Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual''s level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.¹ These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,² lipid-^{3, 4} and RAGE-induced⁵ inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.     

Shc depletion stimulates brown fat activity in vivo and in vitro

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole‐body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well‐established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc‐deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high‐fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin‐dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid‐oxidative enzymes was observed in ShcKO BAT. Levels of brown fat‐specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice.     

Autonomic Cardiovascular Responses in Acclimatized Lowlanders on Prolonged Stay at High Altitude: A Longitudinal Follow Up Study

Acute exposure to hypobaric hypoxia at high altitude is reported to cause sympathetic dominance that may contribute to the pathophysiology of high altitude illnesses. The effect of prolonged stay at high altitude on autonomic functions, however, remains to be explored. Thus, the present study aimed at investigating the effect of high altitude on autonomic neural control of cardiovascular responses by monitoring heart rate variability (HRV) during chronic hypobaric hypoxia. Baseline electrocardiography (ECG) data was acquired from the volunteers at mean sea level (MSL) (<250 m) in Rajasthan. Following induction of the study population to high altitude (4500–4800 m) in Ladakh region, ECG data was acquired from the volunteers after 6 months (ALL 6) and 18 months of induction (ALL 18). Out of 159 volunteers who underwent complete investigation during acquisition of baseline data, we have only included the data of 104 volunteers who constantly stayed at high altitude for 18 months to complete the final follow up after 18 months. HRV parameters, physiological indices and biochemical changes in serum were investigated. Our results show sympathetic hyperactivation along with compromise in parasympathetic activity in ALL 6 and ALL 18 when compared to baseline data. Reduction of sympathetic activity and increased parasympathetic response was however observed in ALL 18 when compared to ALL 6. Our findings suggest that autonomic response is regulated by two distinct mechanisms in the ALL 6 and ALL 18. While the autonomic alterations in the ALL 6 group could be attributed to increased sympathetic activity resulting from increased plasma catecholamine concentration, the sympathetic activity in ALL 18 group is associated with increased concentration of serum coronary risk factors and elevated homocysteine. These findings have important clinical implications in assessment of susceptibility to cardio-vascular risks in acclimatized lowlanders staying for prolonged duration at high altitude.     

Comparison of the CopB systems of plasmids R1 and Co1V2-K94: A single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid CoIV2-K94

Summary We have isolated a deletion mutation and a point mutation in the copB gene of the replication region Repl of the IncFI plasmid Co1V2-K94. Subsequently, this copB gene with and without point mutation was cloned and sequenced, and the point mutation was mapped in the coding region of copB with a change of one amino acid from arginine to serine. Furthermore, this copB mutant had an approximately 10-fold increase in copy number. The CopB-phenotype of Co1V2-K94 could be complemented in trans by the copB gene of coresident IncFII plasmids such as R1 and R538, but not R100, suggesting that ColV2-K94 and R1 or R538 contain the same copB allele.     

Development of a polyclonal antibody-based AC-ELISA and its comparison with PCR for diagnosis of canine parvovirus infection

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 10^2.8 TCID₅₀/mL whereas PCR sensitivity was 10^0.8 TCID₅₀/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

Contrasting effects of natural selection on human and chimpanzee CC chemokine receptor 5

Human immunodeficiency virus type 1 (HIV-1) evolved via cross-species transmission of simian immunodeficiency virus (SIVcpz) from chimpanzees (Pan troglodytes). Chimpanzees, like humans, are susceptible to infection by HIV-1. However, unlike humans, infected chimpanzees seldom develop immunodeficiency when infected with SIVcpz or HIV-1. SIVcpz and most strains of HIV-1 require the cell-surface receptor CC chemokine receptor 5 (CCR5) to infect specific leukocyte subsets, and, subsequent to infection, the level of CCR5 expression influences the amount of HIV-1 entry and the rate of HIV-1 replication. Evidence that variants in the 5' cis-regulatory region of CCR5 (5'CCR5) affect disease progression in humans suggests that variation in CCR5 might also influence the response of chimpanzees to HIV-1/SIVcpz. To determine whether patterns of genetic variation at 5'CCR5 in chimpanzees are similar to those in humans, we analyzed patterns of DNA sequence variation in 37 wild-born chimpanzees (26 P. t. verus, 9 P. t. troglodytes, and 2 P. t. schweinfurthii), along with previously published 5'CCR5 data from 112 humans and 50 noncoding regions in the human and chimpanzee genomes. These analyses revealed that patterns of variation in 5'CCR5 differ dramatically between chimpanzees and humans. In chimpanzees, 5'CCR5 was less diverse than 80% of noncoding regions and was characterized by an excess of rare variants. In humans, 5'CCR5 was more diverse than 90% of noncoding regions and had an excess of common variants. Under a wide range of demographic histories, these patterns suggest that, whereas human 5'CCR5 has been subject to balancing selection, chimpanzee 5'CCR5 has been influenced by a selective sweep. This result suggests that chimpanzee 5'CCR5 might harbor or be linked to functional variants that influence chimpanzee resistance to disease caused by SIVcpz/HIV-1.