Use of a Bacteroides thetaiotaomicron-specific α-1-6, mannanase quantitative PCR to detect human faecal pollution in water

Aims: The aims of this work were to develop a quantitative test, based on Bacteroides thetaiotaomicron, for human faecal pollution in water and to evaluate test performance. Methods and Results: qPCR primers, based on the complete genomic sequence of B. thetaiotaomicron VPI 5482, were designed and tested. The single-copy putative mannanase homologue, α-1-6 mannanase, was selected as the particular target and sequences within this gene chosen as the qPCR primers by Blast search for specificity to B. thetaiotaomicron. The average concentration of B. thetaiotaomicron in human faeces was 1·39 × 10⁸ cells per gram faeces and the detection limit was 9·3 B. thetaiotaomicron copies per qPCR procedure. Comparison of B. thetaiotaomicron content in sewage vs pooled nonhuman faecal samples indicated that the current assay is specific for sewage. Conclusion: The subject assay is potentially useful for quantification of sewage pollution in water. Significance and Impact of the Study: Bacteroides-associated markers, proposed for faecal source tracking, have exclusively been based on gene sequences related to generally classified and uncultured bacteria. However, genes associated with host-microbe interaction have been suggested as more specific markers. The present assay targets such a gene of B. thetaiotaomicron which is considered to be a symbiont in the human gut.     

Telomere length analysis of human mesenchymal stem cells by quantitative PCR

Human mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells are required, which necessitates extensive sub-culturing and expansion of hMSCs. Throughout ex vivo expansion, the cells undergo telomere shortening, and critically short telomeres can trigger loss of cell viability. Telomeres are nucleoprotein structures that cap the ends of chromosomes, and serve to protect the DNA from the degradation which occurs due to the end-replication problem in all eukaryotes. As hMSCs have only a finite ability for self-renewal like most somatic cells, assaying for telomere length in hMSCs provides critical information on the replicative capacity of the cells, an important criterion in the selection of hMSCs for therapy. Telomere length is generally quantified by Southern blotting and fluorescence in situ hybridization, and more recently by PCR-based methods. Here we describe the quantification of hMSC telomere length by real-time PCR; our results demonstrate the effect of telomere shortening on the proliferation and clonogenicity of hMSCs. Thus, this assay constitutes a useful tool for the determination of relative telomere length in hMSCs.     

Rainfall homogenizes while fruiting increases diversity of spore deposition in Mediterranean conditions

There is a lack of knowledge regarding the main factors modulating fungal spore deposition in forest ecosystems. We have described the local spatio-temporal dynamics of fungal spore deposition along a single fruiting season and its relation with fruit body emergence and rainfall events. Passive spore traps were weekly sampled during autumn and analysed by metabarcoding of the ITS2 region in combination with qPCR. There were larger compositional changes of deposited spores across sampling weeks than amongst sampling plots. Spore diversity and abundance correlated with mushroom emergence and weekly rainfall. Spore compositional changes were related to rainfall, with lower spatial compositional heterogeneity across plots during weeks with higher rainfall. Soil saprotrophs, and amongst them, puffball species, showed the strongest positive correlation with rainfall across fungal guilds. We saw high fine-scale temporal changes of deposited spores, and both mushroom emergence and rainfall may be important factors driving airborne spore deposition.     

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed,Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.     

EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE): NITROGEN‐METABOLISM GENES AND THEIR EXPRESSION IN RESPONSE TO EXTERNAL NITROGEN SOURCES1

The availability and composition of dissolved nitrogen in ocean waters are factors that influence species composition in natural phytoplankton communities. The same factors affect the ratio of organic to inorganic carbon incorporation in calcifying species, such as the coccolithophore Emiliania huxleyi (Lohman) W. W. Hay et H. Mohler. E. huxleyi has been shown to thrive on various nitrogen sources, including dissolved organic nitrogen. Nevertheless, assimilation of dissolved nitrogen under nitrogen‐replete and ‐limited conditions is not well understood in this ecologically important species. In this study, the complete amino acid sequences for three functional genes involved in nitrogen metabolism in E. huxleyi were identified: a putative formamidase, a glutamine synthetase (GSII family), and assimilatory nitrate reductase. Expression patterns of the three enzymes in cells grown on inorganic as well as organic nitrogen sources indicated reduced expression levels of nitrate reductase when cells were grown on NH₄⁺ and a reduced expression level of the putative formamidase when growth was on NO₃^?. The data reported here suggest the presence of a nitrogen preference hierarchy in E. huxleyi. In addition, the gene encoding for a phosphate repressible phosphate permease was more highly expressed in cells growing on formamide than in cells growing on inorganic nitrogen sources. This finding suggests a coupling between phosphate and nitrogen metabolism, which might give this species a competitive advantage in nutrient‐depleted environments. The potential of using expression of genes investigated here as indicators of specific nitrogen‐metabolism strategies of E. huxleyi in natural populations of phytoplankton is discussed.     

Poorly differentiated XX/XY sex chromosomes are widely shared across skink radiation

Differentiated sex chromosomes are believed to be evolutionarily stable, while poorly differentiated sex chromosomes are considered to be prone to turnovers. With around 1700 currently known species forming ca 15% of reptile species diversity, skinks (family Scincidae) are a very diverse group of squamates known for their large ecological and morphological variability. Skinks generally have poorly differentiated and cytogenetically indistinguishable sex chromosomes, and their sex determination was suggested to be highly variable. Here, we determined X-linked genes in the common sandfish (Scincus scincus) and demonstrate that skinks have shared the same homologous XX/XY sex chromosomes across their wide phylogenetic spectrum for at least 85 million years, approaching the age of the highly differentiated ZZ/ZW sex chromosomes of birds and advanced snakes. Skinks thus demonstrate that even poorly differentiated sex chromosomes can be evolutionarily stable. The conservation of sex chromosomes across skinks allows us to introduce the first molecular sexing method widely applicable in this group.