DRhoGEF2 Regulates Cellular Tension and Cell Pulsations in the Amnioserosa during Drosophila Dorsal Closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells

The Prevalence of Norovirus in returning international travelers with diarrhea

Background: There is a high incidence of diarrhea in traveling populations. Norovirus (NV) infection is a common cause of diarrhea and is associated with 7% of all diarrhea related deaths in the US. However, data on the overall prevalence of NV infection in traveling populations is limited. Furthermore, the prevalence of NV amongst travelers returning to Europe has not been reported. This study determined the prevalence of NV among international travelers returning to Germany from over 50 destinations in and outside Europe. Methods: Stool samples of a total of 104 patients with a recent (< 14days) history of international travel (55 male, mean age 37 yrs.) were tested for the presence of NV genogroup (GG) I and II infection using a sensitive and well established quantitative RT PCR method. 57 patients experienced diarrhea at the time of presentation at the Department of Infectious Diseases & Tropical Medicine. The remaining 47 patients had no experience of diarrhea or other gastrointestinal symptoms for at least 14 days prior to their date of presentation at our institute. Results: In our cohort, NV infection was detected in 15.7% of returning travelers with diarrhea. The closer to the date of return symptoms appeared, the higher the incidence of NV, ranging as high as 21.2% within the first four days after return. Conclusions: In our cohort, NV infection was shown to be frequent among returning travelers especially in those with diarrhea, with over 1/5 of diarrhea patients tested positive for NV within the first four days after their return to Germany. Due to this prevalence, routine testing for NV infection and hygienic precautions may be warranted in this group. This is especially applicable to patients at an increased risk of spreading the disease, such as healthcare workers, teachers or food-handlers

Circulating endothelial cells are an early predictor in renal cell carcinoma for tumor response to sunitinib

<p>Abstract</p> <p>Background</p> <p>Tyrosine kinase inhibitors (TKI) have enriched the therapeutic options in patients with renal cell carcinoma (RCC), which frequently induce morphological changes in tumors. However, only little is known about the biological activity of TKI. Circulating endothelial cells (CEC) have been associated with endothelial damage and, hence, may serve as a putative marker for the biological activity of TKI. The main objective of our study was to evaluate the predictive value of CEC, monocytes, and soluble vascular endothelial growth factor receptor (sVEGFR)-2 in RCC patients receiving sunitinib treatment.</p> <p>Methods</p> <p>Analyses of CEC, monocytes, and sVEGFR-2 were accomplished for twenty-six consecutive patients with metastatic RCC who received treatment with sunitinib (50 mg, 4 wks on 2 wks off schedule) at our institution in 2005 and 2006.</p> <p>Results</p> <p>In RCC patients CEC are elevated to 49 ± 44/ml (control 8 ± 8/ml; P = 0.0001). Treatment with sunitinib is associated with an increase in CEC within 28 days of treatment in patients with a Progression free survival (PFS) above the median to 111 ± 61 (P = 0.0109), whereas changes in patients with a PFS below the median remain insignificant 69 ± 61/ml (P = 0.1848). Monocytes and sVEGFR2 are frequently altered upon sunitinib treatment, but fail to correlate with clinical response, defined by PFS above or below the median.</p> <p>Conclusions</p> <p>Sunitinib treatment is associated with an early increase of CEC in responding patients, suggesting superior endothelial cell damage in these patients as a putative predictive biomarker.</p

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3−/− and LtapLp/+ mutants, Dvl3+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant

Cabozantinib versus everolimus, nivolumab, axitinib, sorafenib and best supportive care: A network meta-analysis of progression-free survival and overall survival in second line treatment of advanced renal cell carcinoma

