A versatile transgenic allele for mouse overexpression studies

For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.

     

Identification of chloroplast genome loci suitable for high‐resolution phylogeographic studies of Colocasia esculenta (L.) Schott (Araceae) and closely related taxa

Recently, we reported the chloroplast genome‐wide association of oligonucleotide repeats, indels and nucleotide substitutions in aroid chloroplast genomes. We hypothesized that the distribution of oligonucleotide repeat sequences in a single representative genome can be used to identify mutational hotspots and loci suitable for population genetic, phylogenetic and phylogeographic studies. Using information on the location of oligonucleotide repeats in the chloroplast genome of taro (Colocasia esculenta), we designed 30 primer pairs to amplify and sequence polymorphic loci. The primers have been tested in a range of intra‐specific to intergeneric comparisons, including ten taro samples (Colocasia esculenta) from diverse geographical locations, four other Colocasia species (C. affinis, C. fallax, C. formosana, C. gigantea) and three other aroid genera (represented by Remusatia vivipara, Alocasia brisbanensis and Amorphophallus konjac). Multiple sequence alignments for the intra‐specific comparison revealed nucleotide substitutions (point mutations) at all 30 loci and microsatellite polymorphisms at 14 loci. The primer pairs reported here reveal levels of genetic variation suitable for high‐resolution phylogeographic and evolutionary studies of taro and other closely related aroids. Our results confirm that information on repeat distribution can be used to identify loci suitable for such studies, and we expect that this approach can be used in other plant groups.     

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.     

Estimating Leptospirosis Incidence Using Hospital-Based Surveillance and a Population-Based Health Care Utilization Survey in Tanzania

Background

The incidence of leptospirosis, a neglected zoonotic disease, is uncertain in Tanzania and much of sub-Saharan Africa, resulting in scarce data on which to prioritize resources for public health interventions and disease control. In this study, we estimate the incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania.

Methodology/Principal Findings

We conducted a population-based household health care utilization survey in two districts in the Kilimanjaro Region of Tanzania and identified leptospirosis cases at two hospital-based fever sentinel surveillance sites in the Kilimanjaro Region. We used multipliers derived from the health care utilization survey and case numbers from hospital-based surveillance to calculate the incidence of leptospirosis. A total of 810 households were enrolled in the health care utilization survey and multipliers were derived based on responses to questions about health care seeking in the event of febrile illness. Of patients enrolled in fever surveillance over a 1 year period and residing in the 2 districts, 42 (7.14%) of 588 met the case definition for confirmed or probable leptospirosis. After applying multipliers to account for hospital selection, test sensitivity, and study enrollment, we estimated the overall incidence of leptospirosis ranges from 75–102 cases per 100,000 persons annually.

Conclusions/Significance

We calculated a high incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania, where leptospirosis incidence was previously unknown. Multiplier methods, such as used in this study, may be a feasible method of improving availability of incidence estimates for neglected diseases, such as leptospirosis, in resource constrained settings.     

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.     

Effects of transgenic Bt cotton on insecticide use and abundance of two generalist predators

Considerable effort has been expended to determine if crops genetically engineered to produce Bacillus thuringiensis (Bt) toxins harm non‐target arthropods. However, if Bt crops kill target pests and thereby reduce insecticide use, this could benefit some non‐target arthropods. We analyzed data from 21 commercial cotton fields in Arizona to test the effects of Bt cotton on insecticide use and abundance of two non‐target arthropods, the generalist predators Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) and Orius tristicolor (White) (Heteroptera: Anthocoridae). The number of insecticide sprays was more than double for non‐Bt cotton compared with Bt cotton that produced Cry1Ac. The abundance of both predators was negatively associated with the number of insecticide sprays, although significantly so for only one of two sampling periods for each species tested. With the effects of insecticides statistically removed, field type (Bt or non‐Bt cotton) did not affect the abundance of either predator. Accordingly, without adjusting for the effects of insecticide sprays, the abundance of C. carnea was higher in Bt cotton fields than in non‐Bt cotton fields, but significantly so during only one of two sampling periods. The abundance of O. tristicolor did not differ between field types, even without adjusting for effects of insecticide sprays. The results indicate that Bt crops can affect insecticide use, which in turn can affect the relative abundance of non‐target arthropods in Bt and non‐Bt fields. Thus, environmental impact assessment should incorporate analysis of the effects of transgenic crops on management practices, as well as evaluation of the direct effects of such crops.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.