Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT _m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K_a = 16.2±0.6×10⁷ M⁻¹ where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K_a = 3.1±0.4×10⁷ M⁻¹) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

Uniform selection as a primary force reducing population genetic differentiation of cavitation resistance across a species range

Background

Cavitation resistance to water stress-induced embolism determines plant survival during drought. This adaptive trait has been described as highly variable in a wide range of tree species, but little is known about the extent of genetic and phenotypic variability within species. This information is essential to our understanding of the evolutionary forces that have shaped this trait, and for evaluation of its inclusion in breeding programs.

Methodology

We assessed cavitation resistance (P ₅₀), growth and carbon isotope composition in six Pinus pinaster populations in a provenance and progeny trial. We estimated the heritability of cavitation resistance and compared the distribution of neutral markers (F _ST) and quantitative genetic differentiation (Q _ST), for retrospective identification of the evolutionary forces acting on these traits.

Results/Discussion

In contrast to growth and carbon isotope composition, no population differentiation was found for cavitation resistance. Heritability was higher than for the other traits, with a low additive genetic variance (h² _ns = 0.43±0.18, CV_A = 4.4%). Q _ST was significantly lower than F _ST, indicating uniform selection for P ₅₀, rather than genetic drift. Putative mechanisms underlying Q_STST are discussed.     

HIV-1 disease progression is associated with bile-salt stimulated lipase (BSSL) gene polymorphism

Background

DC-SIGN expressed by dendritic cells captures HIV-1 resulting in trans-infection of CD4⁺ T-lymphocytes. However, BSSL (bile-salt stimulated lipase) binding to DC-SIGN interferes with HIV-1 capture. DC-SIGN binding properties of BSSL associate with the polymorphic repeated motif of BSSL exon 11. Furthermore, BSSL binds to HIV-1 co-receptor CXCR4. We hypothesized that BSSL modulates HIV-1 disease progression and emergence of CXCR4 using HIV-1 (X4) variants.

Results

The relation between BSSL genotype and HIV-1 disease progression and emergence of X4 variants was studied using Kaplan Meier and multivariate Cox proportional hazard analysis in a cohort of HIV-1 infected men having sex with men (n = 334, with n = 130 seroconverters). We analyzed the association of BSSL genotype with set-point viral load and CD4 cell count, both pre-infection and post-infection at viral set-point. The number of repeats in BSSL exon 11 were highly variable ranging from 10 to 18 in seropositive individuals and from 5–17 in HRSN with 16 repeats being dominant (>80% carry at least one allele with 16 repeats). We defined 16 to 18 repeats as high (H) and less than 16 repeats as low (L) repeat numbers. Homozygosity for the high (H) repeat number BSSL genotype (HH) correlated with high CD4 cell numbers prior to infection (p = 0.007). In HIV-1 patients, delayed disease progression was linked to the HH BSSL genotype (RH = 0.462 CI = 0.282–0.757, p = 0.002) as was delayed emergence of X4 variants (RH = 0.525, 95% CI = 0.290–0.953, p = 0.034). The LH BSSL genotype, previously found to be associated with enhanced DC-SIGN binding of human milk, was identified to correlate with accelerated disease progression in our cohort of HIV-1 infected MSM (RH = 0.517, 95% CI = 0.328–0.818, p = 0.005).

Conclusion

We identify BSSL as a marker for HIV-1 disease progression and emergence of X4 variants. Additionally, we identified a relation between BSSL genotype and CD4 cell counts prior to infection.     

α1Proteinase inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease

Background

The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE_CS), it acts not as a proteinase, but as a receptor for α₁proteinase inhibitor (α₁PI, α₁antitrypsin, SerpinA1). Binding of α₁PI to HLE_CS forms a motogenic complex. We previously demonstrated that α₁PI deficiency attends HIV-1 disease and that α₁PI augmentation produces increased numbers of immunocompetent circulating CD4⁺ lymphocytes. Herein we investigated the mechanism underlying the α₁PI deficiency that attends HIV-1 infection.

Methods and Findings

Active α₁PI in HIV-1 subjects (median 17 µM, n = 35) was significantly below normal (median 36 µM, p<0.001, n = 30). In HIV-1 uninfected subjects, CD4⁺ lymphocytes were correlated with the combined factors α₁PI, HLE_CS ⁺ lymphocytes, and CXCR4⁺ lymphocytes (r² = 0.91, p<0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with >220 CD4 cells/µl, CD4⁺ lymphocytes were correlated solely with active α₁PI (r² = 0.93, p<0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human α₁PI. Chimpanzee α₁PI differs from human α₁PI by a single amino acid within the 3F5-binding epitope. Unlike human α₁PI, chimpanzee α₁PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4⁺ lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-α₁PI immune complexes correlated with decreased CD4⁺ lymphocytes in HIV-1 subjects.

Conclusions

This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.     

Genome-wide association study identifies single nucleotide polymorphism in DYRK1A associated with replication of HIV-1 in monocyte-derived macrophages

Background

HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages.

Methodology/Principal Findings

Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16×10⁻⁵). While the association was not genome-wide significant (p<1×10⁻⁷), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84×10⁻⁶). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048).

Conclusions/Significance

These findings suggest that the kinase DYRK1A is involved in the replication of HIV-1, in vitro in macrophages as well as in vivo.     

Evidence for more than one Parkinson's disease-associated variant within the HLA region

Parkinson''s disease (PD) was recently found to be associated with HLA in a genome-wide association study (GWAS). Follow-up GWAS''s replicated the PD-HLA association but their top hits differ. Do the different hits tag the same locus or is there more than one PD-associated variant within HLA? We show that the top GWAS hits are not correlated with each other (0.00≤r²≤0.15). Using our GWAS (2000 cases, 1986 controls) we conducted step-wise conditional analysis on 107 SNPs with P<10⁻³ for PD-association; 103 dropped-out, four remained significant. Each SNP, when conditioned on the other three, yielded P_SNP1 = 5×10⁻⁴, P_SNP2 = 5×10⁻⁴, P_SNP3 = 4×10⁻³ and P_SNP4 = 0.025. The four SNPs were not correlated (0.01≤r²≤0.20). Haplotype analysis (excluding rare SNP2) revealed increasing PD risk with increasing risk alleles from OR = 1.27, P = 5×10⁻³ for one risk allele to OR = 1.65, P = 4×10⁻⁸ for three. Using additional 843 cases and 856 controls we replicated the independent effects of SNP1 (P_{conditioned-on-SNP4} = 0.04) and SNP4 (P_{conditioned-on-SNP1} = 0.04); SNP2 and SNP3 could not be replicated. In pooled GWAS and replication, SNP1 had OR_{conditioned-on-SNP4} = 1.23, P_{conditioned-on-SNP4} = 6×10⁻⁷; SNP4 had OR_{conditioned-on-SNP1} = 1.18, P_{conditioned-on-SNP1} = 3×10⁻³; and the haplotype with both risk alleles had OR = 1.48, P = 2×10⁻¹². Genotypic OR increased with the number of risk alleles an individual possessed up to OR = 1.94, P = 2×10⁻¹¹ for individuals who were homozygous for the risk allele at both SNP1 and SNP4. SNP1 is a variant in HLA-DRA and is associated with HLA-DRA, DRB5 and DQA2 gene expression. SNP4 is correlated (r² = 0.95) with variants that are associated with HLA-DQA2 expression, and with the top HLA SNP from the IPDGC GWAS (r² = 0.60). Our findings suggest more than one PD-HLA association; either different alleles of the same gene, or separate loci.     

Lack of evidence for changing virulence of HIV-1 in North America

Background

Several long-term cohort studies and in-vitro fitness assays have resulted in inconsistent reports on changes in HIV-1 virulence, including reports of decreasing, stable, and increasing virulence over the course of the AIDS pandemic. We tested the hypothesis of changing HIV-1 virulence by examining trends in prognostic clinical markers of disease progression from 1984 to 2005 among nearly 400 antiretroviral-naïve participants in the United States Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV infection in men who have sex with men (MSM).

Methodology/Principal Findings

Because clinical AIDS endpoints could not be used (due to antiretroviral therapies and prophylaxis), three prognostic markers of disease progression were used as proxies for HIV-1 virulence: plasma viral RNA load and CD4+ T cell count at “set point” (between ∼9 and ∼15 months after seroconversion), and rate of CD4 cell decline within three years after seroconversion. We performed multivariate analyses of the association between these markers and seroconversion year, with covariates including MACS site, race/ethnic group, seroconversion age, and CCR5Δ32 status. No statistically significant association was found between year of seroconversion and “set point” plasma viral load (at ∼9 months after seroconversion: slope = −0.004 log₁₀ copies/mL/year, p = 0.76; at ∼15 months: slope = −0.005 log₁₀ copies/mL/year, p = 0.71), CD4 cell count after seroconversion (at ∼9 months: slope = −0.112 cells/µL/year, p = 0.22; at ∼15 months: slope = −0.047 cells/µL/year, p = 0.64), or rate of CD4 cell decline over the first three years after seroconversion (slope = −0.010 cells/ul/yr², p = 0.88).

Conclusions/Significance

The lack of significant trends from 1984 to 2005 in these prognostic markers of HIV disease progression suggests no major change in HIV-1 virulence over the AIDS pandemic in MSM in the US.     

The fate of assimilated carbon during drought: impacts on respiration in Amazon rainforests

Interannual variations in CO₂ exchange across Amazonia, as deduced from atmospheric inversions, correlate with El Niño occurrence. They are thought to result from changes in net ecosystem exchange and fire incidence that are both related to drought intensity. Alterations to net ecosystem production (NEP) are caused by changes in gross primary production (GPP) and ecosystem respiration (R_eco). Here, we analyse observations of the components of R_eco (leaves, live and dead woody tissue, and soil) to provide first estimates of changes in R_eco during short-term (seasonal to interannual) moisture limitation. Although photosynthesis declines if moisture availability is limiting, leaf dark respiration is generally maintained, potentially acclimating upwards in the longer term. If leaf area is lost, then short-term canopy-scale respiratory effluxes from wood and leaves are likely to decline. Using a moderate short-term drying scenario where soil moisture limitation leads to a loss of 0.5 m² m⁻² yr⁻¹ in leaf area index, we estimate a reduction in respiratory CO₂ efflux from leaves and live woody tissue of 1.0 (±0.4) t C ha⁻¹ yr⁻¹. Necromass decomposition declines during drought, but mortality increases; the median mortality increase following a strong El Niño is 1.1% (n=46 tropical rainforest plots) and yields an estimated net short-term increase in necromass CO₂ efflux of 0.13–0.18 t C ha⁻¹ yr⁻¹. Soil respiration is strongly sensitive to moisture limitation over the short term, but not to associated temperature increases. This effect is underestimated in many models but can lead to estimated reductions in CO₂ efflux of 2.0 (±0.5) t C ha⁻¹ yr⁻¹. Thus, the majority of short-term respiratory responses to drought point to a decline in R_eco, an outcome that contradicts recent regional-scale modelling of NEP. NEP varies with both GPP and R_eco but robust moisture response functions are clearly needed to improve quantification of the role of R_eco in influencing regional-scale CO₂ emissions from Amazonia.     

Beyond Parkinson disease: amyotrophic lateral sclerosis and the axon guidance pathway

Background

We recently described a genomic pathway approach to study complex diseases. We demonstrated that models constructed using single nucleotide polymorphisms (SNPs) within axon guidance pathway genes were highly predictive of Parkinson disease (PD) susceptibility, survival free of PD, and age at onset of PD within two independent whole-genome association datasets. We also demonstrated that several axon guidance pathway genes represented by SNPs within our final models were differentially expressed in PD.

