GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

GABA_A receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABA_A receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABA_A receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.     

Baseline Natural Killer and T Cell Populations Correlation with Virologic Outcome after Regimen Simplification to Atazanavir/Ritonavir Alone (ACTG 5201)

Objectives

Simplified maintenance therapy with ritonavir-boosted atazanavir (ATV/r) provides an alternative treatment option for HIV-1 infection that spares nucleoside analogs (NRTI) for future use and decreased toxicity. We hypothesized that the level of immune activation (IA) and recovery of lymphocyte populations could influence virologic outcomes after regimen simplification.

Methods

Thirty-four participants with virologic suppression ≥48 weeks on antiretroviral therapy (2 NRTI plus protease inhibitor) were switched to ATV/r alone in the context of the ACTG 5201 clinical trial. Flow cytometric analyses were performed on PBMC isolated from 25 patients with available samples, of which 24 had lymphocyte recovery sufficient for this study. Assessments included enumeration of T-cells (CD4/CD8), natural killer (NK) (CD3⁺CD56⁺CD16⁺) cells and cell-associated markers (HLA-DR, CD''s 38/69/94/95/158/279).

Results

Eight of the 24 patients had at least one plasma HIV-1 RNA level (VL) >50 copies/mL during the study. NK cell levels below the group median of 7.1% at study entry were associated with development of VL >50 copies/mL following simplification by regression and survival analyses (p = 0.043 and 0.023), with an odds ratio of 10.3 (95% CI: 1.92–55.3). Simplification was associated with transient increases in naïve and CD25⁺ CD4⁺ T-cells, and had no impact on IA levels.

Conclusions

Lower NK cell levels prior to regimen simplification were predictive of virologic rebound after discontinuation of nucleoside analogs. Regimen simplification did not have a sustained impact on markers of IA or T lymphocyte populations in 48 weeks of clinical monitoring.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00084019","term_id":"NCT00084019"}}NCT00084019     

Neither the HIV Protease Inhibitor Lopinavir-Ritonavir nor the Antimicrobial Trimethoprim-Sulfamethoxazole Prevent Malaria Relapse in Plasmodium cynomolgi-Infected Non-Human Primates

Plasmodium vivax malaria causes significant morbidity and mortality worldwide, and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax relapses. HIV and P. vivax malaria geographically overlap in many areas of the world, including South America and Asia. Despite the increasing body of knowledge regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no data regarding the effects of these treatments on P. vivax''s hypnozoite form and clinical relapses of malaria. We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remains untested. Separately, although other antifolates have been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole, commonly used in HIV infection and exposure management, has not been evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we investigated the antimalarial activity of these drugs on Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested. Because HIV and malaria geographically overlap, and more patients are being managed for HIV infection and exposure, understanding HIV drug impact on malaria infection is important.     

Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site

An unusually thick (~1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to “Ferromicrobium acidophilus,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.     

Effect of clinician information sessions on diagnostic testing for Chagas disease

BackgroundChagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.Methodology/Principal findingsWe conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).Conclusion/SignificanceIn this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.     

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge <Emphasis Type="Italic">Xestospongia testudinaria</Emphasis>, with Characterization of Stability in Human Serum

Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC₅₀ 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC₅₀ 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2?±?0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.     

Hydrophobic mannosides act as acceptors for trypanosome {alpha}-mannosyltransferases

A series of hydrophobic mannosides were synthesized and testedfor their ability to act as acceptor substrates for mannosyltransferasesin a Trypanosoma brucei cell-free system. The thiooctyl -mannosidesand octyl -mannosides all accepted single mannose residues in-linkage, as judged by thin layer chromatography of the productsbefore and after jack bean -mannosidase digestion. The mannosylationreactions were inhibited by amphomycin, suggesting that theimmediate donor was dolicholphosphate-mannose (Dol-P-Man) inall cases. The transferred -mannose residues were shown to beboth 1-2 and 1-6 linked by Aspergillus phoenicis -mannosidaseand acetolysis treatments, respectively. These data suggestthat the compounds can act as acceptor substrates for the Dol-P-Mandependent 1-2 and 1-6 mannosyltransferases of the GPI biosyntheticpathway and/or the dolichol-cycle of protein N-glycosylation.One of the compounds, Man1-6Man1-O-(CH₂)₇CH₃, inhibited endogenousGPI biosynthesis in the cell-free system, suggesting that itcould be a substrate for the trypanosome Dol-P-Man:Man₂GlcN-Pl1-2 mannosyltransferase. dolichol glycosylphosphatidylinositol mannosyltransferase trypanosome