To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral ...suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.
It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as ...the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.
Copy number variation (CNV) is a common form of structural variation detected in human genomes, occurring as both constitutional and somatic events. Cytogenetic techniques like chromosomal microarray ...(CMA) are widely used in analyzing CNVs. However, CMA techniques cannot resolve the full nature of these structural variations (i.e. the orientation and location of associated breakpoint junctions) and must be combined with other cytogenetic techniques, such as karyotyping or FISH, to do so. This makes the development of a next‐generation sequencing (NGS) approach capable of resolving both CNVs and breakpoint junctions desirable. Mate‐pair sequencing (MPseq) is a NGS technology designed to find large structural rearrangements across the entire genome. Here we present an algorithm capable of performing copy number analysis from mate‐pair sequencing data. The algorithm uses a step‐wise procedure involving normalization, segmentation, and classification of the sequencing data. The segmentation technique combines both read depth and discordant mate‐pair reads to increase the sensitivity and resolution of CNV calls. The method is particularly suited to MPseq, which is designed to detect breakpoint junctions at high resolution. This allows for the classification step to accurately calculate copy number levels at the relatively low read depth of MPseq. Here we compare results for a series of hematological cancer samples that were tested with CMA and MPseq. We demonstrate comparable sensitivity to the state‐of‐the‐art CMA technology, with the benefit of improved breakpoint resolution. The algorithm provides a powerful analytical tool for the analysis of MPseq results in cancer.
Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein ...complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-1 complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-1 complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.
•Comparison of junctions reported by mate-pair sequencing with SVAtools, karyotype, FISH and CMA.•Breakpoint resolution by mate-pair sequencing using SVAtools is less than 1000 bps, usually less than ...200 bps.•SVAtools also provides gene fusion and disruption detection.
Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10 kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions and gene fusions, and characterize the genomic structure of complex rearrangements.
To illustrate the power of SVAtools' junction detection methods to provide comprehensive molecular karyotypes, MPseq data were compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, FISH and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools' junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.
Quality of life among prostate cancer survivors varies by socio-demographic factors and treatment type received; however, less in known about differences in functional outcomes by method of ...presentation. We investigate differences in reported urinary, bowel, sexual and hormone-related problems between symptomatic and PSA-detected prostate cancer survivors.
A UK wide cross-sectional postal survey of prostate cancer survivors conducted 18-42 months post-diagnosis. Questions were included on presentation method and treatment. Functional outcome was determined using the EPIC-26 questionnaire. Reported outcomes were compared for symptomatic and PSA-detected survivors using ANOVA and multivariable log-linear regression.
Thirty-five thousand eight hundred twenty-three men responded (response rate: 60.8%). Of these, 31.3% reported presenting via PSA test and 59.7% symptomatically. In multivariable analysis, symptomatic men reported more difficulty with urinary incontinence (Adjusted mean ratio (AMR): 0.96, 95% CI: 0.96-0.97), urinary irritation (AMR: 0.95, 95% CI: 0.95-0.96), bowel function (AMR: 0.97, 95% CI: 0.97-0.98), sexual function (AMR: 0.90, 95% CI: 0.88-0.92), and vitality/hormonal function (AMR: 0.96, 95% CI: 0.96-0.96) than PSA-detected men. Differences were consistent across respondents of differing age, stage, Gleason score and treatment type.
Prostate cancer survivors presenting symptomatically report poorer functional outcomes than PSA-detected survivors. Differences were not explained by socio-demographic or clinical factors. Clinicians should be aware that men presenting with symptoms are more likely to report functional difficulties after prostate cancer treatment and may need additional aftercare if these difficulties persist. Method of presentation should be considered as a covariate in patient-reported outcome studies of prostate cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected ...over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.
Abstract
Objectives
Chromosome (G-banding) and fluorescence in situ hybridization (FISH) serve as the primary methodologies utilized for detecting genetic aberrations in hematologic neoplasms. ...Chromosomal microarray can detect copy number aberrations (CNAs) with greater resolution when compared to G-banding and FISH, and can also identify copy-neutral loss of heterozygosity (CN-LOH). The purpose of our review is to highlight a preselected group of hematologic neoplasms for which chromosomal microarray has the greatest clinical utility.
Methods
A case-based approach and review of the literature was performed to identify the advantages and disadvantages of utilizing chromosomal microarray for specific hematologic neoplasms.
Results
Chromosomal microarray identified CNAs and CN-LOH of clinical significance, and could be performed on fresh or paraffin-embedded tissue and liquid neoplasms. Microarray studies could not detect balanced rearrangements, low-level clones, or distinguish independent clones.
Conclusions
When utilized appropriately, chromosomal microarray can provide clinically significant information that complements traditional cytogenetic testing methodologies.
Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles ...of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Chromosome (G-banding) and fluorescence in situ hybridization (FISH) serve as the primary methodologies utilized for detecting genetic aberrations in hematologic neoplasms. Chromosomal microarray can ...detect copy number aberrations (CNAs) with greater resolution when compared to G-banding and FISH, and can also identify copy-neutral loss of heterozygosity (CN-LOH). The purpose of our review is to highlight a preselected group of hematologic neoplasms for which chromosomal microarray has the greatest clinical utility.
A case-based approach and review of the literature was performed to identify the advantages and disadvantages of utilizing chromosomal microarray for specific hematologic neoplasms.
Chromosomal microarray identified CNAs and CN-LOH of clinical significance, and could be performed on fresh or paraffin-embedded tissue and liquid neoplasms. Microarray studies could not detect balanced rearrangements, low-level clones, or distinguish independent clones.
When utilized appropriately, chromosomal microarray can provide clinically significant information that complements traditional cytogenetic testing methodologies.
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