Methodology/Principal Findings

Here we employed our genomic pathway approach to analyze data from a whole-genome association dataset of amyotrophic lateral sclerosis (ALS); and demonstrated that models constructed using SNPs within axon guidance pathway genes were highly predictive of ALS susceptibility (odds ratio = 1739.73, p = 2.92×10⁻⁶⁰), survival free of ALS (hazards ratio = 149.80, p = 1.25×10⁻⁷⁴), and age at onset of ALS (R² = 0.86, p = 5.96×10⁻⁶⁶). We also extended our analyses of a whole-genome association dataset of PD, which shared 320,202 genomic SNPs in common with the whole-genome association dataset of ALS. We compared for ALS and PD the genes represented by SNPs in the final models for susceptibility, survival free of disease, and age at onset of disease and noted that 52.2%, 37.8%, and 34.9% of the genes were shared respectively.

Conclusions/Significance

Our findings for the axon guidance pathway and ALS have prior biological plausibility, overlap partially with PD, and may provide important insight into the causes of these and related neurodegenerative disorders.     

Allergic asthmatics show divergent lipid mediator profiles from healthy controls both at baseline and following birch pollen provocation

Background

Asthma is a respiratory tract disorder characterized by airway hyper-reactivity and chronic inflammation. Allergic asthma is associated with the production of allergen-specific IgE and expansion of allergen-specific T-cell populations. Progression of allergic inflammation is driven by T-helper type 2 (Th2) mediators and is associated with alterations in the levels of lipid mediators.

Objectives

Responses of the respiratory system to birch allergen provocation in allergic asthmatics were investigated. Eicosanoids and other oxylipins were quantified in the bronchoalveolar lumen to provide a measure of shifts in lipid mediators associated with allergen challenge in allergic asthmatics.

Methods

Eighty-seven lipid mediators representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS following off-line extraction of bronchoalveolar lavage fluid (BALF). Multivariate statistics using OPLS were employed to interrogate acquired oxylipin data in combination with immunological markers.

Results

Thirty-two oxylipins were quantified, with baseline asthmatics possessing a different oxylipin profile relative to healthy individuals that became more distinct following allergen provocation. The most prominent differences included 15-LOX-derived ω-3 and ω-6 oxylipins. Shared-and-Unique-Structures (SUS)-plot modeling showed a correlation (R² = 0.7) between OPLS models for baseline asthmatics (R²Y[cum] = 0.87, Q²[cum] = 0.51) and allergen-provoked asthmatics (R²Y[cum] = 0.95, Q²[cum] = 0.73), with the majority of quantified lipid mediators and cytokines contributing equally to both groups. Unique structures for allergen provocation included leukotrienes (LTB₄ and 6-trans-LTB₄), CYP-derivatives of linoleic acid (epoxides/diols), and IL-10.

Conclusions

Differences in asthmatic relative to healthy profiles suggest a role for 15-LOX products of both ω-6 and ω-3 origin in allergic inflammation. Prominent differences at baseline levels indicate that non-symptomatic asthmatics are subject to an underlying inflammatory condition not observed with other traditional mediators. Results suggest that oxylipin profiling may provide a sensitive means of characterizing low-level inflammation and that even individuals with mild disease display distinct phenotypic profiles, which may have clinical ramifications for disease.     

Incidence and correlates of HIV-1 RNA detection in the breast milk of women receiving HAART for the prevention of HIV-1 transmission

Background

The incidence and correlates of breast milk HIV-1 RNA detection were determined in intensively sampled women receiving highly active antiretroviral therapy (HAART) for the prevention of mother-to-child HIV-1 transmission.

Methods

Women initiated HAART at 34 weeks of pregnancy. Breast milk was collected every 2–5 days during 1 month postpartum for measurements of cell-associated HIV DNA and cell-free HIV RNA. Plasma and breast milk were also collected at 2 weeks, 1, 3 and 6 months for concurrent HIV-1 RNA and DNA measurements. Regression was used to identify cofactors for breast milk HIV-1 RNA detection.

Results

Of 259 breast milk specimens from 25 women receiving HAART, 34 had detectable HIV-1 RNA (13%, incidence 1.4 episodes/100 person-days 95% CI = 0.97–1.9). Fourteen of 25 (56%) women had detectable breast milk HIV-1 RNA [mean 2.5 log₁₀ copies/ml (range 2.0–3.9)] at least once. HIV-1 DNA was consistently detected in breast milk cells despite HAART, and increased slowly over time, at a rate of approximately 1 copy/10⁶ cells per day (p = 0.02). Baseline CD4, plasma viral load, HAART duration, and frequency of breast problems were similar in women with and without detectable breast milk HIV-1 RNA. Women with detectable breast milk HIV-1 RNA were more likely to be primiparous than women without (36% vs 0%, p = 0.05). Plasma HIV-1 RNA detection (OR = 9.0, 95%CI = 1.8–44) and plasma HIV-1 RNA levels (OR = 12, 95% CI = 2.5–56) were strongly associated with concurrent detection of breast milk HIV-1 RNA. However, no association was found between breast milk HIV-1 DNA level and concurrent breast milk HIV-1 RNA detection (OR = 0.96, 95%CI = 0.54–1.7).

Conclusions

The majority of women on HAART had episodic detection of breast milk HIV-1 RNA. Breast milk HIV-1 RNA detection was associated with systemic viral burden rather than breast milk HIV-1 DNA.     

Comprehensive genotyping in two homogeneous Graves' disease samples reveals major and novel HLA association alleles

Background

Graves'' disease (GD) is the leading cause of hyperthyroidism and thyroid eye disease inherited as a complex trait. Although geoepidemiology studies showed relatively higher prevalence of GD in Asians than in Caucasians, previous genetic studies were contradictory concerning whether and/or which human leukocyte antigen (HLA) alleles are associated with GD in Asians.

Methodology/Principal Findings

We conducted a case-control association study (499 unrelated GD cases and 504 controls) and a replication in an independent family sample (419 GD individuals and their 282 relatives in 165 families). To minimize genetic and phenotypic heterogeneity, we included only ethnic Chinese Han population in Taiwan and excluded subjects with hypothyroidism. We performed direct and comprehensive genotyping of six classical HLA loci (HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1) to 4-digit resolution. Combining the data of two sample populations, we found that B*46:01 (odds ratio under dominant model [OR]  = 1.33, Bonferroni corrected combined P [P_Bc]  = 1.17×10⁻²), DPB1*05:01 (OR  = 2.34, P_Bc = 2.58×10⁻¹⁰), DQB1*03:02 (OR  = 0.62, P_Bc  = 1.97×10⁻²), DRB1*15:01 (OR  = 1.68, P_Bc = 1.22×10⁻²) and DRB1*16:02 (OR  = 2.63, P_Bc  = 1.46×10⁻⁵) were associated with GD. HLA-DPB1*05:01 is the major gene of GD in our population and singly accounts for 48.4% of population-attributable risk.

Conclusions/Significance

These GD-associated alleles we identified in ethnic Chinese Hans, and those identified in other Asian studies, are totally distinct from the known associated alleles in Caucasians. Identification of population-specific association alleles is the critical first step for individualized medicine. Furthermore, comparison between different susceptibility/protective alleles across populations could facilitate generation of novel hypothesis about GD pathophysiology and indicate a new direction for future investigation.     

Functional stability of unliganded envelope glycoprotein spikes among isolates of human immunodeficiency virus type 1 (HIV-1)

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T₉₀ values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T₉₀ (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1_ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1_JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.     

HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes

HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1_Ba-L infection of monocyte-derived macrophages (MDM) on phagocytosis of opsonised P. falciparum infected erythrocytes (IE) and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR) (10 (0–28) versus (34 (27–108); IE internalised/100 MDM; p = 0.001) and decreased secretion of IL-6 (1,116 (352–3,387) versus 1,552 (889–6,331); pg/mL; p = 0.0078) and IL-1β (16 (7–21) versus 33 (27–65); pg/mL; p = 0.0078). Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.     

Genetic Modulation of Lipid Profiles following Lifestyle Modification or Metformin Treatment: The Diabetes Prevention Program

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10⁻¹⁷). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10⁻⁵–1×10⁻¹⁹). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10⁻⁵, P _interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P _interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P _interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P _interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.     

Increased rate of CD4+ T-cell decline and faster time to antiretroviral therapy in HIV-1 subtype CRF01_AE infected seroconverters in Singapore

Background

It remains controversial as to whether HIV-1 subtypes influence disease progression. Singapore offers a unique opportunity to address this issue due to the presence of co-circulating subtypes. We compared subtype CRF01_AE and non-CRF01_AE infected patients, with regards to estimated annual rate of CD4+ T-cell loss and time from estimated data of seroconversion (EDS) to antiretroviral therapy (ART).

Methods

We recruited ART-naive patients with known dates of seroconversion between October 2002 and December 2007 at the Singapore Communicable Disease Centre, the national reference treatment centre. Multilevel mixed-effects models were used to analyse the rate of CD4+ T-cell decline. Time from EDS to ART was analyzed with the Kaplan-Meier survival method and compared with Cox proportional hazards models.

Results

54 patients with previously assigned HIV-1 subtypes (24 CRF01_AE, 17 B, 8 B'', 1 CRF33_01B, 3 CRF34_01B and 1 G) were observed for 89 patient-years. Subtype CRF01_AE and non-CRF01_AE infected patients did not differ in age, gender, risk factor, rate of symptomatic seroconversion, baseline CD4+ T-cell count, log₁₀ viral load or haemoglobin concentration. The estimated annual rate of CD4+ T-cell loss was 58 cells/mm³/year (95% CI: 7 to 109; P = 0.027) greater in subtype CRF01_AE infected patients compared to non-CRF01_AE patients, after adjusting for age, baseline CD4+ T-cell count and baseline log₁₀ viral load. The median time from EDS to ART was 1.8 years faster comparing CRF01_AE to non-CRF01_AE infected patient with a 2.5 times (95% CI: 1.2-5.0; P = 0.013) higher hazard for ART initiation, after controlling for age, baseline CD4+ T-cell count and baseline log₁₀ viral load.

Conclusions

Infecting subtype significantly impacted the rate of CD4+ T-cell loss and time to treatment in this cohort. Studies to understand the biological basis for this difference could further our understanding of HIV pathogenesis.     

Large-scale pathway-based analysis of bladder cancer genome-wide association data from five studies of European background

Pathway analysis of genome-wide association studies (GWAS) offer a unique opportunity to collectively evaluate genetic variants with effects that are too small to be detected individually. We applied a pathway analysis to a bladder cancer GWAS containing data from 3,532 cases and 5,120 controls of European background (n = 5 studies). Thirteen hundred and ninety-nine pathways were drawn from five publicly available resources (Biocarta, Kegg, NCI-PID, HumanCyc, and Reactome), and we constructed 22 additional candidate pathways previously hypothesized to be related to bladder cancer. In total, 1421 pathways, 5647 genes and ∼90,000 SNPs were included in our study. Logistic regression model adjusting for age, sex, study, DNA source, and smoking status was used to assess the marginal trend effect of SNPs on bladder cancer risk. Two complementary pathway-based methods (gene-set enrichment analysis [GSEA], and adapted rank-truncated product [ARTP]) were used to assess the enrichment of association signals within each pathway. Eighteen pathways were detected by either GSEA or ARTP at P≤0.01. To minimize false positives, we used the I² statistic to identify SNPs displaying heterogeneous effects across the five studies. After removing these SNPs, seven pathways (‘Aromatic amine metabolism’ [P_GSEA = 0.0100, P_ARTP = 0.0020], ‘NAD biosynthesis’ [P_GSEA = 0.0018, P_ARTP = 0.0086], ‘NAD salvage’ [P_ARTP = 0.0068], ‘Clathrin derived vesicle budding’ [P_ARTP = 0.0018], ‘Lysosome vesicle biogenesis’ [P_GSEA = 0.0023, P_ARTP<0.00012], ’Retrograde neurotrophin signaling’ [P_GSEA = 0.00840], and ‘Mitotic metaphase/anaphase transition’ [P_GSEA = 0.0040]) remained. These pathways seem to belong to three fundamental cellular processes (metabolic detoxification, mitosis, and clathrin-mediated vesicles). Identification of the aromatic amine metabolism pathway provides support for the ability of this approach to identify pathways with established relevance to bladder carcinogenesis.