At the early stage of infection, human immunodeficiency virus (HIV)-1 predominantly uses the CCR5 coreceptor for host cell entry. The subsequent emergence of HIV variants that use the CXCR4 ...coreceptor in roughly half of all infections is associated with an accelerated decline of CD4+ T-cells and rate of progression to AIDS. The presence of a 'fitness valley' separating CCR5- and CXCR4-using genotypes is postulated to be a biological determinant of whether the HIV coreceptor switch occurs. Using phylogenetic methods to reconstruct the evolutionary dynamics of HIV within hosts enables us to discriminate between competing models of this process. We have developed a phylogenetic pipeline for the molecular clock analysis, ancestral reconstruction, and visualization of deep sequence data. These data were generated by next-generation sequencing of HIV RNA extracted from longitudinal serum samples (median 7 time points) from 8 untreated subjects with chronic HIV infections (Amsterdam Cohort Studies on HIV-1 infection and AIDS). We used the known dates of sampling to directly estimate rates of evolution and to map ancestral mutations to a reconstructed timeline in units of days. HIV coreceptor usage was predicted from reconstructed ancestral sequences using the geno2pheno algorithm. We determined that the first mutations contributing to CXCR4 use emerged about 16 (per subject range 4 to 30) months before the earliest predicted CXCR4-using ancestor, which preceded the first positive cell-based assay of CXCR4 usage by 10 (range 5 to 25) months. CXCR4 usage arose in multiple lineages within 5 of 8 subjects, and ancestral lineages following alternate mutational pathways before going extinct were common. We observed highly patient-specific distributions and time-scales of mutation accumulation, implying that the role of a fitness valley is contingent on the genotype of the transmitted variant.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using ...variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSM(NSI/SI) and geno2pheno(coreceptor) to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage. We analyzed PBMC and serum samples obtained from eight HIV-1-infected individuals at three-month intervals up to one year prior to the first phenotypic detection of CXCR4-using variants in the MT-2 assay. Between 3,482 and 10,521 reads were generated from each sample. In all individuals, V3 sequences of predicted CXCR4-using HIV-1 were detected at least three months prior to phenotypic detection of CXCR4-using variants in the MT-2 assay. Subsequent analysis of the genetic relationships of these V3 sequences using minimum spanning trees revealed that the transition in coreceptor usage followed a stepwise mutational pathway involving sequential intermediate variants, which were generally present at relatively low frequencies compared to the major predicted CCR5- and CXCR4-using variants. In addition, we observed differences between individuals with respect to the number of predicted CXCR4-using variants, the diversity among major predicted CCR5-using variants, and the presence or absence of intermediate variants with discordant phenotype predictions. These results provide the first detailed description of the mutational pathways in V3 during the transition from CCR5- to CXCR4-usage in natural HIV-1 infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The interplay between C-C chemokine receptor type 5 (CCR5) host genetic background, disease progression, and intrahost HIV-1 evolutionary dynamics remains unclear because differences in viral ...evolution between hosts limit the ability to draw conclusions across hosts stratified into clinically relevant populations. Similar inference problems are proliferating across many measurably evolving pathogens for which intrahost sequence samples are readily available. To this end, we propose novel hierarchical phylogenetic models (HPMs) that incorporate fixed effects to test for differences in dynamics across host populations in a formal statistical framework employing stochastic search variable selection and model averaging. To clarify the role of CCR5 host genetic background and disease progression on viral evolutionary patterns, we obtain gp120 envelope sequences from clonal HIV-1 variants isolated at multiple time points in the course of infection from populations of HIV-1-infected individuals who only harbored CCR5-using HIV-1 variants at all time points. Presence or absence of a CCR5 wt/Δ32 genotype and progressive or long-term nonprogressive course of infection stratify the clinical populations in a two-way design. As compared with the standard approach of analyzing sequences from each patient independently, the HPM provides more efficient estimation of evolutionary parameters such as nucleotide substitution rates and d(N)/d(S) rate ratios, as shown by significant shrinkage of the estimator variance. The fixed effects also correct for nonindependence of data between populations and results in even further shrinkage of individual patient estimates. Model selection suggests an association between nucleotide substitution rate and disease progression, but a role for CCR5 genotype remains elusive. Given the absence of clear d(N)/d(S) differences between patient groups, delayed onset of AIDS symptoms appears to be solely associated with lower viral replication rates rather than with differences in selection on amino acid fixation.