Background Relative effect of therapies indicated for the treatment of advanced renal cell carcinoma (aRCC) after failure of first line treatment is currently not known. The objective of the present study is to evaluate progression-free survival (PFS) and overall survival (OS) of cabozantinib compared to everolimus, nivolumab, axitinib, sorafenib, and best supportive care (BSC) in aRCC patients who progressed after previous VEGFR tyrosine-kinase inhibitor (TKI) treatment. Methodology & findings Systematic literature search identified 5 studies for inclusion in this analysis. The assessment of the proportional hazard (PH) assumption between the survival curves for different treatment arms in the identified studies showed that survival curves in two of the studies did not fulfil the PH assumption, making comparisons of constant hazard ratios (HRs) inappropriate. Consequently, a parametric survival network meta-analysis model was implemented with five families of functions being jointly fitted in a Bayesian framework to PFS, then OS, data on all treatments. The comparison relied on data digitized from the Kaplan-Meier curves of published studies, except for cabozantinib and its comparator everolimus where patient level data were available. This analysis applied a Bayesian fixed-effects network meta-analysis model to compare PFS and OS of cabozantinib versus its comparators. The log-normal fixed-effects model displayed the best fit of data for both PFS and OS, and showed that patients on cabozantinib had a higher probability of longer PFS and OS than patients exposed to comparators. The survival advantage of cabozantinib increased over time for OS. For PFS the survival advantage reached its maximum at the end of the first year’s treatment and then decreased over time to zero. Conclusion With all five families of distributions, cabozantinib was superior to all its comparators with a higher probability of longer PFS and OS during the analyzed 3 years, except with the Gompertz model, where nivolumab was preferred after 24 months

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3−/− and LtapLp/+ mutants, Dvl3+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant

Psychometric Curve and Behavioral Strategies for Whisker-Based Texture Discrimination in Rats

The rodent whisker system is a major model for understanding neural mechanisms for tactile sensation of surface texture (roughness). Rats discriminate surface texture using the whiskers, and several theories exist for how texture information is physically sensed by the long, moveable macrovibrissae and encoded in spiking of neurons in somatosensory cortex. However, evaluating these theories requires a psychometric curve for texture discrimination, which is lacking. Here we trained rats to discriminate rough vs. fine sandpapers and grooved vs. smooth surfaces. Rats intermixed trials at macrovibrissa contact distance (nose >2 mm from surface) with trials at shorter distance (nose <2 mm from surface). Macrovibrissae were required for distant contact trials, while microvibrissae and non-whisker tactile cues were used for short distance trials. A psychometric curve was measured for macrovibrissa-based sandpaper texture discrimination. Rats discriminated rough P150 from smoother P180, P280, and P400 sandpaper (100, 82, 52, and 35 µm mean grit size, respectively). Use of olfactory, visual, and auditory cues was ruled out. This is the highest reported resolution for rodent texture discrimination, and constrains models of neural coding of texture information

Critical Review of Norovirus Surrogates in Food Safety Research: Rationale for Considering Volunteer Studies

The inability to propagate human norovirus (NoV) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses, like feline calicivirus (FCV) and murine norovirus-1 (MNV), which are propagatable in cell culture. The use of surrogates is predicated on the assumption that they generally mimic the viruses they represent; however, studies are proving this concept invalid. In direct comparisons between FCV and MNV, their susceptibility to temperatures, environmental and food processing conditions, and disinfectants are dramatically different. Differences have also been noted between the inactivation of NoV and its surrogates, thus questioning the validity of surrogates. Considerable research funding is provided globally each year to conduct surrogate studies on NoVs; however, there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies. Human challenge studies are needed to determine which processing techniques are effective in reducing NoVs in foods. A major obstacle to clinical trials on NoVs is the perception that such trials are too costly and risky, but in reality, there is far more cost and risk in allowing millions of unsuspecting consumers to contract NoV illness each year, when practical interventions are only a few volunteer studies away. A number of clinical trials have been conducted, providing important insights into NoV inactivation. A shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic, practical, and scientifically valid processing approaches to improve food safety

Production and characterization of murine models of classic and intermediate maple syrup urine disease

BACKGROUND: Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. METHODS: To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. RESULTS: By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. CONCLUSION: These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease