Murine mCLCA6 is an integral apical membrane protein of non-goblet cell enterocytes and co-localizes with the cystic fibrosis transmembrane conductance regulator.

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine.     

Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.     

IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

Validation of Cell-Cycle Arrest Biomarkers for Acute Kidney Injury after Pediatric Cardiac Surgery

Background

The lack of early biomarkers for acute kidney injury (AKI) seriously inhibits the initiation of preventive and therapeutic measures for this syndrome in a timely manner. We tested the hypothesis that insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, function as early biomarkers for AKI after congenital heart surgery with cardiopulmonary bypass (CPB).

Methods

We prospectively studied 51 children undergoing cardiac surgery with CPB. Serial urine samples were analyzed for [TIMP-2]•[IGFBP7]. The primary outcome measure was AKI defined by the pRIFLE criteria within 72 hours after surgery.

Results

12 children (24%) developed AKI within 1.67 (SE 0.3) days after surgery. Children who developed AKI after cardiac surgery had a significant higher urinary [TIMP-2]•[IGFBP7] as early as 4 h after the procedure, compared to children who did not develop AKI (mean of 1.93 ((ng/ml)²/1000) (SE 0.4) vs 0.47 ((ng/ml)²/1000) (SE 0.1), respectively; p<0.05). Urinary [TIMP-2]•[IGFBP7] 4 hours following surgery demonstrated an area under the receiver-operating characteristic curve of 0.85. Sensitivity was 0.83, and specificity was 0.77 for a cutoff value of 0.70 ((ng/ml)²/1000).

Conclusions

Urinary [TIMP-2]•[IGFBP7] represent sensitive, specific, and highly predictive early biomarkers for AKI after surgery for congenital heart disease.

Trial Registration

www.germanctr.de/, DRKS00005062     

Depletion of beta-COP reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1

We have utilized small interfering RNA (siRNA)-mediated depletion of the beta-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in beta-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the gamma-subunit of COP-I, suggesting that Cav1 is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1.     

Fitness,risk taking,and spatial behavior covary with boldness in experimental vole populations

Individuals of a population may vary along a pace‐of‐life syndrome from highly fecund, short‐lived, bold, dispersive “fast” types at one end of the spectrum to less fecund, long‐lived, shy, plastic “slow” types at the other end. Risk‐taking behavior might mediate the underlying life history trade‐off, but empirical evidence supporting this hypothesis is still ambiguous. Using experimentally created populations of common voles (Microtus arvalis)—a species with distinct seasonal life history trajectories—we aimed to test whether individual differences in boldness behavior covary with risk taking, space use, and fitness. We quantified risk taking, space use (via automated tracking), survival, and reproductive success (via genetic parentage analysis) in 8 to 14 experimental, mixed‐sex populations of 113 common voles of known boldness type in large grassland enclosures over a significant part of their adult life span and two reproductive events. Populations were assorted to contain extreme boldness types (bold or shy) of both sexes. Bolder individuals took more risks than shyer ones, which did not affect survival. Bolder males but not females produced more offspring than shy conspecifics. Daily home range and core area sizes, based on 95% and 50% Kernel density estimates (20 ± 10 per individual, n = 54 individuals), were highly repeatable over time. Individual space use unfolded differently for sex‐boldness type combinations over the course of the experiment. While day ranges decreased for shy females, they increased for bold females and all males. Space use trajectories may, hence, indicate differences in coping styles when confronted with a novel social and physical environment. Thus, interindividual differences in boldness predict risk taking under near‐natural conditions and have consequences for fitness in males, which have a higher reproductive potential than females. Given extreme inter‐ and intra‐annual fluctuations in population density in the study species and its short life span, density‐dependent fluctuating selection operating differently on the sexes might maintain (co)variation in boldness, risk taking, and pace‐of‐life.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Beta2-integrin-induced p38 MAPK activation is a key mediator in the CD14/TLR4/MD2-dependent uptake of lipopolysaccharide by hepatocytes

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.