Genetic Diversity and Distribution of the Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in Coastal Areas of Japan

Background

The marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.

Methods/Principal Finding

A total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.

Conclusions/Significance

This study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.     

Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.     

New Insights of Microsporidial Infection among Asymptomatic Aboriginal Population in Malaysia

Background

Studies on microsporidial infection mostly focus on immunodeficiency or immunosuppressive individuals. Therefore, this cross-sectional study describes the prevalence and risk factors of microsporidiosis among asymptomatic individuals in Malaysia.

Methods/Findings

Four hundred and forty seven stool samples were collected and examined for microsporidia after staining with Gram-chromotrope Kinyoun. Demographic, socioeconomic, environmental, and behavioral information were collected by using a pre-tested questionnaire. Overall, 67 (15%) samples were positive for microsporidia. The prevalence of infection was significantly higher among individuals aged more than 15 years compared to those aged <15 years (OR = 1.97, 95% CI = 1.08, 3.62; P = 0.028). Furthermore, logistic regression analysis confirmed that the presence of other family members infected with microsporidia (OR = 8.45; 95% CI = 4.30, 16.62; P<0.001) and being a consumer of raw vegetables (OR = 2.05; 95% CI = 1.15, 3.66; P = 0.016) were the significant risk factors of this infection.

Conclusions

These findings clearly show that exposure to microsporidia is common among Aboriginal population. Further studies using molecular approach on microsporidia isolates from asymptomatic individuals is needed to determine species-specific. The risk factors associated with microsporidiosis will help in identifying more clearly the sources of the infection in the environment that pose a risk for transmission so that preventive strategies can be implemented.     

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.     

Urinary and Dietary Analysis of 18,470 Bangladeshis Reveal a Correlation of Rice Consumption with Arsenic Exposure and Toxicity

Background

We utilized data from the Health Effects of Arsenic Longitudinal Study (HEALS) in Araihazar, Bangladesh, to evaluate the association of steamed rice consumption with urinary total arsenic concentration and arsenical skin lesions in the overall study cohort (N=18,470) and in a subset with available urinary arsenic metabolite data (N=4,517).

Methods

General linear models with standardized beta coefficients were used to estimate associations between steamed rice consumption and urinary total arsenic concentration and urinary arsenic metabolites. Logistic regression models were used to estimate prevalence odds ratios (ORs) and their 95% confidence intervals (CIs) for the associations between rice intake and prevalent skin lesions at baseline. Discrete time hazard models were used to estimate discrete time (HRs) ratios and their 95% CIs for the associations between rice intake and incident skin lesions.

Results

Steamed rice consumption was positively associated with creatinine-adjusted urinary total arsenic (β=0.041, 95% CI: 0.032-0.051) and urinary total arsenic with statistical adjustment for creatinine in the model (β=0.043, 95% CI: 0.032-0.053). Additionally, we observed a significant trend in skin lesion prevalence (P-trend=0.007) and a moderate trend in skin lesion incidence (P-trend=0.07) associated with increased intake of steamed rice.

Conclusions

This study suggests that rice intake may be a source of arsenic exposure beyond drinking water.     

ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.)

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

     

Treatment of nitric oxide supplemented with nitrogen and sulfur regulates photosynthetic performance and stomatal behavior in mustard under salt stress

Nitric oxide (NO) is a hormone that connects numerous reactions in plant cells under normal and environmental stress conditions. The application of 100 µM NO as sodium nitroprusside (SNP; NO donor) applied individually or in combination with N or S in different combinations (i.e. 100 mg N or S kg⁻¹ soil applied at the time of sowing [100 N + 100S]_0d or with split, 50 mg N or S kg⁻¹ soil at the time of sowing and similar dose at 20 d after sowing [50 N + 50S]_0d + [50 N + 50S]_20d) was tested to alleviate salt stress in mustard (Brassica juncea L.). Application of 100 µM NO plus split application of N and S more significantly promoted stomatal behavior, photosynthetic and growth performance in the absence of salt stress and maximally alleviated effects of salt stress through increased N- and S-use efficiency, proline and antioxidant system. The combined application of N and S at the time of sowing was lesser effective in promoting photosynthesis and growth under salt or no salt stress conditions in presence or absence of NO. The study suggests that salt stress effects on the photosynthetic performance are mitigated more efficiently when NO was applied together with the split application of N and S given at two stages, and the photosynthetic activity was promoted under salt stress through increased N and S assimilation and antioxidant system. This strategy may be adopted in agricultural system for overcoming salt stress effects on performance of mustard.     

Reduction of nuclear Y654-p-β-catenin expression through SH3GL2-meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-β-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-β-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10–32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-β-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-β-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-β-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.