Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo

Benthic pH gradients across a range of shelf sea sediment types linked to sediment characteristics and seasonal variability

This study used microelectrodes to record pH profiles in fresh shelf sea sediment cores collected across a range of different sediment types within the Celtic Sea. Spatial and temporal variability was captured during repeated measurements in 2014 and 2015. Concurrently recorded oxygen microelectrode profiles and other sedimentary parameters provide a detailed context for interpretation of the pH data. Clear differences in profiles were observed between sediment type, location and season. Notably, very steep pH gradients exist within the surface sediments (10–20 mm), where decreases greater than 0.5 pH units were observed. Steep gradients were particularly apparent in fine cohesive sediments, less so in permeable sandier matrices. We hypothesise that the gradients are likely caused by aerobic organic matter respiration close to the sediment–water interface or oxidation of reduced species at the base of the oxic zone (NH4+, Mn2+, Fe2+, S−). Statistical analysis suggests the variability in the depth of the pH minima is controlled spatially by the oxygen penetration depth, and seasonally by the input and remineralisation of deposited organic phytodetritus. Below the pH minima the observed pH remained consistently low to maximum electrode penetration (ca. 60 mm), indicating an absence of sub-oxic processes generating H+ or balanced removal processes within this layer. Thus, a climatology of sediment surface porewater pH is provided against which to examine biogeochemical processes. This enhances our understanding of benthic pH processes, particularly in the context of human impacts, seabed integrity, and future climate changes, providing vital information for modelling benthic response under future climate scenarios

Quantitative trait loci mapping for canine hip dysplasia and its related traits in UK Labrador Retrievers

BACKGROUND: Canine hip dysplasia (CHD) is characterised by a malformation of the hip joint, leading to osteoarthritis and lameness. Current breeding schemes against CHD have resulted in measurable but moderate responses. The application of marker-assisted selection, incorporating specific markers associated with the disease, or genomic selection, incorporating genome-wide markers, has the potential to dramatically improve results of breeding schemes. Our aims were to identify regions associated with hip dysplasia or its related traits using genome and chromosome-wide analysis, study the linkage disequilibrium (LD) in these regions and provide plausible gene candidates. This study is focused on the UK Labrador Retriever population, which has a high prevalence of the disease and participates in a recording program led by the British Veterinary Association (BVA) and The Kennel Club (KC). RESULTS: Two genome-wide and several chromosome-wide QTLs affecting CHD and its related traits were identified, indicating regions related to hip dysplasia. CONCLUSION: Consistent with previous studies, the genetic architecture of CHD appears to be based on many genes with small or moderate effect, suggesting that genomic selection rather than marker-assisted selection may be an appropriate strategy for reducing this disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-833) contains supplementary material, which is available to authorized users

A high density linkage map of the bovine genome

<p>Abstract</p> <p>Background</p> <p>Recent technological advances have made it possible to efficiently genotype large numbers of single nucleotide polymorphisms (SNPs) in livestock species, allowing the production of high-density linkage maps. Such maps can be used for quality control of other SNPs and for fine mapping of quantitative trait loci (QTL) via linkage disequilibrium (LD).</p> <p>Results</p> <p>A high-density bovine linkage map was constructed using three types of markers. The genotypic information was obtained from 294 microsatellites, three milk protein haplotypes and 6769 SNPs. The map was constructed by combining genetic (linkage) and physical information in an iterative mapping process. Markers were mapped to 3,155 unique positions; the 6,924 autosomal markers were mapped to 3,078 unique positions and the 123 non-pseudoautosomal and 19 pseudoautosomal sex chromosome markers were mapped to 62 and 15 unique positions, respectively. The linkage map had a total length of 3,249 cM. For the autosomes the average genetic distance between adjacent markers was 0.449 cM, the genetic distance between unique map positions was 1.01 cM and the average genetic distance (cM) per Mb was 1.25.</p> <p>Conclusion</p> <p>There is a high concordance between the order of the SNPs in our linkage map and their physical positions on the most recent bovine genome sequence assembly (Btau 4.0). The linkage maps provide support for fine mapping projects and LD studies in bovine populations. Additionally, the linkage map may help to resolve positions of unassigned portions of the bovine genome.</p

Human Immunodeficiency Virus type 1 Endocytic Trafficking Through Macrophage Bridging Conduits Facilitates Spread of Infection

Bridging conduits (BC) sustain communication and homeostasis between distant tethered cells. These are also exploited commonly for direct cell-to-cell transfer of microbial agents. Conduits efficiently spread infection, effectively, at speeds faster than fluid phase exchange while shielding the microbe against otherwise effective humoral immunity. Our laboratory has sought to uncover the mechanism(s) for these events for human immunodeficiency virus type one (HIV-1) infection. Indeed, in our prior works HIV-1 Env and Gag antigen and fluorescent virus tracking were shown sequestered into endoplasmic reticulum-Golgi organelles but the outcomes for spreading viral infection remained poorly defined. Herein, we show that HIV-1 specifically traffics through endocytic compartments contained within BC and directing such macrophage-to-macrophage viral transfers. Following clathrin-dependent viral entry, HIV-1 constituents bypass degradation by differential sorting from early to Rab11+ recycling endosomes and multivesicular bodies. Virus-containing endocytic viral cargoes propelled by myosin II through BC spread to neighboring uninfected cells. Disruption of endosomal motility with cytochalasin D, nocodasole and blebbistatin diminish intercellular viral spread. These data lead us to propose that HIV-1 hijacks macrophage endocytic and cytoskeletal machineries for high-speed cell-to-cell spread

