Myosin-1C augments secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane.

Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPB). Actin-associated motor proteins regulate this secretory pathway at multiple points. Prior to fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures allowing maturation of content. Post-fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here we provide the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited post-fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity as inhibition of class I myosins using the inhibitor Pentachloropseudilin or expression of an ATPase deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments

Distribution of cytochrome P450 2C, 2E1, 3A4, and 3A5 in human colon mucosa

BACKGROUND: Despite the fact that the alimentary tract is part of the body's first line of defense against orally ingested xenobiotica, little is known about the distribution and expression of cytochrome P450 (CYP) enzymes in human colon. Therefore, expression and protein levels of four representative CYPs (CYP2C(8), CYP2E1, CYP3A4, and CYP3A5) were determined in human colon mucosa biopsies obtained from ascending, descending and sigmoid colon. METHODS: Expression of CYP2C, CYP2E1, CYP3A4, and CYP3A5 mRNA in colon mucosa was determined by RT-PCR. Protein concentration of CYPs was determined using Western blot methods. RESULTS: Extensive interindividual variability was found for the expression of most of the genes. However, expression of CYP2C mRNA levels were significantly higher in the ascending colon than in the sigmoid colon. In contrast, mRNA levels of CYP2E1 and CYP3A5 were significantly lower in the ascending colon in comparison to the descending and sigmoid colon. In sigmoid colon protein levels of CYP2C8 were significantly higher by ~73% than in the descending colon. In contrast, protein concentration of CYP2E1 was significantly lower by ~81% in the sigmoid colon in comparison to the descending colon. CONCLUSION: The current data suggest that the expression of CYP2C, CYP2E1, and CYP3A5 varies in different parts of the colon

Multiple ITS Copies Reveal Extensive Hybridization within Rheum (Polygonaceae), a Genus That Has Undergone Rapid Radiation

During adaptive radiation events, characters can arise multiple times due to parallel evolution, but transfer of traits through hybridization provides an alternative explanation for the same character appearing in apparently non-sister lineages. The signature of hybridization can be detected in incongruence between phylogenies derived from different markers, or from the presence of two divergent versions of a nuclear marker such as ITS within one individual.In this study, we cloned and sequenced ITS regions for 30 species of the genus Rheum, and compared them with a cpDNA phylogeny. Seven species contained two divergent copies of ITS that resolved in different clades from one another in each case, indicating hybridization events too recent for concerted evolution to have homogenised the ITS sequences. Hybridization was also indicated in at least two further species via incongruence in their position between ITS and cpDNA phylogenies. None of the ITS sequences present in these nine species matched those detected in any other species, which provides tentative evidence against recent introgression as an explanation. Rheum globulosum, previously indicated by cpDNA to represent an independent origin of decumbent habit, is indicated by ITS to be part of clade of decumbent species, which acquired cpDNA of another clade via hybridization. However decumbent and glasshouse morphology are confirmed to have arisen three and two times, respectively.These findings suggested that hybridization among QTP species of Rheum has been extensive, and that a role of hybridization in diversification of Rheum requires investigation

Socioeconomic Inequalities in Newborn Care During Facility and Home Deliveries: A Cross Sectional Analysis of Data from Demographic Surveillance Sites in Rural Bangladesh, India and Nepal

Background: In Bangladesh, India and Nepal, neonatal outcomes of poor infants are considerably worse than those of better-off infants. Understanding how these inequalities vary by country and place of delivery (home or facility) will allow targeting of interventions to those who need them most. We describe socio-economic inequalities in newborn care in rural areas of Bangladesh, Nepal and India for all deliveries and by place of delivery. Methods: We used data from surveillance sites in Bangladesh, India and from Makwanpur and Dhanusha districts in Nepal, covering periods from 2001 to 2011. We used literacy (ability to read a short text) as indicator of socioeconomic status. We developed a composite score of nine newborn care practices (score range 0–9 indicating infants received no newborn care to all nine newborn care practices). We modeled the effect of literacy and place of delivery on the newborn care score and on individual practices. Results: In all study sites (60,078 deliveries in total), use of facility delivery was higher among literate mothers. In all sites, inequalities in newborn care were observed: the difference in new born care between literate and illiterate ranged 0.35–0.80. The effect of literacy on the newborn care score reduced after adjusting for place of delivery (range score difference literate-illiterate: 0.21–0.43). Conclusion: Socioeconomic inequalities in facility care greatly contribute to inequalities in newborn care. Improving newborn care during home deliveries and improving access to facility care are a priority for addressing inequalities in newborn care and newborn mortality

Depression and sickness behavior are Janus-faced responses to shared inflammatory pathways

