The fine-scale and complex architecture of human copy-number variation

Despite considerable excitement over the potential functional significance of copy-number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at approximately 1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1020 of 1153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of currently known common human CNVs is likely smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with interindividual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously uncharacterized complexity.     

Host location in flow by larvae of the symbiotic barnacle Trevathana dentata using odour-gated rheotaxis

The detection and location of specific organisms in the aquatic environment, whether they are mates, prey or settlement sites, are two of the most important challenges facing aquatic animals. Large marine invertebrates such as lobsters have been found to locate specific organisms by navigating in the plume of chemicals emitted by the target. However, active plume tracking in flow by small organisms such as marine larvae has received little scientific attention. Here, we present results from a study examining host location in flow by nauplius larvae of the barnacle Trevathana dentata, which inhabits the stony reef coral Cyphastrea chalcidicum. The experiments included analysis of larval motion in an annular flume under four conditions: (i) still water, (ii) in flow, (iii) in still water with waterborne host metabolites and (iv) in flow with host metabolites. Our results show that T. dentata nauplii are unable to locate their target organism in still water using chemotaxis, but are capable of efficient host location in flow using odour-gated rheotaxis. This technique may enable host location by earlier, less-developed larval stages.     

Alginate coating of Xenopus laevis embryos

Xenopus laevis eggs were coated, immediately after squeeze-stripping and fertilization, with a thin layer ( approximately 50 microm) of film based on one of three different types of alginates which varied in their mannuronic/guluronic acid ratio. The alginate was cross-linked with either Ca or Ba ions at three different concentrations. The developmental, survival, and hatching of these embryos and the swelling of their natural jelly coats or hydrocolloid coatings were studied over 7 days, while embryos were maintained in flowing aerated water at a ratio of 85 mL per embryo or at a very diminished ratio of 1.6 mL of sterile or nonsterile MMR solution per embryo. All experiments were conducted in triplicate at 20+/-1 degrees C. Oxygen was monitored continuously. Mineral content was determined in the alginate-jelly coat and within the embryos over time. The coating conferred major advantages when the ratio between the embryos and the surrounding medium was at a minimum under nonsterile conditions, perhaps as a result of the film's resistance to diffusion. In the studied systems, the coating seemed to postpone embryo hatching to a more developed stage. In addition, the coating served as a barrier to microbial contamination and thus improved survival prospects.     

Diversity of Bacterial Biofilm Communities on Sprinklers from Dairy Farm Cooling Systems in Israel

On dairy farms in hot climates worldwide, cows suffer from heat stress, which is alleviated by the use of water cooling systems. Sprinklers and showerheads are known to support the development of microbial biofilms, which can be a source of infection by pathogenic microorganisms. The aim of this study was to investigate the presence of microbial biofilms in dairy cooling systems, and to analyze their population compositions using culture-independent technique, 16S rRNA gene sequencing. Biofilm samples were collected on eight dairy farms from 40 sprinklers and the microbial constituents were identified by deep sequencing of the 16S rRNA gene. A total of 9,374 operational taxonomic units (OTUs) was obtained from all samples. The mean richness of the samples was 465 ± 268 OTUs which were classified into 26 different phyla; 76% of the reads belonged to only three phyla: Proteobacteria, Actinobacteria and Firmicutes. Although the most prevalent OTUs (Paracoccus, Methyloversatilis, Brevundimonas, Porphyrobacter, Gp4, Mycobacterium, Hyphomicrobium, Corynebacterium and Clostridium) were shared by all farms, each farm formed a unique microbial pattern. Some known potential human and livestock pathogens were found to be closely related to the OTUs found in this study. This work demonstrates the presence of biofilm in dairy cooling systems which may potentially serve as a live source for microbial pathogens.     

Nephroprotective Effect of Heparanase in Experimental Nephrotic Syndrome

Background

Heparanase, an endoglycosidase that cleaves heparan sulfate (HS), is involved in various biologic processes. Recently, an association between heparanase and glomerular injury was suggested. The present study examines the involvement of heparanase in the pathogenesis of Adriamycin-induced nephrotic syndrome (ADR-NS) in a mouse model.

Methods

BALB/c wild-type (wt) mice and heparanase overexpressing transgenic mice (hpa-TG) were tail-vein injected with either Adriamycin (ADR, 10 mg/kg) or vehicle. Albuminuria was investigated at days 0, 7, and 14 thereafter. Mice were sacrificed at day 15, and kidneys were harvested for various analyses: structure and ultrastructure alterations, podocyte proteins expression, and heparanase enzymatic activity.

Results

ADR-injected wt mice developed severe albuminuria, while ADR-hpa-TG mice showed only a mild elevation in urinary albumin excretion. In parallel, light microscopy of stained cross sections of kidneys from ADR-injected wt mice, but not hpa-TG mice, showed mild to severe glomerular and tubular damage. Western blot and immunofluorescence analyses revealed significant reduction in nephrin and podocin protein expression in ADR-wt mice, but not in ADR-hpa-TG mice. These results were substantiated by electron-microscopy findings showing massive foot process effacement in injected ADR-wt mice, in contrast to largely preserved integrity of podocyte architecture in ADR-hpa-TG mice.

Conclusions

Our results suggest that heparanase may play a nephroprotective role in ADR-NS, most likely independently of HS degradation. Moreover, hpa-TG mice comprise an invaluable in vivo platform to investigate the interplay between heparanase and glomerular injury.     

A Population-Structured HIV Epidemic in Israel: Roles of Risk and Ethnicity

Background

HIV in Israel started with a subtype-B epidemic among men who have sex with men, followed in the 1980s and 1990s by introductions of subtype C from Ethiopia (predominantly acquired by heterosexual transmission) and subtype A from the former Soviet Union (FSU, most often acquired by intravenous drug use). The epidemic matured over the last 15 years without additional large influx of exogenous infections. Between 2005 and 2013 the number of infected men who have sex with men (MSM) increased 2.9-fold, compared to 1.6-fold and 1.3-fold for intravenous drug users (IVDU) and Ethiopian-origin residents. Understanding contemporary spread is essential for effective public health planning.

Methods

We analyzed demographic and virologic data from 1,427 HIV-infected individuals diagnosed with HIV-I during 1998–2012. HIV phylogenies were reconstructed with maximum-likelihood and Bayesian methods.

Results

Subtype-B viruses, but not A or C, demonstrated a striking number of large clusters with common ancestors having posterior probability ≥0.95, including some suggesting presence of transmission networks. Transmitted drug resistance was highest in subtype B (13%). MSM represented a frequent risk factor in cross-ethnic transmission, demonstrated by the presence of Israeli-born with non-B virus infections and FSU immigrants with non-A subtypes.

Conclusions

Reconstructed phylogenetic trees demonstrated substantial grouping in subtype B, but not in non-MSM subtype-A or in subtype-C, reflecting differences in transmission dynamics linked to HIV transmission categories. Cross-ethnic spread occurred through multiple independent introductions, with MSM playing a prevalent role in the transmission of the virus. Such data provide a baseline to track epidemic trends and will be useful in informing and quantifying efforts to reduce HIV transmission.     

Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells

Osteopontin (OPN), a major component of the bone matrix, is expressed at different stages of bone formation. To determine possible relationships between OPN expression and stages of osteogenic cell differentiation, we have performed single cell analyses of intracellular OPN in early (proliferating), subconfluent (differentiating), and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC) using a combination of flow cytometry and confocal microscopy. At each culture stage, a high proportion (60–98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a small proportion of OPN-ve cells which were characterized by their small size, low granularity, high proliferative capacity, and enhanced osteogenic potential. The OPN+ve cells displayed two distinct patterns of intracellular immunostaining: a perinuclear distribution typical of secreted proteins and a perimembrane distribution in which patches of OPN were concentrated at the cell surface. Perimembranous staining predominated in migrant cells, which contained greater than tenfold higher levels of OPN than nonmigrant cells as separated in a Boyden chamber. When cell proliferation was high (day 2), most cells were OPN+ve. At all culture stages the intensity of OPN staining was increased as cells progressed through the cell cycle. As cells differentiated and started to form matrix (days 4 and 6), the mean cell expression of OPN was also increased (fourfold), independent of changes in total cell protein. However, despite the association of OPN with osteogenic cells, we were surprised to find that a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these cell populations was confirmed by RT-PCR, and a strong correlation was observed between the quantitative flow cytometry data and Western blot analysis of cell extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies, therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: small OPN-ve cell populations enriched in osteogenic precursors, differentiating osteogenic cells that synthesize and secrete OPN, and migrating stromal cells characterized by a perimembranous OPN staining pattern. J Cell Physiol 170:88–100, 1997 © 1997 Wiley-Liss, Inc.     

A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation‐based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin‐ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin‐induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full‐genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.