HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN

Bioconductor workflow for microbiome data analysis: from raw reads to community analyses [version 1; referees: 2 approved]

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package

Effect of thermal treatment and storage on bioactive compounds, organic acids and antioxidant activity of baobab fruit (Adansonia digitata) pulp from Malawi

Bioactive compounds of baobab (Adansonia digitata) pulp from Malawi were investigated. The effect of thermal treatment and storage on selected quality attributes of the juice was also evaluated. Organic compounds were analysed by HPLC; total phenol content (TPC) and total antioxidant activity (FRAP, ABTS and DPPH) were measured by spectrophotometry. Malawi baobab pulp contains high levels of procyanidin B2 (533 ± 22.6 mg/100 g FW), vitamin C (AA + DHA) (466 ± 2.5 mg/100 g FW), gallic acid (68.5 ± 12.4 mg/100 g FW) and (−)-epicatechin (43.0 ± 3.0 mg/100 g FW) and showed a maximum TPC of 1.89 × 103 ± 1.61 mg GAE/100 g FW. The maximum antioxidant activity was 2.81 × 103 ± 92.8 mg TEAC/100 g FW for FRAP, 1.52 × 103 ± 17.1 mg TEAC/100 g FW for ABTS and 50.9 ± 0.43% DPPH for DPPH. Thermal pasteurisation (72 °C, 15 s) retained vitamin C which further showed extended half-life under refrigeration temperature (6 °C). Procyanidin B2, (−)-epicatechin, TPC and antioxidant activity fluctuated during storage. Antioxidant activity was significantly correlated (p ≤ 0.05) with bioactive compounds and TPC

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT3 receptor antagonists

PURPOSE: The ferret cisplatin emesis model has been used for ~30 years and enabled identification of clinically used anti-emetics. We provide an objective assessment of this model including efficacy of 5-HT(3) receptor antagonists to assess its translational validity. METHODS: A systematic review identified available evidence and was used to perform meta-analyses. RESULTS: Of 182 potentially relevant publications, 115 reported cisplatin-induced emesis in ferrets and 68 were included in the analysis. The majority (n = 53) used a 10 mg kg(−1) dose to induce acute emesis, which peaked after 2 h. More recent studies (n = 11) also used 5 mg kg(−1), which induced a biphasic response peaking at 12 h and 48 h. Overall, 5-HT(3) receptor antagonists reduced cisplatin (5 mg kg(−1)) emesis by 68% (45–91%) during the acute phase (day 1) and by 67% (48–86%) and 53% (38–68%, all P < 0.001), during the delayed phase (days 2, 3). In an analysis focused on the acute phase, the efficacy of ondansetron was dependent on the dosage and observation period but not on the dose of cisplatin. CONCLUSION: Our analysis enabled novel findings to be extracted from the literature including factors which may impact on the applicability of preclinical results to humans. It reveals that the efficacy of ondansetron is similar against low and high doses of cisplatin. Additionally, we showed that 5-HT(3) receptor antagonists have a similar efficacy during acute and delayed emesis, which provides a novel insight into the pharmacology of delayed emesis in the ferret

Using ion mobility spectrometry-mass spectrometry to decipher the conformational and assembly characteristics of the hepatitis B capsid protein.

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150-183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50-111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis

Observing the Evolution of the Universe

How did the universe evolve? The fine angular scale (l>1000) temperature and polarization anisotropies in the CMB are a Rosetta stone for understanding the evolution of the universe. Through detailed measurements one may address everything from the physics of the birth of the universe to the history of star formation and the process by which galaxies formed. One may in addition track the evolution of the dark energy and discover the net neutrino mass. We are at the dawn of a new era in which hundreds of square degrees of sky can be mapped with arcminute resolution and sensitivities measured in microKelvin. Acquiring these data requires the use of special purpose telescopes such as the Atacama Cosmology Telescope (ACT), located in Chile, and the South Pole Telescope (SPT). These new telescopes are outfitted with a new generation of custom mm-wave kilo-pixel arrays. Additional instruments are in the planning stages.Comment: Science White Paper submitted to the US Astro2010 Decadal Survey. Full list of 177 author available at http://cmbpol.uchicago.ed

Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

A Diverse Group of Previously Unrecognized Human Rhinoviruses Are Common Causes of Respiratory Illnesses in Infants

Human rhinoviruses (HRVs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious.To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples). Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group ("C"). None of these new strains could be cultured in traditional cell lines.By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group