Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.     

Sperm whale depredation of sablefish longline gear in the northeast Pacific Ocean

Interactions between marine mammals and fisheries include competition for prey (catch), marine mammal entanglement in fishing gear, and catch removal off fishing gear (depredation). We estimated the magnitude of sperm whale depredation on a major North Pacific longline fishery (sablefish) using data collected during annual longline surveys. Sperm whale depredation occurs while the longline gear is off‐bottom during retrieval. Sperm whales were observed on 16% of longline survey sampling days, mostly (95% of sightings) over the continental slope. Sightings were most common in the central and eastern Gulf of Alaska (98% of sightings), occasional in the western Gulf of Alaska and Aleutian Islands, and absent in the Bering Sea. Longline survey catches were commonly preyed upon when sperm whales were present (65% of sightings), as evidenced by damaged fish. Neither sperm whale presence (P = 0.71) nor depredation rate (P = 0.78) increased significantly from 1998 to 2004. Longline survey catch rates were about 2% less at locations where depredation was observed, but the effect was not significant (P = 0.34). Estimated sperm whale depredation was <1% of the annual sablefish longline fishery catch off Alaska during 1998 to 2004.     

Cystatin C--a paradigm of evidence based laboratory medicine

Cystatin C is a 13-kDa protein, of the cysteine proteinase inhibitor superfamily, produced by all nucleated cells. Its production rate is constant throughout the ages of 1 to 50 years. It is freely filtered at the glomerulus and then resorbed and fully catabolised by proximal renal tubules, making it an ideal marker of glomerular filtration rate (GFR). Serum creatinine, the most established marker of renal function, is affected by age, gender, muscle mass, nutritional status and analytical interference. The abbreviated Modifiation of Diet in Renal Diseases (MDRD) equation has recently been introduced in an attempt to overcome these shortcomings, but still has many limitations. Cystatin C is not affected by gender, muscle mass, malignancy, its production rate is usually constant and its plasma concentration therefore is dependent only on GFR. Cystatin C has been demonstrated to be more accurate than serum creatinine in the detection of early renal impairment and in specific populations may allow for early detection of renal disease. Cystatin C has also been found to be a strong predictor of long-term clinical outcomes in patients with cardiovascular diseases. Although cystatin C may have advantages in detection of early renal impairment there is a paucity of evidence that it significantly improves clinical decision making over creatinine. This coupled with assay cost may be the reason why cystatin C, although well recognised, has not been introduced into routine operational use, although that may eventuate with emerging evidence.     

Population genetics and phylogeography of freshwater mussels in North America, Elliptio dilatata and Actinonaias ligamentina (Bivalvia: Unionidae)

Extrinsic and intrinsic forces combined shape the population structure of every species differently. Freshwater mussels are obligate parasites to a host fish during a juvenile stage (glochidia). Elliptio dilatata (ED) and Actinonaias ligamentina (AL) are co-occurring freshwater mussel taxa with similar North American distribution and share some potential host fish. Using mitochondrial DNA, we determined the genotypes of 190 + individuals from collection sites in at least two tributaries in the Lake Erie and Ohio River watersheds, along with the Ouachita and Strawberry rivers in the southeast. Both species had followed a stepping-stone model of dispersal, with greater pairwise genetic structure among collection sites of ED. Also, phylogeographical analysis for ED found significant geographical structuring of haplotype diversity. Overall, within-population variation increased significantly from north to south, with low genetic diversity in the Strawberry River. We calculated significant among-population structure for both species (ED: Phi(ST) = 0.62, P < 0.001; AL: Phi(ST) = 0.16, P < 0.001). Genetic analysis identified the Ouachita River as an area of significant reproductive isolation for both species. Results for AL indicated dispersal into northern areas from two genetically distinct glacial refugia, where results for ED indicated dispersal followed by low gene flow in northern areas. The conservation strategies for mussels that co-occur in the same 'bed' could be species specific. Species such as ED have management units on the population scale, where AL has a more homogeneous genetic structure across its range.     

A modified alkaline comet assay for measuring DNA repair capacity in human populations

Trzeciak, A. R., Barnes, J. and Evans, M. K. A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations. Radiat. Res. 169, 110-121 (2008). Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.     

Thrombin mediates the extraintestinal thrombosis associated with experimental colitis

Recent evidence implicating tissue factor and the protein C pathway in the hypercoagulable state associated with intestinal inflammation suggests that thrombin is likely to contribute to this response. The objective of this study was to assess the role of thrombin in the extraintestinal thrombosis associated with experimental colitis. Thrombus formation was quantified in microvessels of the cremaster muscle in mice with dextran sodium sulfate (DSS)-induced colonic inflammation. The light/dye endothelial injury model was used to elicit thrombus formation in DSS colitic mice treated with either hirudin, heparin, or antithrombin III. The initiation and propagation/stabilization phases of thrombus formation were quantified using the time of onset of the thrombus and time to blood flow cessation, respectively. Thrombus formation was accelerated in arterioles of DSS colitic mice, as exhibited by significant reductions in the time of thrombus initiation and propagation/stabilization. Colitic mice treated with hirudin, heparin, or antithrombin III did not exhibit a significant change in the time of onset of the thrombus compared with untreated colitic mice. However, all three antithrombin agents largely prevented the DSS-induced reduction in the time to flow cessation following light/dye injury, with hirudin offering complete protection. These findings indicate that thrombin plays a major role in the extraintestinal thrombus formation associated with experimental colitis. Thrombin appears to contribute to the propagation/stabilization, rather than initiation, phase of the colitis-associated thrombogenesis at the distant vascular site. The results support the therapeutic use of antithrombin agents for reducing the risk of thromboembolism in patients with inflammatory bowel disease.     

