Physiological consequences of rising water salinity for a declining freshwater turtle.

Sea-level rise, drought and water diversion can all lead to rapid salinization of freshwater habitats, especially in coastal areas. Increased water salinities can in turn alter the geographic distribution and ecology of freshwater species including turtles. The physiological consequences of salinization for freshwater turtles, however, are poorly known. Here, we compared the osmoregulatory response of two geographically separate populations of the freshwater Western Pond Turtle (Actinemys marmorata)-a species declining across its range in western North America-to three constant salinities: 0.4 ppt, 10 ppt and 15 ppt over 2 weeks. We found that turtles from a coastal estuarine marsh population regulated their plasma osmolality at lower levels than their conspecifics from an inland freshwater creek population 45 km away. Plasma osmolalities were consistently lower in estuarine marsh turtles than the freshwater creek turtles over the entire 2-week exposure to 10 ppt and 15 ppt water. Furthermore, estuarine marsh turtles maintained plasma osmolalities within 1 SD of their mean field osmolalities over the 2-week exposure, whereas freshwater creek turtles exceeded their field values within the first few days after exposure to elevated salinities. However, individuals from both populations exhibited body mass loss in 15 ppt water, with significantly greater loss in estuarine turtles. We speculate that the greater ability to osmoregulate by the estuarine marsh turtles may be explained by their reduced feeding and drinking in elevated salinities that was not exhibited by the freshwater creek population. However, due to mass loss in both populations, physiological and behavioural responses exhibited by estuarine marsh turtles may only be effective adaptations for short-term exposures to elevated salinities, such as those from tides and when traversing saline habitats, and are unlikely to be effective for long-term exposure to elevated salinity as is expected under sea-level rise

The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

<p>Abstract</p> <p>Background</p> <p>Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of <it>CCW12 </it>results in severe cell wall damage and reduced mating efficiency.</p> <p>Results</p> <p>In order to explore the function of <it>CCW12</it>, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of <it>CCW12</it>. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are <it>PFD1</it>, <it>WHI3</it>, <it>SRN2</it>, <it>PAC10</it>, <it>FEN1 </it>and <it>YDR417C</it>, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant <it>ccw12</it>Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are <it>BCK1</it>, <it>CHS3</it>, <it>EDE1</it>, <it>PFD1</it>, <it>SLT2 </it>and <it>SLA1 </it>that were also identified in the SGA. In contrast, a specific feature of mutant <it>ccw12</it>Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection.</p> <p>Conclusions</p> <p>The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of <it>CCW12</it>. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.</p> <p>The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.</p

Anandamide Induces Sperm Release from Oviductal Epithelia through Nitric Oxide Pathway in Bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR– 92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer