Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

Loss of the SPHF homologue Slr1768 leads to a catastrophic failure in the maintenance of thylakoid membranes in Synechocystis sp. PCC 6803

Background

In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified.

Methodology/Principal Findings

We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Δslr1768 mutant with the wild-type, we found that Δslr1768 has a conditional phenotype; specifically under high light conditions (130 µmol m⁻² s⁻¹) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 µmol m⁻² s⁻¹) the phenotype is significantly reduced, with a growth rate similar to the wild-type and only a low frequency of cells with evident thylakoid disruption.

Conclusions/Significance

This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids.     

Genetic Variability in Stress Tolerance and Fitness among Natural Populations of Steinernema carpocapsae

Genetic variability in stress tolerance (heat, desiccation, and hypoxia) and fitness (virulence and reproduction potential) among natural populations of Steinernema carpocapsae was assessed by estimating phenotypic differences. Significant differences were observed in stress tolerance among populations. Populations isolated from North Carolina showed significantly more stress tolerance than those isolated from Ohio. Significant differences were also observed in populations isolated from the same locality. Survival of infective juveniles after exposure to 40°C for 2 h ranged from 37 to 82%. A threefold difference was observed in infective juvenile survival following exposure to osmotic desiccation or hypoxic condition. Several populations tested were superior to the most widely used strain (‘All’ strain) in stress tolerance traits, with one population KMD33, being superior to the ‘All’ strain in all traits. Fitness as expressed by virulence and reproductive potential differed significantly among populations but showed less variability than the stress tolerance traits. All populations tested had a reproductive potential greater than or similar to that of the ‘All’ strain and most of them caused >60% insect mortality of the wax moth larvae, Galleria mellonella. The high genetic variability in stress tolerance among natural populations suggests the feasibility of using selection for genetic improvement of these traits.     

Evolution of allometries in the worker caste of Dorylus army ants

We investigated the effect of local environment on the demography and population dynamics of arctic ground squirrels ( Spermophilus parryii plesius ) by comparing reproduction, survival, and population trends of squirrels living in low elevation boreal forest and high elevation alpine tundra sites in southwestern Yukon Territory, Canada. Contrary to the trend for most birds and mammals, reproduction was significantly lower at the lower elevation and females living at higher elevation did not delay the age at which they first reproduced. Even though survival in the boreal forest was lower in summer than in the alpine, it was higher over winter so annual adult female survival was similar between sites.
Sensitivity analysis of model parameters revealed that in the forest, population growth rate (λ) was most sensitive to small changes in adult active season survival whereas for the alpine population, λ was most sensitive to changes in juvenile winter survival. In their respective habitats, these parameters also showed high year to year variation and thus contributed greatly to the population trends observed. Even though ground squirrels persisted in the boreal forest, the measured demographic rates indicate the forest was sink habitat (λ<1) and may have relied on nearby grassy meadows for immigrants. In contrast, the alpine habitat maintained a ground squirrel population in the absence of immigration (λ=1).
The variation in demographic rates between ground squirrels living at high and low elevation may arise from phenotypic responses of squirrels to different habitat structure. Arctic ground squirrels rely on sight to detect predators from a safe distance, and the boreal forest, with its lower visibility and higher predator density, appears to be suboptimal habitat.     

Coupling Met to specific pathways results in distinct developmental outcomes.

Receptor tyrosine kinases (RTKs) mediate distinct biological responses by stimulating similar intracellular signaling pathways. Whether the specificity of the response is determined by qualitative or quantitative differences in signaling output is not known. We addressed this question in vivo by replacing the multifunctional docking sites of Met, the receptor for hepatocyte growth factor, with specific binding motifs for phosphatidylinositol-3 kinase, Src tyrosine kinase, or Grb2 (Met(2P), Met(2S), and Met(2G), respectively). All three mutants retained normal signaling through the multiadaptor Gab1, but differentially recruited specific effectors. While Met(2G) mice developed normally, Met(2P) and Met(2S) mice were loss-of-function mutants displaying different phenotypes and rescue of distinct tissues. These data indicate that RTK-mediated activation of specific signaling pathways is required to fulfill cell-specific functions in vivo.     

Ultrasound anatomy of the sciatic nerve of the thigh]

Ultrasound examinations of the sciatic nerve were performed using high-resolution transducers (7.5, 10 to 20 MHz) both in anatomical specimens and in healthy volunteers. The ultrasonographic anatomy (sono-anatomy) of the nerve, its course along the thigh and its echogenicity in comparison with muscles, tendons and adipose tissue were investigated in 10 isolated muscle/nerve preparations. In addition, the influence of the angle of the applied transducer on the various different tissues was evaluated. In the clinical part of the study, the sciatic nerve was identified ultrasonographically in both thighs of 50 sex-matched healthy volunteers aged between 2 and 76 years. The normal sciatic nerve presents as a tubular echogenic structure with parallel linear internal echoes in the longitudinal section, and as a punctiform moderately echoic structure in cross-section, with the perineurium producing bright boundary echoes. Varying the insonating angle of the transducer reduced echogenicity, but to a smaller degree than in muscles and tendons. Unequivocal identified of the sciatic nerve from the level of the gluteal fold to its bifurcation in the distal thigh was possible in all but one case. We conclude that the course of the sciatic nerve along the thigh can be reliably identified and imaged with high-resolution ultrasound.