We evaluated the role of rituximab during remission induction chemotherapy in relapsed aggressive CD20+ non-Hodgkin lymphoma. Of 239 patients, 225 were evaluable for analysis. Randomized to DHAP ...(cisplatin-cytarabine-dexamethasone)-VIM (etoposide-ifosfamide-methotrexate)-DHAP (cisplatin-cytarabine-dexamethasone) chemotherapy with rituximab (R; R-DHAP arm) were 119 patients (113 evaluable) and to chemotherapy without rituximab (DHAP arm) 120 patients (112 evaluable). Patients in complete remission (CR) and partial remission (PR) after 2 chemotherapy courses were eligible for autologous stem-cell transplantation. After the second chemotherapy cycle, 75% of the patients in the R-DHAP arm had responsive disease (CR or PR) versus 54% in the DHAP arm (P = .01). With a median follow-up of 24 months, there was a significant difference in failure-free survival (FFS24; 50% vs 24% vs, P < .001), and progression free survival (PFS24; 52% vs 31% P < .002) in favor of the R-DHAP arm. Cox-regression analysis demonstrated a significant effect of rituximab treatment on FFS24 (HR 0.41, 95% confidence interval CI 0.29-0.57 versus 0.51, 95% CI 0.37-0.70) and overall-survival (OS24: HR 0.60 0.41-0.89 vs 0.76 0.52-1.10) when adjusted for time since upfront treatment, age, World Health Organization performance status, and secondary age-adjusted international prognostic index. These results demonstrate improved FFS and PFS for relapsed aggressive B-cell NHL if rituximab is added to the re-induction chemotherapy regimen.
Abstract To trace the evolutionary patterns underlying evolution of coreceptor use within a host, we studied an HIV-1 transmission pair involving a donor who exclusively harbored CCR5-using (R5) ...variants throughout his entire disease course and a recipient who developed CXCR4-using variants. Over time, R5 variants in the donor optimized coreceptor use, which was associated with an increased number of potential N-linked glycosylation sites (PNGS) and elevated V3 charge in the viral envelope. Interestingly, R5 variants that were transmitted to the recipient preserved the viral characteristics of this late stage genotype and phenotype. Following a selective sweep, CXCR4-using variants subsequently emerged in the recipient coinciding with a further increase in the number of PNGS and V3 charge in the envelope of R5 viruses. Although described in a single transmission pair, the transmission and subsequent persistence of R5 variants with late stage characteristics demonstrate the potential for coreceptor use adaptation at the population level.
Ptychographical iterative phase retrieval is a promising new transmission imaging technique which uses a set of measured intensities from the consecutive illumination of overlapping regions of the ...specimen to form an image of the transmission function in phase and amplitude. Although the technique has been shown to work very effectively in both the light-optical and X-ray domains, electron ptychography poses significant difficulties, not least of which is the uncertainty in probe position due to drift and other instabilities. We demonstrate three methods for deriving the relative positions of the illumination spot on the specimen a-posteriori.
Abstract The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 ...variants in plasma that are considered to be recently produced. Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals. Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.
This study investigated the relationship between oxidation and antioxidation parameters before superovulation (SOV) treatment and embryo recovery in donor cows. The relative redox status of the 61 ...donor cows was evaluated based on the median values of diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) measurements (d-ROMs of 100 U.CARR; BAP of 2413 µmol L−1) before SOV treatment. Following this, the animals were divided into four groups: cows with low d-ROMs but high BAP were assigned to the “LH” group (n=11), cows with high d-ROMs and BAP were assigned to the “HH” group (n=20), cows with high d-ROMs but low BAP were assigned to the “HL” group (n=10), and cows with low d-ROMs and BAP were assigned to the “LL” group (n=20). Embryos were collected from superovulated cows 6 d after artificial insemination. The percentages of fertilised, transferable, and Code-1 embryos were significantly higher (P<0.05) in the HH group than those in the HL group. The HH group had the highest mean values for all embryo recovery results. Our results suggest that the redox status of donor cows before SOV treatment affects embryo recovery, as cows with high levels of both oxidative and antioxidative status have better embryo production.
Ptychography is a wavelength-limited phase retrieval method that promises to provide sub-0.5Å resolution in the electron microscope, even if the lens employed (which is required only to form an ...illumination spot) can itself only achieve rather modest resolution. The fidelity of a practical specimen reconstruction using ptychography hinges on various experimental factors, the major one of which is the source brightness. Achieving a high degree of coherence of an electron beam requires source demagnification which reduces the electron counts reaching the detector for a given exposure time. Specimen drift, damage and contamination also limits the practical exposure time. In this paper we investigate the effect of the counting statistics required for a good quality ptychographic reconstruction
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