Lrp Acts as Both a Positive and Negative Regulator for Type 1 Fimbriae Production in Salmonella enterica Serovar Typhimurium

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment

Effects of Mountain Pine Beetle on Fuels and Expected Fire Behavior in Lodgepole Pine Forests, Colorado, USA

In Colorado and southern Wyoming, mountain pine beetle (MPB) has affected over 1.6 million ha of predominantly lodgepole pine forests, raising concerns about effects of MPB-caused mortality on subsequent wildfire risk and behavior. Using empirical data we modeled potential fire behavior across a gradient of wind speeds and moisture scenarios in Green stands compared three stages since MPB attack (Red [1–3 yrs], Grey [4–10 yrs], and Old-MPB [∼30 yrs]). MPB killed 50% of the trees and 70% of the basal area in Red and Grey stages. Across moisture scenarios, canopy fuel moisture was one-third lower in Red and Grey stages compared to the Green stage, making active crown fire possible at lower wind speeds and less extreme moisture conditions. More-open canopies and high loads of large surface fuels due to treefall in Grey and Old-MPB stages significantly increased surface fireline intensities, facilitating active crown fire at lower wind speeds (>30–55 km/hr) across all moisture scenarios. Not accounting for low foliar moistures in Red and Grey stages, and large surface fuels in Grey and Old-MPB stages, underestimates the occurrence of active crown fire. Under extreme burning conditions, minimum wind speeds for active crown fire were 25–35 km/hr lower for Red, Grey and Old-MPB stands compared to Green. However, if transition to crown fire occurs (outside the stand, or within the stand via ladder fuels or wind gusts >65 km/hr), active crown fire would be sustained at similar wind speeds, suggesting observed fire behavior may not be qualitatively different among MPB stages under extreme burning conditions. Overall, the risk (probability) of active crown fire appears elevated in MPB-affected stands, but the predominant fire hazard (crown fire) is similar across MPB stages and is characteristic of lodgepole pine forests where extremely dry, gusty weather conditions are key factors in determining fire behavior

Protein Translation and Cell Death: The Role of Rare tRNAs in Biofilm Formation and in Activating Dormant Phage Killer Genes

We discovered previously that the small Escherichia coli proteins Hha (hemolysin expression modulating protein) and the adjacent, poorly-characterized YbaJ are important for biofilm formation; however, their roles have been nebulous. Biofilms are intricate communities in which cell signaling often converts single cells into primitive tissues. Here we show that Hha decreases biofilm formation dramatically by repressing the transcription of rare codon tRNAs which serves to inhibit fimbriae production and by repressing to some extent transcription of fimbrial genes fimA and ihfA. In vivo binding studies show Hha binds to the rare codon tRNAs argU, ileX, ileY, and proL and to two prophage clusters D1P12 and CP4-57. Real-time PCR corroborated that Hha represses argU and proL, and Hha type I fimbriae repression is abolished by the addition of extra copies of argU, ileY, and proL. The repression of transcription of rare codon tRNAs by Hha also leads to cell lysis and biofilm dispersal due to activation of prophage lytic genes rzpD, yfjZ, appY, and alpA and due to induction of ClpP/ClpX proteases which activate toxins by degrading antitoxins. YbaJ serves to mediate the toxicity of Hha. Hence, we have identified that a single protein (Hha) can control biofilm formation by limiting fimbriae production as well as by controlling cell death. The mechanism used by Hha is the control of translation via the availability of rare codon tRNAs which reduces fimbriae production and activates prophage lytic genes. Therefore, Hha acts as a toxin in conjunction with co-transcribed YbaJ (TomB) that attenuates Hha toxicity

Continuum of care in maternal, newborn and child health in Pakistan: analysis of trends and determinants from 2006 to 2012

