Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions

Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K _M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k _cat) for the hydrolysis of GTP than EF-G1A; 0.2 s^-1 vs. 0.04 s^-1. These values resulted in specificity constants (k _cat ^obs/K _M) for EF-G1A and EF-G1B of 0.5 x 10³ s^-1 M^-1 and 3.0 x 10³ s^-1 M^-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.     

Improved Activity of a Thermophilic Cellulase,Cel5A,from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.     

Factors Associated with Dengue Shock Syndrome: A Systematic Review and Meta-Analysis

Background

The pathogenesis of dengue shock syndrome (DSS, grade 3 and 4) is not yet completely understood. Several factors are reportedly associated with DSS, a more severe form of dengue infection that reportedly causes 50 times higher mortality compared to that of dengue patients without DSS. However, the results from these reports remain inconclusive. To better understand the epidemiology, clinical manifestation, and pathogenesis of DSS for development of new therapy, we systematically reviewed and performed a meta-analysis of relevant studies that reported factors in both DSS and dengue hemorrhagic fever (DHF, grade 1 and 2) patients.

Methods and Findings

PubMed, EMBASE, Scopus, Google Scholar, Dengue Bulletin, Cochrane Library, Virtual Health Library, and a manual search of reference lists of articles published before September 2010 were used to retrieve relevant studies. A meta-analysis using fixed- or random-effects models was used to calculate pooled odds ratios (OR) or event rate with corresponding 95% confidence intervals. Assessment of heterogeneity and publication bias, meta-regression analysis, subgroup analysis, sensitivity analysis, and analysis of factor-specific relationships were further performed. There were 198 studies constituting 203 data sets that met our eligibility criteria. Our meta-regression analysis showed a sustained reduction of DSS/dengue hemorrhagic fever (DHF) ratio over a period of 40 years in Southeast Asia, especially in Thailand. The meta-analysis revealed that age, female sex, neurological signs, nausea/vomiting, abdominal pain, gastrointestinal bleeding, hemoconcentration, ascites, pleural effusion, hypoalbuminemia, hypoproteinemia, hepatomegaly, levels of alanine transaminase and aspartate transaminase, thrombocytopenia, prothrombin time, activated partial thromboplastin time, fibrinogen level, primary/secondary infection, and dengue virus serotype-2 were significantly associated with DSS when pooling all original relevant studies.

Conclusions

The results improve our knowledge of the pathogenesis of DSS by identifying the association between the epidemiology, clinical signs, and biomarkers involved in DSS.     

Psychological and Social Factors Associated with Late Pregnancy Iron Deficiency Anaemia in Rural Viet Nam: A Population-Based Prospective Study

Objectives

The aim of this study was to examine the relationships between psychological and social factors and late pregnancy IDA among pregnant women in rural Viet Nam.

Methods

Pregnant women from 50 randomly-selected communes within Ha Nam province were recruited and assessed at 12 - 20 weeks gestation (Wave 1, W1). They were followed up in the last trimester (Wave 2, W2). IDA was defined as Haemoglobin < 11 g/dL and serum ferritin < 15 ng/mL. Symptoms of Common Mental Disorders (CMD) were assessed by the Edinburgh Postnatal Depression Scale-Vietnam (EPDS-V). Persistent antenatal CMD was defined as having an EPDS-V score ≥ 4 in both W1 and W2. Hypothesis models were tested by Structural Equation Modeling analyses.

Results

A total of 378 women provided complete data at both W1 and W2. The incidence risk of IDA in the third trimester was 13.2% (95% confidence interval (CI): 9.8-16.7). Persistent CMD was found in 16.9% (95% CI: 13.1-20.7) pregnant women and predicted by intimate partner violence, fear of other family members, experience of childhood abuse, coincidental life adversity, and having a preference for the sex of the baby. There was a significant pathway from persistent CMD to IDA in late pregnancy via the length of time that iron supplements had been taken. Receiving advice to take iron supplements and higher household wealth index were indirectly related to lower risk of late pregnancy IDA. Early pregnancy IDA and being multi-parous also contributed to late pregnancy IDA.

Conclusions

Antenatal IDA and CMD are prevalent public health problems among women in Viet Nam. The link between them suggests that while direct recommendations to use iron supplements are important, the social factors associated with common mental disorders should be addressed in antenatal care in order to improve the health of pregnant women and their infants.     

FoxP3+ Regulatory T Cells Attenuate Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3⁺ regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.     

Elimination of Iron Deficiency Anemia and Soil Transmitted Helminth Infection: Evidence from a Fifty-four Month Iron-Folic Acid and De-worming Program

Background

Intermittent iron-folic acid supplementation and regular de-worming are effective initiatives to reduce anemia, iron deficiency, iron deficiency anemia, and soil transmitted helminth infections in women of reproductive age. However, few studies have assessed the long-term effectiveness of population-based interventions delivered in resource-constrained settings.

