These aren’t the loci you’re looking for: Principles of effective SNP filtering for molecular ecologists

Sequencing reduced‐representation libraries of restriction site‐associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures

Conduct Disorder and cognitive functioning: Testing three causal hypotheses

Studied the relation between cognitive functioning, as evidenced by IQ and achievement test performance, and Diagnostic and Statistical Manual of Mental Disorders (DSM-III) categories of conduct disturbance, using data from Black adolescents who were members of collaborative perinatal project from birth to age 7 yrs. At age 17 yrs, Ss and their parents were administered a battery of instruments that included standardized psychiatric diagnostic interviews as part of a call-back study. Analyses were compatible with the hypothesis that deficiencies in cognitive functioning are causally related to adolescent conduct disorder as defined by DSM—III. Results suggest that the relation of cognitive functioning to psychiatric status was specific to conduct disorders. Results were incompatible with the hypothesis that conduct problems lead to deficits in cognitive functioning. The 3 most important factors in accounting for age-17 conduct disorder were cognitive functioning, parent psychopathology, and early aggression

Placental syncytiotrophoblast constitutes a major barrier to vertical transmission of Listeria monocytogenes.

Listeria monocytogenes is an important cause of maternal-fetal infections and serves as a model organism to study these important but poorly understood events. L. monocytogenes can infect non-phagocytic cells by two means: direct invasion and cell-to-cell spread. The relative contribution of each method to placental infection is controversial, as is the anatomical site of invasion. Here, we report for the first time the use of first trimester placental organ cultures to quantitatively analyze L. monocytogenes infection of the human placenta. Contrary to previous reports, we found that the syncytiotrophoblast, which constitutes most of the placental surface and is bathed in maternal blood, was highly resistant to L. monocytogenes infection by either internalin-mediated invasion or cell-to-cell spread. Instead, extravillous cytotrophoblasts-which anchor the placenta in the decidua (uterine lining) and abundantly express E-cadherin-served as the primary portal of entry for L. monocytogenes from both extracellular and intracellular compartments. Subsequent bacterial dissemination to the villous stroma, where fetal capillaries are found, was hampered by further cellular and histological barriers. Our study suggests the placenta has evolved multiple mechanisms to resist pathogen infection, especially from maternal blood. These findings provide a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta

Does mating behaviour affect connectivity in marine fishes? Comparative population genetics of two protogynous groupers (Family Serranidae)

Abstract Pelagic larval duration (PLD) has been hypothesized to be the primary predictor of connectivity in marine fishes; however, few studies have examined the effects that adult reproductive behaviour may have on realized dispersal. We assessed gene flow (connectivity) by documenting variation in microsatellites and mitochondrial DNA sequences in two protogynous species of groupers, the aggregate spawning red hind, Epinephelus guttatus, and the single-male, harem-spawning coney, Cephalopholis fulva, to ask whether reproductive strategy affects connectivity. Samples of both species were obtained from waters off three islands (Puerto Rico, St. Thomas and St. Croix) in the Caribbean Sea. Despite the notion that aggregate spawning of red hind may facilitate larval retention, stronger signals of population structure were detected in the haremspawning coney. Heterogeneity and/or inferred barriers, based on microsatellites, involved St. Croix (red hind and coney) and the west coast of Puerto Rico (coney). Heterogeneity and/or inferred barriers, based on mitochondrial DNA, involved St. Croix (coney only). Genetic divergence in both species was stronger for microsatellites than for mitochondrial DNA, suggesting sex-biased dispersal in both species. Longterm migration rates, based on microsatellites, indicated asymmetric gene flow for both species in the same direction as mean surface currents in the region. Red hind had higher levels of variation in microsatellites and lower levels of variation in mitochondrial DNA. Long-term effective size and effective number of breeders were greater for red hind; estimates of h f , a proxy for long-term effective female size, were the same in both species. Patterns of gene flow in both species appear to stem in part from shared aspects of larval and adult biology, local bathymetry and surface current patterns. Differences in connectivity and levels of genetic variation between the species, however, likely stem from differences in behaviour related to reproductive strategy

Invasive Extravillous Trophoblasts Restrict Intracellular Growth and Spread of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular bacterial pathogen that can infect the placenta, a chimeric organ made of maternal and fetal cells. Extravillous trophoblasts (EVT) are specialized fetal cells that invade the uterine implantation site, where they come into direct contact with maternal cells. We have shown previously that EVT are the preferred site of initial placental infection. In this report, we infected primary human EVT with L. monocytogenes. EVT eliminated ∼80% of intracellular bacteria over 24-hours. Bacteria were unable to escape into the cytoplasm and remained confined to vacuolar compartments that became acidified and co-localized with LAMP1, consistent with bacterial degradation in lysosomes. In human placental organ cultures bacterial vacuolar escape rates differed between specific trophoblast subpopulations. The most invasive EVT—those that would be in direct contact with maternal cells in vivo—had lower escape rates than trophoblasts that were surrounded by fetal cells and tissues. Our results suggest that EVT present a bottleneck in the spread of L. monocytogenes from mother to fetus by inhibiting vacuolar escape, and thus intracellular bacterial growth. However, if L. monocytogenes is able to spread beyond EVT it can find a more hospitable environment. Our results elucidate a novel aspect of the maternal-fetal barrier

Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alveolar epithelial cells are known as progenitor cells for the restoration from the damage in the lung. Calcitonin gene-related peptide (CGRP) has been reported to play an important role in the proliferation of various types of epithelial and endothelial cells. We investigated the effects of CGRP on the proliferation of alveolar epithelial cells <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>A549 cells were cultured in Dulbecco Modified Eagle Medium with 5% fatal bovin serum for 24 hours, then CGRP was added <it>in vitro</it>. The proliferation of DNA synthesis was measured using 5-bromo-2-deoxyuridine, an analog of thymidine, by enzyme-linked immunosorbent assay.</p> <p>As one intracellular response to CGRP, we examined activation of p44/42- extracellular signal-regulated kinase (ERK) pathway by adding CGRP, using western blotting method.</p> <p>Recombinant adenovirus encoding nuclear-targeted-human β-CGRP (rhCGRP) was administered into Male Wister rat (n = 5, 10 weeks old) lungs by intratracheal instillation <it>in vivo</it>. 7 days after the administration of CGRP, rat lungs were harvested and histological findings and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) were evaluated to examine cell proliferation.</p> <p>Results</p> <p><it>In vitro </it>study, CGRP increased the proliferation of A549 cells in a dose and time dependent manner. CGRP8-37 (inhibitor of CGRP receptor) decreased CGRP induced proliferation of DNA synthesis. Phosphorylation of ERK pathway was observed within 15 minutes and peaked in one hour. U0126 (inhibitor of ERK pathway) decreased CGRP induced proliferation of DNA synthesis.<it>In vivo </it>study, histological examination of the lung indicated proliferation of alveolar epithelial cells in the rhCGRP-treated group and the nuclei of alveolar epithelial cells were positive for PCNA immunostaining.</p> <p>Conclusion</p> <p>In this study, we conclude that CGRP stimulates proliferation of human alveolar epithelial cells <it>in vivo </it>and <it>in vitro</it>.</p

Environmental variables, habitat discontinuity and life history shaping the genetic structure of Pomatoschistus marmoratus

Coastal lagoons are semi-isolated ecosystems exposed to wide fluctuations of environmental conditions and showing habitat fragmentation. These features may play an important role in separating species into different populations, even at small spatial scales. In this study, we evaluate the concordance between mitochondrial (previous published data) and nuclear data analyzing the genetic variability of Pomatoschistus marmoratus in five localities, inside and outside the Mar Menor coastal lagoon (SE Spain) using eight microsatellites. High genetic diversity and similar levels of allele richness were observed across all loci and localities, although significant genic and genotypic differentiation was found between populations inside and outside the lagoon. In contrast to the FST values obtained from previous mitochondrial DNA analyses (control region), the microsatellite data exhibited significant differentiation among samples inside the Mar Menor and between lagoonal and marine samples. This pattern was corroborated using Cavalli-Sforza genetic distances. The habitat fragmentation inside the coastal lagoon and among lagoon and marine localities could be acting as a barrier to gene flow and contributing to the observed genetic structure. Our results from generalized additive models point a significant link between extreme lagoonal environmental conditions (mainly maximum salinity) and P. marmoratus genetic composition. Thereby, these environmental features could be also acting on genetic structure of coastal lagoon populations of P. marmoratus favoring their genetic divergence. The mating strategy of P. marmoratus could be also influencing our results obtained from mitochondrial and nuclear DNA. Therefore, a special consideration must be done in the selection of the DNA markers depending on the reproductive strategy of the species

Ctr2 Links Copper Homeostasis to Polysaccharide Capsule Formation and Phagocytosis Inhibition in the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is a human opportunistic fungal pathogen responsible for ∼1/3 of HIV/AIDS deaths worldwide. This budding yeast expresses a polysaccharide capsule necessary for virulence. Capsule production inhibits phagocytosis by macrophages. Here we describe results that link copper homeostasis to capsule production and the inhibition of phagocytosis. Specifically, using Agrobacterium-mediated insertional mutagenesis, we identified an insertion in the promoter region of the putative copper transporter-encoding gene CTR2 that results in reduced expression of CTR2 and increased phagocytosis by murine RAW264.7 macrophages. The mutant also displayed sensitivity to copper starvation and defects in polysaccharide capsule production and melanization. These defects were all reversed by genetic correction of the promoter insertion by homologous targeting. Several melanization-defective mutants identified previously, those in the RIM20, RIM101, and VPS25 genes, also display sensitivity to copper starvation, reduced capsule production and increased phagocytosis. Together these results indicate a previously undescribed link between copper homeostasis to polysaccharide capsule production and phagocytosis inhibition in Cryptococcus neoformans

Adipose Tissue Serves as a Reservoir for Recrudescent Rickettsia prowazekii Infection in a Mouse Model

Brill-Zinsser disease, the relapsing form of epidemic typhus, typically occurs in a susceptible host years or decades after the primary infection; however, the mechanisms of reactivation and the cellular reservoir during latency are poorly understood. Herein we describe a murine model for Brill-Zinsser disease, and use PCR and cell culture to show transient rickettsemia in mice treated with dexamethasone >3 months after clinical recovery from the primary infection. Treatment of similarly infected mice with cyclosporine failed to produce recrudescent bacteremia. Therapy with doxycycline for the primary infection prevented recrudescent bacteremia in most of these mice following treatment with dexamethasone. Rickettsia prowazekii (the etiologic agent of epidemic typhus) was detected by PCR, cell culture, and immunostaining methods in murine adipose tissue, but not in liver, spleen, lung, or central nervous system tissues of mice 4 months after recovery from the primary infection. The lungs of dexamethasone-treated mice showed impaired expression of β-defensin transcripts that may be involved in the pathogenesis of pulmonary lesions. In vitro, R. prowazekii rickettsiae infected and replicated in the murine adipocyte cell line 3T3-L1. Collectively these data suggest a role for adipose tissue as a potential reservoir for dormant infections with R. prowazekii