Iqbal S, Maqsood S, Zakar R, Zakria Zakar M, Fischer F. Continuum of care in maternal, newborn and child health in Pakistan: analysis of trends and determinants from 2006 to 2012. BMC Health Services Research. 2017;17(1): 189.Background Pakistan, being a developing country, presents the dismal picture of maternal and neonatal mortality and morbidity. The majority of maternal and neonatal deaths could be avoided if Continuum of Care (CoC) is provided in a structured pathway from pregnancy to birth and to the first week of life of the newborn child. This study aimed to analyse the trends of CoC at all three levels (antenatal care, skilled delivery and postpartum care) and to identify various factors affecting the continuation in receiving CoC in Pakistan during 2006 to 2012. Methods Secondary data analysis was performed on nationally representative data from the last two iterations of Pakistan Demographic and Health Survey (PDHS), conducted during 2006/07 to 2012/13. The analysis is limited to women of the reproductive age group (15–49 years) who gave birth during the last five years preceding both surveys. This leads to a sample size of 5,724 and 7,461 respondents from PDHS 2006/07 and 2012/13 respectively. The association between CoC and several factors, including individual attributes (reproductive status), family influences, community context, as well as cultural and social values was assessed in bivariate analyses in a first step. Furthermore, odds ratios and adjusted odds ratios with 95% confidence intervals using a binary and multivariable logistic regression were calculated. Results Our research presents the trends of a composite measure of CoC including antenatal care, delivery assistance and postpartum care. The largest gap in CoC was observed at antenatal care followed by delivery and postnatal care within 48 h after delivery. Results show that CoC completion rate has increased from 15% to 27% amongst women in Pakistan over time from 2006 to 2012. Women with high age at first birth, having less number of children, with higher education, belonging to richest quintile, living in Sindh province and urban areas, having high autonomy and exposure to mass media were most likely to avail complete CoC. Conclusions The findings show that women in Pakistan still lack the CoC. This calls for attention to develop and implement tailored interventions, focusing on the needs of women in Pakistan to provide CoC in an integrated manner, involving both public and private sectors by appropriately addressing the factors hindering CoC completion rates

Neonatal Mortality Levels for 193 Countries in 2009 with Trends since 1990: A Systematic Analysis of Progress, Projections, and Priorities

Mikkel Oestergaard and colleagues develop annual estimates for neonatal mortality rates and neonatal deaths for 193 countries for 1990 to 2009, and forecasts into the future

Population structure and genetic diversity of native and invasive populations of Solanum rostratum (Solanaceae)

Aims: We investigate native and introduced populations of Solanum rostratum, an annual, self-compatible plant that has been introduced around the globe. This study is the first to compare the genetic diversity of Solanum rostratum between native and introduced populations. We aim to (1) determine the level of genetic diversity across the studied regions; (2) explore the likely origins of invasive populations in China; and (3) investigate whether there is the evidence of multiple introductions into China. Methods: We genotyped 329 individuals at 10 microsatellite loci to determine the levels of genetic diversity and to investigate population structure of native and introduced populations of S. rostratum. We studied five populations in each of three regions across two continents: Mexico, the U.S.A. and China. Important Findings: We found the highest genetic diversity among Mexican populations of S. rostratum. Genetic diversity was significantly lower in Chinese and U.S.A. populations, but we found no regional difference in inbreeding coefficients (FIS) or population differentiation (FST). Population structure analyses indicate that Chinese and U.S.A. populations are more closely related to each other than to sampled Mexican populations, revealing that introduced populations in China share an origin with the sampled U.S.A. populations. The distinctiveness between some introduced populations indicates multiple introductions of S. rostratum into China

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

The cholera toxin B pentamer (CtxB5), which belongs to the AB5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB5 and the related heat labile enterotoxin LTB5 have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts

Gene Expression and Biological Pathways in Tissue of Men with Prostate Cancer in a Randomized Clinical Trial of Lycopene and Fish Oil Supplementation

Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial.Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients.Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001).We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients.ClinicalTrials.gov NCT00402285