Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.     

A novel series of benzimidazole NR2B-selective NMDA receptor antagonists

A series of novel benzimidazoles are discussed as NR2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists. High throughput screening (HTS) efforts identified a number of potent and selective NR2B antagonists such as 1. Exploration of the substituents around the core of this template identified a number of compounds with high potency for NR2B (pIC(50) >7) and good selectivity against the NR2A subunit (pIC(50) <4.3) as defined by FLIPR-Ca(2+) and radioligand binding studies. These agents offer potential for the development of therapeutics for a range of nervous system disorders including chronic pain, neurodegeneration, migraine and major depression.     

Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.     

A multi-locus phylogeny reveals a complex pattern of diversification related to climate and habitat heterogeneity in southern African white-eyes

The recent, rapid radiation of Zosteropidae, coupled with their high levels of colonizing ability and phenotypic diversity, makes species delimitation within this family problematic. Given these problems, challenges to establish the mechanisms driving diversity and speciation within this group have arisen. Four morphologically distinct southern African Zosterops taxa, with a contentious taxonomic past, provide such a challenge. Here, supplemented with morphological and environmental analytical techniques, a combination of mitochondrial and nuclear markers were analyzed using Bayesian and Likelihood methods to determine their speciation patterns and to establish the phylogenetic relationships of these four morphologically diverse southern African Zosterops taxa. Nearly all individuals were phenotypically diagnosable, even those individuals collected in areas of contact between taxa. Localities where two or more taxa co-occur appear to possess intermediate environmental characteristics. Initial Bayesian and Likelihood mitochondrial DNA analyses and Bayesian structure analyses of the combined nuclear markers indicated levels of hybridization in areas of sympatry. A combined mtDNA and nuclear DNA analysis and a species tree analysis (with hybrids excluded) placed Z. pallidus as sister to the other southern African taxa, with Z. senegalensis the putative sister taxon to a clade comprising Z. capensis and Z. virens. The grouping of taxon-specific sampling localities and the apparent intermediate nature of birds from areas of sympatry points toward an influence of habitat type and the associated climatic conditions in driving Zosterops diversification in southern Africa.     

Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones

Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.     

Chronic glutamine supplementation increases nasal but not salivary IgA during 9 days of interval training.

Oral glutamine supplementation during and after exercise abolishes exercise-induced decreases in plasma glutamine concentration but does not affect secretory IgA (sIgA) salivary output. Whether chronic glutamine supplementation during high-intensity interval training influences salivary and nasal sIgA concentration is unknown. The purpose of this study was examine the effects of chronic glutamine supplementation on sIgA during intense running training. Runners (n = 13, body mass 69.9 +/- 2.8 kg, peak whole body oxygen uptake 55.5 +/- 2 ml.kg(-1).min(-1), age 29.1 +/- 2.8 yr) participated in twice-daily interval training for 9-9.5 days, followed by recovery (5-7 days). Oral glutamine supplement (0.1 g/kg) or placebo was given four times daily for the first 14 days. After an overnight fast, venous blood, nasal washes, and stimulated saliva were collected at baseline (T1), midtraining (T2), posttraining (T3), and after recovery (T4). Mood states were assessed by using Profile of Mood States (POMS) inventories. We found that glutamine concentration in resting subjects decreased from T1 to T4 (P < 0.05) and was not altered by supplementation. Salivary IgA concentration and output were unchanged by training or supplementation. Mean nasal IgA across the study period was greater in runners receiving glutamine (264.7 +/- 35.0 microg/mg protein) vs. placebo (172.4 +/- 33.7 microg/mg protein; P < 0.05). POMS analyses indicated that vigor was lower at T3 vs. T1 (P < 0.05) and fatigue was higher at T2 vs. T1 and T4 (P < 0.05). We conclude that chronic glutamine supplementation during interval training results in higher nasal IgA than placebo but does not affect salivary IgA concentration or output.     

Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNA<Superscript><Emphasis Type="Italic">Lys3</Emphasis></Superscript> but promotes intracellular budding of virus-like particles

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA ^Lys3 . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA ^Lys3 . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.     

Trehalose and dry dipalmitoylphosphatidylcholine revisited

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum T_m of 24°C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105–112°C for dry pure DPPC and of 42°C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.