Methodology/Principal Findings

The objectives were to evaluate the impact of weekly iron-folic acid supplementation and de-worming on mean hemoglobin and the prevalence of anaemia, iron deficiency, and soil transmitted helminth infection in a rural population of women in northern Vietnam and to identify predictive factors for hematological outcomes. A prospective cohort design was used to evaluate a population-based supplementation and deworming program over 54 months. The 389 participants were enrolled just prior to commencement of the intervention. After 54 months 76% (95% CI [68%, 84%]) were taking the iron-folic acid supplement and 95% (95% CI [93%, 98%]) had taken the most recently distributed deworming treatment. Mean hemoglobin rose from 122 g/L (95% CI [120, 124]) to 131 g/L (95% CI [128, 134]) and anemia prevalence fell from 38% (95% CI [31%, 45%]) to 18% (95% CI [12%, 23%]); however, results differed significantly between ethnic groups. Iron deficiency fell from 23% (95% CI [17%, 29%]) to 8% (95% CI [4%, 12%]), while the prevalence of iron deficiency anemia was reduced to 4% (95% CI [1%, 7%]). The prevalence of hookworm infection was reduced from 76% (95% CI [68%, 83%]) to 11% (95% CI [5%, 18%]). The level of moderate or heavy infestation of any soil-transmitted helminth was reduced to less than 1%.

Conclusions/Significance

Population-based interventions can efficiently and effectively reduce anemia and practically eliminate iron deficiency anemia and moderate to heavy soil transmitted helminth infections, maintaining them below the level of public health concern.     

Cholera in Pregnancy: Outcomes from a Specialized Cholera Treatment Unit for Pregnant Women in Léogane,Haiti

Background

The association between cholera in pregnancy and negative fetal outcome has been described since the 19^th century. However, there is limited published literature on the subject. We describe pregnancy outcomes from a specialized multidisciplinary hospital unit at the onset of a large cholera outbreak in Haiti in 2010 and 2011.

Methods

Pregnant women with cholera were hospitalized in a specialized unit within the MSF hospital compound in Léogâne and treated using standard cholera treatment guidelines but with earlier, more intense fluid replacement. All women had intravenous access established at admission regardless of their hydration status, and all received antibiotic treatment. Data were collected on patient demographics, pregnancy and cholera status, and pregnancy outcome. In this analysis we calculated risk ratios for fetal death and performed logistic regression analysis to control for confounding factors.

Results

263 pregnant women with cholera were hospitalized between December 2010 and July 2011. None died during hospitalization, 226 (86%) were discharged with a preserved pregnancy and 16 (6%) had live fullterm singleton births, of whom 2 died within the first 5 days postpartum. The remaining 21 pregnancies (8%) resulted in intrauterine fetal death. The risk of fetal death was associated with factors reflecting severity of the cholera episode: after adjusting for confounding factors, the strongest risk factor for fetal death was severe maternal dehydration (adjusted risk ratio for severe vs. mild dehydration was 9.4, 95% CI 2.5–35.3, p = 0.005), followed by severe vomiting (adjusted risk ratio 5.1, 95% 1.1–23.8, p = 0.041).

Conclusion

This is the largest cohort of pregnant women with cholera described to date. The main risk factor identified for fetal death was severity of dehydration. Our experience suggests that establishing specialized multidisciplinary units which facilitate close follow-up of both pregnancy and dehydration status due to cholera could be beneficial for patients, especially in large epidemics.     

Effects of Landscape Fragmentation and Climate on Lyme Disease Incidence in the Northeastern United States

Lyme disease is the most frequently reported vector borne illness in the United States, and incidences are increasing steadily year after year. This study explores the influence of landscape (e.g., land use pattern and landscape fragmentation) and climatic factors (e.g., temperature and precipitation) at a regional scale on Lyme disease incidence. The study area includes thirteen states in the Northeastern United States. Lyme disease incidence at county level for the period of 2002–2006 was linked with several key landscape and climatic variables in a negative binomial regression model. Results show that Lyme disease incidence has a relatively clear connection with regional landscape fragmentation and temperature. For example, more fragmentation between forests and residential areas results in higher local Lyme disease incidence. This study also indicates that, for the same landscape, some landscape variables derived at a particular scale show a clearer connection to Lyme disease than do others. In general, the study sheds more light on connections between Lyme disease incidence and climate and landscape patterns at the regional scale. Integrating findings of this regional study with studies at a local scale will further refine understanding of the pattern of Lyme disease as well as increase our ability to predict, prevent, and respond to disease.