Replication of HIV-1 subtype C V3–V5 region chimeras in primary peripheral blood T-lymphocytes (PBL)

<p><b>Copyright information:</b></p><p>Taken from "Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages"</p><p>http://www.virologyj.com/content/4/1/126</p><p>Virology Journal 2007;4():126-126.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2216014.</p><p></p> PBL (1 × 10cells/well) were stimulated with PHA and infected with equal amounts (reverse transcriptase counts) of subtype C V3–V5 region chimeras, subtype B V3 region chimeras, primary subtype B isolates, primary subtype C isolates and parental HIV-1. Cells were fed every 3 days with appropriate medium and virus production was measured in the culture supernatant by RT assay. The data are presented as cpm/ml ± SD on triplicate experiments and are based on PBL from five different donors

Replication of HIV-1 subtype C V3–V5 region chimeras in T-lymphocyte (A3

<p><b>Copyright information:</b></p><p>Taken from "Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages"</p><p>http://www.virologyj.com/content/4/1/126</p><p>Virology Journal 2007;4():126-126.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2216014.</p><p></p>01) cell line. A3.01 cells (1 × 10cells/well) were infected with equal amounts (RT counts) subtype C chimeras (171 to 1014), parental HIV-1and HIV-1. Virus production was measured by reverse transcriptase (RT) assay in culture media harvested every 3 days and the cells fed with appropriate media. The results are presented as cpm/ml ± SD of five separate triplicate experiments. The subtype C chimeras were unable to replicate in A3.01 cell line

Comparison of envelope (V3 to V5 regions) from subtype C chimeras with subtype B (X4 and R5) and C envelope sequences

<p><b>Copyright information:</b></p><p>Taken from "Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages"</p><p>http://www.virologyj.com/content/4/1/126</p><p>Virology Journal 2007;4():126-126.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2216014.</p><p></p> The sequences of subtype C chimeras used in this study were analyzed by performing multiple sequence alignment with parental clone HIV-1as a reference and HIV-1and known HIV-1 subtype C envelope regions for comparison. Subtype C chimeras are designated by numbers. Dots indicate a match with the reference sequence whereas substitutions are indicated by the single letter code for the changed amino acid. Gaps are shown as dashes. Structural elements of the envelope are indicated by spanning arrowheads and glycosylation sites are indicated by asterisk. Amino acid positions are indicated to denote the amino acid numbers of the complete envelope gp120

Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring

<div><p>Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8–12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on <i>Per1</i>, <i>Per 2</i> and <i>Clock</i> circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces food intake and energy expenditure, increases anxiety like behavior and disturbs sleep patterns but not circadian rhythm in adult male offspring.</p></div

Inhibiting EBV BART miRNA levels affect NKTCL growth rate without affecting cell viability.

<p>(<b>A</b>) SNK6 cells were transfected with antisense to the indicated EBV miRNAs and cell numbers counted every 24 hours for three days. Cell growth rate was calculated as difference in cell numbers between the 24 hour and 72 hour time point and compared with cells transfected with control Scramble miRNA. Data shown are the average ± SD from three independent experiments. (* represents p value of ≤0.05 in a paired t-test). (<b>B</b>) SNK6 transfected with antisense EBV miRNAs were analyzed for viability by Trypan blue exclusion in a Vi-CELL counter every 24 hours for three days. The data presented is the cell viability at 72 hours post-transfection and is the average ± SD from three independent experiments. (<b>C</b>) SNT16 cells were transfected with antisense to the indicated EBV miRNAs and cell growth rate analyzed as described above. Data shown are the average ± SD from three independent experiments. (* represents p value of <0.05 in a paired t-test).</p

Precursor EBV-BART9 miRNA increases LMP1 protein and mRNA levels in SNK6 cells.

<p>(<b>A</b>) SNK6 cells were transfected with precursor EBV-BART9 or control miRNA. The cells were collected 96 hours post-transfection and cell lysates prepared for immunoblot analysis. Quantification of immunoblots showed a ∼33% increase in LMP1 protein levels in cells transfected with EBV miRNA compared to control miRNA transfected cells when normalized to loading control. Data shown is a representative immunoblot from three independent experiments. (<b>B</b>) Total RNA was extracted from SNK6 cells transfected with precursor EBV-BART9 or control miRNA. Following cDNA synthesis, LMP1 mRNA levels were analyzed by Q-RT-PCR. Data presented is the average ± SD from three independent experiments. (* represents p value of <0.05 in a paired t-test).</p

Immunoblot and Q-RT-PCR analysis of LMP1 expression in SNK6 following inhibition of EBV-BART9 miRNA.

<p>(<b>A</b>) SNK6 cells were transfected with anti-EBV-BART9 miRNA or Scramble control miRNA and cell lysates prepared 96 hours post-transfection. LMP1 protein expression was analyzed in immunoblots. When compared to cells transfected with control miRNA and normalized to β-actin loading control, quantification of immunoblots showed that BART9 inhibition reduced LMP1 protein levels by ∼50%. (<b>B</b>) SNK6 cells were transfected with control or anti-EBV-BART9 miRNA and samples collected every 24 hours in a time-course experiment. Cell lysates were prepared and immunoblot analysis carried out to determine LMP1 expression. Quantification of LMP1 levels using Image J as described above showed that LMP1 protein levels are reduced only at later time-point. (<b>C</b>) SNK6 cells were transfected with either anti-EBV-BART9 or control miRNA and cells collected 96 hours post-transfection. Total RNA was extracted and cDNA synthesized using iScript cDNA synthesis kit. Using LMP1 specific primers, Q-PCR was carried out and data analyzed using the ΔΔCt method. Data shown is the average ± SD from three independent experiments. (** represents p value of <0.005 in a paired t-test).</p

Characterization of NK T cell lymphoma cell lines by immunoblot analysis.

<p>Cell lysates were prepared from two NK-like (SNK6, SNK10) and three T cell-like (SNT8, SNT15 and SNT16) NKTCLs. Immunoblots were performed to analyze the expression of the indicated EBV latent and lytic proteins. EBV negative DG75 cells were used as a negative control and EBV positive MHK cells, which maintain Latency III gene expression and express all of the latent proteins, were used as a positive control.</p

Increasing EBV-BART9 miRNA level has a subtle effect on SNK6 growth rate.

<p>(<b>A</b>) Precursor EBV-BART9 miRNA or control miRNA were transfected into SNK6 cells and samples collected every 24 hours for three days. Growth rate of SNK6 cells was determined by calculating cell numbers. When normalized to cell numbers in control miRNA transfected cells, there was ∼8% reduction in SNK6 growth rate. The data shown is the average ± SD from three independent experiments. (<b>B</b>) In the experiments described above, SNK6 were analyzed for viability by Trypan blue exclusion in a Vi-CELL counter at every time point. The data shown is the cell viability at 72 hours post-transfection and is the average ± SD from three independent experiments.</p

Body composition.

<p>(A): Body weight; (B) Body fat as percent of total body mass in male offspring (8–12 weeks age) from dams exposed to either control or MLP diet (n = 7 each). Bars are mean ± SEM and P<0.05 statistically significant by student t-test.</p