Murine Leukemia Virus Spreading in Mice Impaired in the Biogenesis of Secretory Lysosomes and Ca2+-Regulated Exocytosis

Retroviruses have been observed to bud intracellularly into multivesicular bodies (MVB), in addition to the plasma membrane. Release from MVB is thought to occur by Ca(2+)-regulated fusion with the plasma membrane.To address the role of the MVB pathway in replication of the murine leukemia virus (MLV) we took advantage of mouse models for the Hermansky-Pudlak syndrome (HPS) and Griscelli syndrome. In humans, these disorders are characterized by hypopigmentation and immunological alterations that are caused by defects in the biogenesis and trafficking of MVBs and other lysosome related organelles. Neonatal mice for these disease models lacking functional AP-3, Rab27A and BLOC factors were infected with Moloney MLV and the spread of virus into bone marrow, spleen and thymus was monitored. We found a moderate reduction in MLV infection levels in most mutant mice, which differed by less than two-fold compared to wild-type mice. In vitro, MLV release form bone-marrow derived macrophages was slightly enhanced. Finally, we found no evidence for a Ca(2+)-regulated release pathway in vitro. Furthermore, MLV replication was only moderately affected in mice lacking Synaptotagmin VII, a Ca(2+)-sensor regulating lysosome fusion with the plasma membrane.Given that MLV spreading in mice depends on multiple rounds of replication even moderate reduction of virus release at the cellular level would accumulate and lead to a significant effect over time. Thus our in vivo and in vitro data collectively argue against an essential role for a MVB- and secretory lysosome-mediated pathway in the egress of MLV

Sampling, separation, and quantification of N-acyl homoserine lactones from marine intertidal sediments

N-acyl homoserine lactones (AHLs) are molecules produced by many Gram-negative bacteria as mediators of cell-cell signaling in a mechanism known as quorum sensing (QS). QS is widespread in marine bacteria regulating diverse processes, such as virulence or excretion of polymers that mediate biofilm formation. Associated eukaryotes, such as microalgae, respond to these cues as well, leading to an intricate signaling network. To date, only very few studies attempted to measure AHL concentrations in phototrophic microbial communities, which are hot spots for bacteria-bacteria as well as microalgae-bacteria interactions. AHL quantification in environmental samples is challenging and requires a robust and reproducible sampling strategy. However, knowing about AHL concentrations opens up multiple perspectives from answering fundamental ecological questions to deriving guidelines for manipulation and control of biofilms. Here, we present a method for sampling and AHL identification and quantification from marine intertidal sediments. The use of contact cores for sediment sampling ensures reproducible sample surface area and volume at each location. Flash-freezing of the samples with liquid nitrogen prevents enzymatic AHL degradation between sampling and extraction. After solvent extraction, samples were analyzed with an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method that allows to baseline-separate 16 different AHLs in less than 10 min. The sensitivity of the method is sufficient for detection and quantification of AHLs in environmental samples of less than 16 cm(3)

Recent and historical recombination in the admixed Norwegian Red cattle breed

<p>Abstract</p> <p>Background</p> <p>Comparison of recent patterns of recombination derived from linkage maps to historical patterns of recombination from linkage disequilibrium (LD) could help identify genomic regions affected by strong artificial selection, appearing as reduced recent recombination. Norwegian Red cattle (NRF) make an interesting case study for investigating these patterns as it is an admixed breed with an extensively recorded pedigree. NRF have been under strong artificial selection for traits such as milk and meat production, fertility and health.</p> <p>While measures of LD is also crucial for determining the number of markers required for association mapping studies, estimates of recombination rate can be used to assess quality of genomic assemblies.</p> <p>Results</p> <p>A dataset containing more than 17,000 genome-wide distributed SNPs and 2600 animals was used to assess recombination rates and LD in NRF. Although low LD measured by r²was observed in NRF relative to some of the breeds from which this breed originates, reports from breeds other than those assessed in this study have described more rapid decline in r²at short distances than what was found in NRF. Rate of decline in r²for NRF suggested that to obtain an expected r²between markers and a causal polymorphism of at least 0.5 for genome-wide association studies, approximately one SNP every 15 kb or a total of 200,000 SNPs would be required. For well known quantitative trait loci (QTLs) for milk production traits on <it>Bos Taurus </it>chromosomes 1, 6 and 20, map length based on historic recombination was greater than map length based on recent recombination in NRF.</p> <p>Further, positions for 130 previously unpositioned contigs from assembly of the bovine genome sequence (Btau_4.0) found using comparative sequence analysis were validated by linkage analysis, and 28% of these positions corresponded to extreme values of population recombination rate.</p> <p>Conclusion</p> <p>While LD is reduced in NRF compared to some of the breeds from which this admixed breed originated, it is elevated over short distances compared to some other cattle breeds. Genomic regions in NRF where map length based on historic recombination was greater than map length based on recent recombination coincided with some well known QTL regions for milk production traits.</p> <p>Linkage analysis in combination with comparative sequence analysis and detection of regions with extreme values of population recombination rate proved to be valuable for detecting problematic regions in the Btau_4.0 genome assembly.</p