Rice ORMDL Controls Sphingolipid Homeostasis Affecting Fertility Resulting from Abnormal Pollen Development

The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.     

Prediction of Percentage Body Fat in Rural Thai Population Using Simple Anthropometric Measurements

Objective: To develop and validate sex‐specific equations for predicting percentage body fat (%BF) in rural Thai population, based on BMI and anthropometric measurements. Research Methods and Procedures: %BF (DXA; GE Lunar Corp., Madison, WI) was measured in 181 men and 255 women who were healthy and between 20 and 84 years old. Anthropometric measures such as weight (kilograms), height (centimeters), BMI (kilograms per meter squared), waist circumference (centimeters), hip circumference (centimeters), thickness at triceps skinfold (millimeters), biceps skinfold (millimeters), subscapular skinfold (millimeters), and suprailiac skinfold (millimeters) were also measured. The sample was randomly divided into a development group (98 men and 125 women) and a validation group (83 men and 130 women). Regression equations of %BF derived from the development group were then evaluated for accuracy in the validation group. Results: The equation for estimating %BF in men was: %BF(men) = 0.42 × subscapular skinfold + 0.62 × BMI ? 0.28 × biceps skinfold + 0.17 × waist circumference ? 18.47, and in women: %BF(women) = 0.42 × hip circumference + 0.17 × suprailiac skinfold + 0.46 × BMI ? 23.75. The coefficient of determination (R²) for both equations was 0.68. Without anthropometric variables, the predictive equation using BMI, age, and sex was: %BF = 1.65 × BMI + 0.06 × age ? 15.3 × sex ? 10.67 (where sex = 1 for men and sex = 0 for women), with R² = 0.83. When these equations were applied to the validation sample, the difference between measured and predicted %BF ranged between ±9%, and the positive predictive values were above 0.9. Discussion: These results suggest that simple, noninvasive, and inexpensive anthropometric variables may provide an accurate estimate of %BF and could potentially aid the diagnosis of obesity in rural Thais.     

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.     

Time to Update and Quantitative Changes in the Results of Cochrane Pregnancy and Childbirth Reviews

Background

The recommended interval between updates for systematic reviews included in The Cochrane Library is 2 years. However, it is unclear whether this interval is always appropriate. Whereas excessive updating wastes time and resources, insufficient updating allows out-of-date or incomplete evidence to guide clinical decision-making. We set out to determine, for Cochrane pregnancy and childbirth reviews, the frequency of updates, factors associated with updating, and whether updating frequency was appropriate.

Methodology/Principal Findings

Cochrane pregnancy and childbirth reviews published in Issue 3, 2007 of the Cochrane Database of Systematic Reviews were retrieved, and data were collected from their original and updated versions. Quantitative changes were determined for one of the primary outcomes (mortality, or the outcome of greatest clinical significance). Potential factors associated with time to update were assessed using the Cox proportional hazard model. Among the 101 reviews in our final sample, the median time before the first update was 3.3 years (95% CI 2.7–3.8). Only 32.7% had been updated within the recommended interval of 2 years. In 75.3% (76/101), a median of 3 new trials with a median of 576 additional participants were included in the updated versions. There were quantitative changes in 71% of the reviews that included new trials (54/76): the median change in effect size was 18.2%, and the median change in 95% CI width was 30.8%. Statistical significance changed in 18.5% (10/54) of these reviews, but conclusions were revised in only 3.7% (2/54). A shorter time to update was associated with the same original review team at updating.

Conclusions/Significance

Most reviews were updated less frequently than recommended by Cochrane policy, but few updates had revised conclusions. Prescribed time to update should be reconsidered to support improved decision-making while making efficient use of limited resources.     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

Current Status of Taeniasis in Thailand

Taeniasis is prevalent in all regions of Thailand, except the South. Infections were more frequently found in males than females of any age from 7-83 years. Taenia saginata is the most common species throughout the country. Taenia asiatica was reported only in the province of Kanchanaburi in the Central region. Co-infections, with Taenia solium and T. asiatica or T. solium and T. saginata, were found. Hybridization between T. asiatica and T. saginata is evidence that co-infection is never found between these 2 species. Finding more than 1 worm in a single patient was not entirely rare. Genetic variation was found without correlation to its geographic distribution in T. saginata, whereas no variation was found in T. asiatica.     

Prospective Study of Avian Influenza Virus Infections among Rural Thai Villagers

Background

In 2008, 800 rural Thai adults living within Kamphaeng Phet Province were enrolled in a prospective cohort study of zoonotic influenza transmission. Serological analyses of enrollment sera suggested this cohort had experienced subclinical avian influenza virus (AIV) infections with H9N2 and H5N1 viruses.

Methods

After enrollment, participants were contacted weekly for 24mos for acute influenza-like illnesses (ILI). Cohort members confirmed to have influenza A infections were enrolled with their household contacts in a family transmission study involving paired sera and respiratory swab collections. Cohort members also provided sera at 12 and 24 months after enrollment. Serologic and real-time RT-PCR assays were performed against avian, swine, and human influenza viruses.

Results

Over the 2 yrs of follow-up, 81 ILI investigations in the cohort were conducted; 31 (38%) were identified as influenza A infections by qRT-PCR. Eighty-three household contacts were enrolled; 12 (14%) reported ILIs, and 11 (92%) of those were identified as influenza infections. A number of subjects were found to have slightly elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2) virus: 21 subjects (2.7%) at 12-months and 40 subjects (5.1%) at 24-months. Among these, two largely asymptomatic acute infections with H9N2 virus were detected by >4-fold increases in annual serologic titers (final titers 1∶80). While controlling for age and influenza vaccine receipt, moderate poultry exposure was significantly associated with elevated H9N2 titers (adjusted OR = 2.3; 95% CI, 1.04–5.2) at the 24-month encounter. One subject had an elevated titer (1∶20) against H5N1 during follow-up.

Conclusions

From 2008–10, evidence for AIV infections was sparse among this rural population. Subclinical H9N2 AIV infections likely occurred, but serological results were confounded by antibody cross-reactions. There is a critical need for improved serological diagnostics to more accurately detect subclinical AIV infections in humans.     

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.     

2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds.

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.