D-Myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P(4)] or phosphatidylinositol 3-kinase (PI 3-kinase) activity acts to inhibit calcium-dependent chloride secretion in T84 colonic epithelial cells. To further distinguish between the contributions of these two signaling pathways to the inhibition of secretion, we studied effects of insulin, because the insulin receptor links to PI 3-kinase but not to pathways postulated to generate Ins(3,4,5,6)P(4). Chloride secretion across T84 cell monolayers was studied in Ussing chambers. Activation of PI 3-kinase was assessed by Western blotting. Basolateral, but not apical, addition of insulin inhibited carbachol- and thapsigargin-induced chloride secretion in a time- and concentration-dependent fashion. Insulin-like growth factor-I (IGF-I) had similar effects. Insulin had no effect on Ins(3,4,5,6)P(4) levels, and the inhibitory effects of insulin and IGF-I on chloride secretion were fully reversed by the PI 3-kinase inhibitors wortmannin and LY-294002. Western blot analysis showed that both insulin and IGF-I recruited the 85-kDa regulatory and 110-kDa catalytic subunits of PI 3-kinase to anti-phosphotyrosine immunoprecipitates. In conclusion, insulin and IGF-I act to inhibit calcium-dependent chloride secretion through a PI 3-kinase-dependent pathway. Because insulin is released in a pulsatile fashion postprandially and IGF-I levels are elevated in pathological settings, our findings may have physiological and/or pathophysiological significance.

Utilizing data from the Million Veteran Program (MVP), we investigated the genetic determinants underlying total testosterone levels via a multi-ancestral analysis of 124,593 individuals of European (n=88,385), African (n=25,235) and Hispanic (n=10,973) ancestry. We identified 46 trans-ancestry variants and 17 ancestry-specific variants, of which 14 trans-ancestry variants and 15 ancestry-specific variants are novel associations with testosterone. Results implicate genes regulating testosterone shared across ancestral groups, which include SHBG, JMJD1C, FXR2, SENP3, TNFSF12-TNFSF13 while also implicating genes such as MSN, DMD, VSIG4, CHEK2, TKTL1 that may underlie ancestry-group differences in testosterone regulation. We also linked testosterone variants on the X chromosome with differential risk of chronic kidney disease and hereditary hemolytic anemias in African and Hispanic ancestry groups, respectively. Lastly, we constructed a polygenic score from our 46 trans-ancestry variants and associated it with testicular dysfunction, hyperlipidemia, gout and prostate cancer with stronger prostate cancer associations in Hispanic and African ancestry groups compared to the European ancestry group. These findings provide insight into ancestry-specific androgen regulation and identify novel variants for disease risk stratification in patients.

Background Studies have shown that healthcare-associated infections (HAIs) due to methicillin-resistant Staphylococcus aureus (MRSA) can lead to substantial healthcare costs in acute care settings. However, little is known regarding the consequences of these infections on patients in long-term care centers (LTCCs). The purpose of this study was to estimate the attributable cost of MRSA HAIs in LTCCs within the Department of Veterans Affairs (VA). Methods We performed a retrospective cohort study of patients admitted to VA LTCCs between 1 January 2009 and 30 September 2015. MRSA HAIs were defined as a positive clinical culture at least 48 hours after LTCC admission so as to exclude community-acquired infections. Positive cultures were further classified by site (sterile or nonsterile). We used multivariable generalized linear models and 2-part models to compare the LTCC and acute care costs between patients with and without an MRSA HAI. Results In our primary analysis, there was no difference in LTCC costs between patients with and without a MRSA HAI. There was, however, a significant increase in the odds of being transferred to an acute care facility (odds ratio, 4.40 [95% confidence interval {CI}, 3.40–5.67]) and in acute care costs ($9711 [95% CI, $6961–$12 462]). Conclusions Our findings of high cost and increased risk of transfer from LTCC to acute care are important because they highlight the substantial clinical and economic impact of MRSA infections in this population.

There is a growing impetus to expand the repository of chassis available to synthetic biologists. The chloroplast genome presents a unique chassis for engineering photosynthetic eukaryotes by virtue of its compact size, lack of epigenetic regulation, and containment within the secluded lipid bilayers of the organelle. The development of the chloroplast as a Synthetic Biology chassis, however, has been limited by a lack of efficient techniques for whole genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117 kb Phaeodactylum tricornutum chloroplast genome that have 90 to 100% efficiency when screening as few as ten Saccharomyces cerevisiae colonies following yeast assembly. The first method directly uses PCR-amplified fragments of the genome for yeast assembly, whereas the second method relies upon the pre-cloning of eight overlapping genomic regions into individual plasmids that they can later be released from. The cloned genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a novel delivery mechanism for bringing DNA directly to the algal chloroplast. As well, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly.