLICATIONS: Specific areas of training for TBAs that were identified and recommended in review including: recognizing delivery complications, community support for TBA practices through policy, evaluation of TBA training programs and increasing collaboration between healthcare facilities and TBAs. Policies focused on improving access to health services and importantly, formal health education training to TBAs, are required to improve maternal health outcomes and underserved communities.

Protistan community analysis: key findings of a large-scale molecular sampling.

Protists are perhaps the most lineage-rich of microbial lifeforms, but remain largely unknown. High-throughput sequencing technologies provide opportunities to screen whole habitats in depth and enable detailed comparisons of different habitats to measure, compare and map protistan diversity. Such comparisons are often limited by low sample numbers within single studies and a lack of standardisation between studies. Here, we analysed 232 samples from 10 sampling campaigns using a standardised PCR protocol and bioinformatics pipeline. We show that protistan community patterns are highly consistent within habitat types and geographic regions, provided that sample processing is standardised. Community profiles are only weakly affected by fluctuations of the abundances of the most abundant taxa and, therefore, provide a sound basis for habitat comparison beyond random short-term fluctuations in the community composition. Further, we provide evidence that distribution patterns are not solely resulting from random processes. Distinct habitat types and distinct taxonomic groups are dominated by taxa with distinct distribution patterns that reflect their ecology with respect to dispersal and habitat colonisation. However, there is no systematic shift of the distribution pattern with taxon abundance.

Association of NOD2 with Crohn's disease in a homogenous Irish population.

Linkage of IBD to the pericentromeric region of chromosome 16 has been widely confirmed by analyses of multiple populations. The NOD2 gene is located in the peak region of linkage on chromosome 16 and thought to be involved in the activation of nuclear factor (NF) kappaB in response to bacterial components. Mutations in the NOD2 gene are found to be strongly associated with susceptibility to Crohn's disease (CD). A total of 65 Irish CD families were genotyped to determine if NOD2 mutations conferred susceptibility to CD and the prevalence of these mutations in sporadic and familial forms of the disease. The Irish population is relatively homogenous and thus may provide advantages in genetic studies of complex diseases. We confirmed the IBD1 locus as a susceptibility locus for IBD within the Irish population by linkage analysis followed by linkage disequilibrium studies. No significant evidence of linkage was observed to the previously identified regions on chromosomes 1, 12 and 14. In all, 131 CD affected families were then genotyped for seven of the previously published NOD2 single-nucleotide polymorphisms (SNPs). Allelic transmission distortion was investigated using the pedigree disequilibrium test (PDT). SNP13 (3020insC) was found to be associated with CD (P=0.0186). Patients who possessed a rare allele of SNP8, 12 or 13 presented earlier when compared to patients without rare variants (mean age, 20.1 vs 24 years, P=0.011) and the rare allele of SNP13 was observed to be predominantly linked to ileal disease (P=0.02). This report confirms the importance of NOD2 as a susceptibility gene for CD within the Irish population.

Organoleptic qualities and acceptability of fortified rice in two Southeast Asian countries.

Fortified rice has the potential to improve the micronutrients status of vulnerable populations. However, fortified rice has to have acceptable organoleptic--the sensory properties of a particular food--qualities. Few data exist on the acceptability of fortified rice in Asia. To assess the acceptability of two types of fortified rice (cold and hot extruded) in Vietnam and Cambodia, triangle tests were conducted in Vietnam (53 women) and Cambodia (258 adults), testing fortified rice against conventional rice, with participants being asked to score the organoleptic qualities. In addition, Cambodian schoolchildren (n = 1700) were given conventional rice and two types of fortified rice for two week periods as part of a World Food Program school meal program, with intake monitored. Fortified rice differed significantly in organoleptic qualities from conventional rice, with most subjects correctly identifying fortified rice (P < 0.001). However, fortified rice was found to be highly acceptable in both countries. In Cambodia, schoolchildren consuming fortified rice had higher intakes than when consuming conventional rice (176 g/child/day and 168 g/child/day, respectively; P < 0.05). This study shows that fortified rice is acceptable in two countries in Southeast Asia. However, specific information is needed to explain the organoleptic qualities of fortified rice as perceived by end-users.