Glial processes at the Drosophila larval neuromuscular junction match synaptic growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.     

Systematic evaluation of candidate blood markers for detecting ovarian cancer

Background

Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.

Methods and Findings

We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.

Conclusions

By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.     

Analyzing complex traits with congenic strains

Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this “common-segment” method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call “sequential” analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.     

A Highlights from MBoC Selection: A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin.     

Greater opportunities for sexual selection in male than in female obligate brood parasitic birds

Females are expected to have evolved to be more discriminatory in mate choice than males as a result of greater reproductive investment into larger gametes (eggs vs. sperm). In turn, males are predicted to be more promiscuous than females, showing both a larger variance in the number of mates and a greater increase in reproductive success with more mates, yielding more intense sexual selection on males vs. females (Bateman's Paradigm). However, sex differences in costly parental care strategies can either reinforce or counteract the initial asymmetry in reproductive investment, which may be one cause for some studies failing to conform with predictions of Bateman's Paradigm. For example, in many bird species with small female‐biased initial investment but extensive biparental care, both sexes should be subject to similar strengths of sexual selection because males and females are similarly restricted in their ability to pursue additional mates. Unlike 99% of avian species, however, obligate brood parasitic birds lack any parental care in either sex, predicting a conformation to Bateman's Paradigm. Here we use microsatellite genotyping to demonstrate that in brood parasitic brown‐headed cowbirds (Molothrus ater), per capita annual reproductive success increases with the number of mates in males, but not in females. Furthermore, also as predicted, the variance of the number of mates and offspring is greater in males than in females. Thus, contrary to previous findings in this species, our results conform to predictions of the Bateman's Paradigm for taxa without parental care.     

How could preventive therapy affect the prevalence of drug resistance? Causes and consequences

Various forms of preventive and prophylactic antimicrobial therapies have been proposed to combat HIV (e.g. pre-exposure prophylaxis), tuberculosis (e.g. isoniazid preventive therapy) and malaria (e.g. intermittent preventive treatment). However, the potential population-level effects of preventative therapy (PT) on the prevalence of drug resistance are not well understood. PT can directly affect the rate at which resistance is acquired among those receiving PT. It can also indirectly affect resistance by altering the rate at which resistance is acquired through treatment for active disease and by modifying the level of competition between transmission of drug-resistant and drug-sensitive pathogens. We propose a general mathematical model to explore the ways in which PT can affect the long-term prevalence of drug resistance. Depending on the relative contributions of these three mechanisms, we find that increasing the level of coverage of PT may result in increases, decreases or non-monotonic changes in the overall prevalence of drug resistance. These results demonstrate the complexity of the relationship between PT and drug resistance in the population. Care should be taken when predicting population-level changes in drug resistance from small pilot studies of PT or estimates based solely on its direct effects.     

Synthesis and SAR of sulfoxide substituted carboxyquinolines as NK3 receptor antagonists

The neurokinin-3 (NK3) receptor is regarded as a potential novel target for treating patients with schizophrenia. Herein we report the synthesis and SAR of a series of C3-alkylsulfoxide substituted quinolines as potent NK3 receptor antagonists. These compounds have excellent NK3 functional activity, good selectivity and drug-like properties. Several key compounds have good in vitro/in vivo DMPK characteristics, and are active in a gerbil locomotor activity model.     

A call for more transparent reporting of error rates: the quality of AFLP data in ecological and evolutionary research

Despite much discussion of the importance of quantifying and reporting genotyping error in molecular studies, it is still not standard practice in the literature. This is particularly a concern for amplified fragment length polymorphism (AFLP) studies, where differences in laboratory, peak‐calling and locus‐selection protocols can generate data sets varying widely in genotyping error rate, the number of loci used and potentially estimates of genetic diversity or differentiation. In our experience, papers rarely provide adequate information on AFLP reproducibility, making meaningful comparisons among studies difficult. To quantify the extent of this problem, we reviewed the current molecular ecology literature (470 recent AFLP articles) to determine the proportion of studies that report an error rate and follow established guidelines for assessing error. Fifty‐four per cent of recent articles do not report any assessment of data set reproducibility. Of those studies that do claim to have assessed reproducibility, the majority (~90%) either do not report a specific error rate or do not provide sufficient details to allow the reader to judge whether error was assessed correctly. Even of the papers that do report an error rate and provide details, many (≥23%) do not follow recommended standards for quantifying error. These issues also exist for other marker types such as microsatellites, and next‐generation sequencing techniques, particularly those which use restriction enzymes for fragment generation. Therefore, we urge all researchers conducting genotyping studies to estimate and more transparently report genotyping error using existing guidelines and encourage journals to enforce stricter standards for the publication of genotyping studies.     

CD4+CD25(int) T cells in inflammatory diseases refractory to treatment with glucocorticoids

Up to 30% of patients with autoimmune, allergic, and lymphoproliferative diseases are refractory to glucocorticoid therapy. The present study was undertaken to investigate whether such steroid resistance (SR) is limited to a subpopulation of CD4(+) T cells and, as IL-2 is a putative driver of SR, whether T cell SR is associated with CD25 expression. We show that SR patients have a characteristic subgroup of activated CD4(+) T cells that continue to proliferate despite exposure to high-dose Dexamethasone (Dex), demonstrate that CD4(+)CD25(-) cells are exquisitely sensitive to Dex whereas CD4(+)CD25(int) cells are highly SR, and further find that the combination of an anti-CD25 mAb with Dex enhances suppression of T cell proliferation compared with each agent alone. We therefore conclude that SR is not a general property of all lymphocytes but resides in T cell subpopulations, which are prevalent in SR patients and express intermediary levels of CD25. As a result, we propose a new paradigm for SR disease in which glucocorticoid therapy positively selects SR cells, generating a population of drug-resistant lymphocytes that perpetuate on-going inflammation.