Role for MyD88 signaling in murine gammaherpesvirus 68 latency

Toll-like receptors (TLRs) are known predominantly for their role in activating the innate immune response. Recently, TLR signaling via MyD88 has been reported to play an important function in development of a B-cell response. Since B cells are a major latency reservoir for murine gammaherpesvirus 68 (MHV68), we investigated the role of TLR signaling in the establishment and maintenance of MHV68 latency in vivo. Mice deficient in MyD88 (MyD88(-/-)) or TLR3 (TLR3(-/-)) were infected with MHV68. Analysis of splenocytes recovered at day 16 postinfection from MyD88(-/-) mice compared to those from wild-type control mice revealed a lower frequency of (i) activated B cells, (ii) germinal-center B cells, and (iii) class-switched B cells. Accompanying this substantial defect in the B-cell response was an approximately 10-fold decrease in the establishment of splenic latency. In contrast, no defect in viral latency was observed in TLR3(-/-) mice. Analysis of MHV68-specific antibody responses also demonstrated a substantial decrease in the kinetics of the response in MyD88(-/-) mice. Analysis of wild-type x MyD88(-/-) mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88(-/-) B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. In addition, while MHV68 established latency efficiently in the MyD88-sufficient B cells, there was again a ca. 10-fold reduction in the frequency of MyD88(-/-) B cells harboring latent MHV68. This phenotype indicates that MyD88 is important for the establishment of MHV68 latency and is directly related to the role of MyD88 in the generation of a B-cell response. Furthermore, the generation of a B-cell response to MHV68 was intrinsic to B cells and was independent of the interleukin-1 receptor, a cytokine receptor that also signals through MyD88. These data provide evidence for a unique role for MyD88 in the establishment of MHV68 latency.     

Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses and are required for efficient replication in nonhuman primates

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C', C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C-) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C-) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C-) and rHPIV1-C(F170S), a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-), whereas only the anti-IFN activity is disabled in rHPIV1-C(F170S). In African green monkeys (AGMs), rHPIV1-P(C-) was considerably more attenuated than rHPIV1-C(F170S), suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C-) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.     

Mixed-linkage (1-->3,1-->4)-beta-D-glucan is a major hemicellulose of Equisetum (horsetail) cell walls

Mixed-linkage (1-->3,1-->4)-beta-d-glucan (MLG) is a hemicellulose reputedly confined to certain Poales. Here, the taxonomic distribution of MLG, and xyloglucan, especially in early-diverging pteridophytes, has been re-investigated. Polysaccharides were digested with lichenase and xyloglucan endoglucanase (XEG), which specifically hydrolyse MLG and xyloglucan, respectively. The oligosaccharides produced were analysed by thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and alkaline peeling. Lichenase yielded oligo-beta-glucans from all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum scirpoides, Equisetum sylvaticum and Equisetum xtrachyodon). The major product was the tetrasaccharide beta-glucosyl-(1-->4)-beta-glucosyl-(1-->4)-beta-glucosyl-(1-->3)-glucose (G4G4G3G), which was converted to cellotriose by alkali, confirming its structure. Minor products included G3G, G4G3G and a nonasaccharide. By contrast, poalean MLGs yielded G4G3G > G4G4G3G > nonasaccharide > dodecasaccharide. No other pteridophytes tested contained MLG, including Psilotum and eusporangiate ferns. No MLG was found in lycopodiophytes, bryophytes, Chara or Nitella. XEG digestion showed that Equisetum xyloglucan has unusual repeat units. Equisetum, an exceedingly isolated genus whose closest living relatives diverged > 380 million years ago, has evolved MLG independently of the Poales. Equisetum and poalean MLGs share basic structural motifs but also exhibit clear-cut differences. Equisetum MLG is firmly wall-bound, and may tether neighbouring microfibrils. It is also suggested that MLG acts as a template for silica deposition, characteristic of grasses and horsetails.     

Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions

Antimicrobial hand soaps provide a greater bacterial reduction than nonantimicrobial soaps. However, the link between greater bacterial reduction and a reduction of disease has not been definitively demonstrated. Confounding factors, such as compliance, soap volume, and wash time, may all influence the outcomes of studies. The aim of this work was to examine the effects of wash time and soap volume on the relative activities and the subsequent transfer of bacteria to inanimate objects for antimicrobial and nonantimicrobial soaps. Increasing the wash time from 15 to 30 seconds increased reduction of Shigella flexneri from 2.90 to 3.33 log(10) counts (P = 0.086) for the antimicrobial soap, while nonantimicrobial soap achieved reductions of 1.72 and 1.67 log(10) counts (P > 0.6). Increasing soap volume increased bacterial reductions for both the antimicrobial and the nonantimicrobial soaps. When the soap volume was normalized based on weight (approximately 3 g), nonantimicrobial soap reduced Serratia marcescens by 1.08 log(10) counts, compared to the 3.83-log(10) reduction caused by the antimicrobial soap (P < 0.001). The transfer of Escherichia coli to plastic balls following a 15-second hand wash with antimicrobial soap resulted in a bacterial recovery of 2.49 log(10) counts, compared to the 4.22-log(10) (P < 0.001) bacterial recovery on balls handled by hands washed with nonantimicrobial soap. This indicates that nonantimicrobial soap was less active and that the effectiveness of antimicrobial soaps can be improved with longer wash time and greater soap volume. The transfer of bacteria to objects was significantly reduced due to greater reduction in bacteria following the use of antimicrobial soap.