Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR‐based analyses of low‐concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty—indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics.     

Oocyte growth and follicular hierarchy may be locally controlled by an inhibin-like protein in the lizard Podarcis sicula.

The lizard Podarcis shows an ovarian annual cycle with three to four ovulatory waves between April and July (reproductive period). In August to September, a refractory stage occurs, followed by a nonreproductive period (October to March), during which the oocytes undergo slow growth and prepare themselves for vitellogenesis and ovulation. In the reproductive period, only a certain number of oocytes start growing, giving rise to a follicular hierarchy, which is controlled by still unknown mechanisms. In the present paper, immunoreactive inhibin was detected in previtellogenetic follicles of the reproductive period, and in particular, in the pyriform cells of the follicular epithelium. As the follicle grew and the pyriform cells disappeared, immunostaining shifted to the oocyte cytoplasm. The smaller follicles did not show any immunoreactivity. In the nonreproductive period, no follicles were labeled. We conclude that in the reproductive period, inhibin characterizes the follicles destined to ovulation and might be one of the main factors controlling follicular hierarchy.     

The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts

Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts.     

The giants of the phylum Brachiopoda: a matter of diet?

The species of the brachiopod Gigantoproductus are giants within the Palaeozoic sedentary benthos. This presents a dilemma as living brachiopods have low‐energy lifestyles. Although brachiopod metabolic rates were probably higher during the Palaeozoic than today, the massive size reached by species of Gigantoproductus is nevertheless unusual. By examining the diet of Gigantoproductus species from the Visean (Mississippian, Carboniferous) of Derbyshire (UK), we seek to understand the mechanisms that enabled those low‐metabolism brachiopod species to become giants. Were they suspension feeders, similar to all other brachiopods, or did endosymbiosis provide a lifestyle that allowed them to have higher metabolic rates and become giants? We suggest that the answer to this conundrum may be solved by the identification of the biogeochemical signatures of symbionts, through combined analyses of the carbon and nitrogen‐isotopic compositions of the occluded organic matrix within their calcite shells. The shells are formed of substructured columnar units that are remarkably long and a few hundreds of microns wide, deemed to be mostly pristine based on multiple analyses (petrography, cathodoluminescence (CL), scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), transmission electron microscopy (TEM)); they contain occluded organic fractions detected by TEM, nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC‐MS) analyses. We conclude that the gigantic size reached by the species of Gigantoproductus is probably the result of a mixotroph lifestyle, by which they could rely on the energy and nutrients derived both from photosymbiotic microbes and from filtered particulate food.     

The lipoprotein HP1454 of Helicobacter pylori regulates T‐cell response by shaping T‐cell receptor signalling

Helicobacter pylori (HP) is a Gram‐negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host–pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild‐type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T‐cell antigen receptor‐dependent signalling and lymphocyte proliferation, as well as the CXCL12‐dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T‐cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP‐associated diseases, is dependent on T cells.     

miRNA-regulated gene expression differs in celiac disease patients according to the age of presentation

Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miRNAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0482-2) contains supplementary material, which is available to authorized users.     

Fluid mechanics of the zebrafish embryonic heart trabeculation

Embryonic heart development is a mechanosensitive process, where specific fluid forces are needed for the correct development, and abnormal mechanical stimuli can lead to malformations. It is thus important to understand the nature of embryonic heart fluid forces. However, the fluid dynamical behaviour close to the embryonic endocardial surface is very sensitive to the geometry and motion dynamics of fine-scale cardiac trabecular surface structures. Here, we conducted image-based computational fluid dynamics (CFD) simulations to quantify the fluid mechanics associated with the zebrafish embryonic heart trabeculae. To capture trabecular geometric and motion details, we used a fish line that expresses fluorescence at the endocardial cell membrane, and high resolution 3D confocal microscopy. Our endocardial wall shear stress (WSS) results were found to exceed those reported in existing literature, which were estimated using myocardial rather than endocardial boundaries. By conducting simulations of single intra-trabecular spaces under varied scenarios, where the translational or deformational motions (caused by contraction) were removed, we found that a squeeze flow effect was responsible for most of the WSS magnitude in the intra-trabecular spaces, rather than the shear interaction with the flow in the main ventricular chamber. We found that trabecular structures were responsible for the high spatial variability of the magnitude and oscillatory nature of WSS, and for reducing the endocardial deformational burden. We further found cells attached to the endocardium within the intra-trabecular spaces, which were likely embryonic hemogenic cells, whose presence increased endocardial WSS. Overall, our results suggested that a complex multi-component consideration of both anatomic features and motion dynamics were needed to quantify the trabeculated embryonic heart fluid mechanics.