It is of considerable translational importance whether depression is a form or a consequence of sickness behavior. Sickness behavior is a behavioral complex induced by infections and immune trauma and mediated by pro-inflammatory cytokines. It is an adaptive response that enhances recovery by conserving energy to combat acute inflammation. There are considerable phenomenological similarities between sickness behavior and depression, for example, behavioral inhibition, anorexia and weight loss, and melancholic (anhedonia), physio-somatic (fatigue, hyperalgesia, malaise), anxiety and neurocognitive symptoms. In clinical depression, however, a transition occurs to sensitization of immuno-inflammatory pathways, progressive damage by oxidative and nitrosative stress to lipids, proteins, and DNA, and autoimmune responses directed against self-epitopes. The latter mechanisms are the substrate of a neuroprogressive process, whereby multiple depressive episodes cause neural tissue damage and consequent functional and cognitive sequelae. Thus, shared immuno-inflammatory pathways underpin the physiology of sickness behavior and the pathophysiology of clinical depression explaining their partially overlapping phenomenology. Inflammation may provoke a Janus-faced response with a good, acute side, generating protective inflammation through sickness behavior and a bad, chronic side, for example, clinical depression, a lifelong disorder with positive feedback loops between (neuro)inflammation and (neuro)degenerative processes following less well defined triggers

Single pi+ Electroproduction on the Proton in the First and Second Resonance Regions at 0.25GeV^2 < Q^2 < 0.65GeV^2 Using CLAS

The ep -> e'pi^+n reaction was studied in the first and second nucleon resonance regions in the 0.25 GeV^2 < Q^2 < 0.65 GeV^2 range using the CLAS detector at Thomas Jefferson National Accelerator Facility. For the first time the absolute cross sections were measured covering nearly the full angular range in the hadronic center-of-mass frame. The structure functions sigma_TL, sigma_TT and the linear combination sigma_T+epsilon*sigma_L were extracted by fitting the phi-dependence of the measured cross sections, and were compared to the MAID and Sato-Lee models.Comment: Accepted for publication in PR

Photodisintegration of 4^4He into p+t

The two-body photodisintegration of $^4$He into a proton and a triton has been studied using the CEBAF Large-Acceptance Spectrometer (CLAS) at Jefferson Laboratory. Real photons produced with the Hall-B bremsstrahlung-tagging system in the energy range from 0.35 to 1.55 GeV were incident on a liquid $^4$He target. This is the first measurement of the photodisintegration of $^4$He above 0.4 GeV. The differential cross sections for the $\gamma$$^4$He$\to pt$ reaction have been measured as a function of photon-beam energy and proton-scattering angle, and are compared with the latest model calculations by J.-M. Laget. At 0.6-1.2 GeV, our data are in good agreement only with the calculations that include three-body mechanisms, thus confirming their importance. These results reinforce the conclusion of our previous study of the three-body breakup of $^3$He that demonstrated the great importance of three-body mechanisms in the energy region 0.5-0.8 GeV .Comment: 13 pages submitted in one tgz file containing 2 tex file and 22 postscrip figure

π0\pi^0 photoproduction on the proton for photon energies from 0.675 to 2.875 GeV

Differential cross sections for the reaction $\gamma p \to p \pi^0$ have been measured with the CEBAF Large Acceptance Spectrometer (CLAS) and a tagged photon beam with energies from 0.675 to 2.875 GeV. The results reported here possess greater accuracy in the absolute normalization than previous measurements. They disagree with recent CB-ELSA measurements for the process at forward scattering angles. Agreement with the SAID and MAID fits is found below 1 GeV. The present set of cross sections has been incorporated into the SAID database, and exploratory fits have been extended to 3 GeV. Resonance couplings have been extracted and compared to previous determinations.Comment: 18 pages, 48 figure

Exclusive ρ0\rho^0 electroproduction on the proton at CLAS

The $e p\to e^\prime p \rho^0$ reaction has been measured, using the 5.754 GeV electron beam of Jefferson Lab and the CLAS detector. This represents the largest ever set of data for this reaction in the valence region. Integrated and differential cross sections are presented. The $W$, $Q^2$ and $t$ dependences of the cross section are compared to theoretical calculations based on $t$-channel meson-exchange Regge theory on the one hand and on quark handbag diagrams related to Generalized Parton Distributions (GPDs) on the other hand. The Regge approach can describe at the $\approx$ 30% level most of the features of the present data while the two GPD calculations that are presented in this article which succesfully reproduce the high energy data strongly underestimate the present data. The question is then raised whether this discrepancy originates from an incomplete or inexact way of modelling the GPDs or the associated hard scattering amplitude or whether the GPD formalism is simply inapplicable in this region due to higher-twists contributions, incalculable at present.Comment: 29 pages, 29 figure