Overrepresentation of Injection Drug Use Route of Infection Among Human Immunodeficiency Virus Long-term Nonprogressors: A Nationwide, Retrospective Cohort Study in China, 1989-2016.

BackgroundWhy some persons living with human immunodeficiency virus (HIV) (PLWH) progress quickly and others remain "healthy" for a decade or more without treatment remains a fundamental question of HIV pathology. We aimed to assess the epidemiological characteristics of HIV long-term nonprogressors (LTNPs) based on a cohort of PLWH in China observed between 1989 and 2016.MethodsWe conducted a nationwide, retrospective cohort study among Chinese PLWH with HIV diagnosed before 1 January 2008. Records were extracted from China's national HIV/AIDS database on 30 June 2016. LTNPs were defined as those with AIDS-free, antiretroviral therapy-naive survival, with CD4 cell counts consistently ≥500/μL for ≥8 years after diagnosis. Prevalence was calculated, characteristics were described, and determinants were assessed by means of logistic regression. Potential sources of bias were also investigated.ResultsOur cohort included 89 201 participants, of whom 1749 (2.0%) were categorized as LTNPs. The injection drug use (IDU) route of infection was reported by 70.7% of LTNPs, compared with only 37.1% of non-LTNPs. The odds of LTNP status were greater among those infected via IDU (adjusted odds ratio [95% confidence interval], 2.28 [1.94-2.68]) and with HIV diagnosed in settings with large populations of persons who inject drugs (1.75 [1.51-2.02] for detention centers, 1.61 [1.39-1.87] for Yunnan, 1.94 [1.62-2.31] for Guangdong, and 2.90 [2.09-4.02] for Xinjiang).ConclusionsOverrepresentation of the IDU route of infection among LTNPs is a surprising finding worthy of further study, and this newly defined cohort may be particularly well suited to exploration of the molecular biological mechanisms underlying HIV long-term nonprogression

Machine learning-assisted directed protein evolution with combinatorial libraries

To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning in the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine learning models trained on tested variants provide a fast method for testing sequence space computationally. We validate this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (stereodivergence) of a new-to-nature carbene Si-H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee. By greatly increasing throughput with in silico modeling, machine learning enhances the quality and diversity of sequence solutions for a protein engineering problem.Comment: Corrected best S-selective variant sequence in Figure 4. Corrected less R-selective variant sequences from Round II Input library in Table 2 and Supp Table 4. Corrections may also be found on PNAS version https://www.pnas.org/content/early/2019/12/26/192177011

Covariant polarized radiative transfer on cosmological scales for investigating large-scale magnetic field structures

Polarization of radiation is a powerful tool to study cosmic magnetism and analysis of polarization can be used as a diagnostic tool for large-scale structures. In this paper, we present a solid theoretical foundation for using polarized light to investigate large-scale magnetic field structures: the cosmological polarized radiative transfer (CPRT) formulation. The CPRT formulation is fully covariant. It accounts for cosmological and relativistic effects in a self-consistent manner and explicitly treats Faraday rotation, as well as Faraday conversion, emission, and absorption processes. The formulation is derived from the first principles of conservation of phase-space volume and photon number. Without loss of generality, we consider a flat Friedmann-Robertson-Walker (FRW) space-time metric and construct the corresponding polarized radiative transfer equations. We propose an all-sky CPRT calculation algorithm, based on a ray-tracing method, which allows cosmological simulation results to be incorporated and, thereby, model templates of polarization maps to be constructed. Such maps will be crucial in our interpretation of polarized data, such as those to be collected by the Square Kilometer Array (SKA). We describe several tests which are used for verifying the code and demonstrate applications in the study of the polarization signatures in different distributions of electron number density and magnetic fields. We present a pencil-beam CPRT calculation and an all-sky calculation, using a simulated galaxy cluster or a model magnetized universe obtained from GCMHD+ simulations as the respective input structures. The implications on large-scale magnetic field studies are discussed; remarks on the standard methods using rotation measure are highlighted.Comment: 32 pages, 14 figure

Web-based physiotherapy for people affected by multiple sclerosis: a single blind, randomized controlled feasibility study

Objective: To examine the feasibility of a trial to evaluate web-based physiotherapy compared to a standard home exercise programme in people with multiple sclerosis. Design: Multi-centre, randomized controlled, feasibility study. Setting: Three multiple sclerosis out-patient centres. Participants: A total of 90 people with multiple sclerosis (Expanded Disability Status Scale 4–6.5). Interventions: Participants were randomized to a six-month individualized, home exercise programme delivered via web-based physiotherapy (n = 45; intervention) or a sheet of exercises (n = 45; active comparator). Outcome measures: Outcome measures (0, three, six and nine months) included adherence, two-minute walk test, 25 foot walk, Berg Balance Scale, physical activity and healthcare resource use. Interviews were undertaken with 24 participants and 3 physiotherapists. Results: Almost 25% of people approached agreed to take part. No intervention-related adverse events were recorded. Adherence was 40%–63% and 53%–71% in the intervention and comparator groups. There was no difference in the two-minute walk test between groups at baseline (Intervention-80.4(33.91)m, Comparator-70.6(31.20)m) and no change over time (at six-month Intervention-81.6(32.75)m, Comparator-74.8(36.16)m. There were no significant changes over time in other outcome measures except the EuroQol-5 Dimension at six months which decreased in the active comparator group. For a difference of 8(17.4)m in two-minute walk test between groups, 76 participants/group would be required (80% power, P > 0.05) for a future randomized controlled trial. Conclusion: No changes were found in the majority of outcome measures over time. This study was acceptable and feasible by participants and physiotherapists. An adequately powered study needs 160 participants

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia

DDK dependent regulation of TOP2A at centromeres revealed by a chemical genetics approach

In eukaryotic cells the CDC7/DBF4 kinase, also known as DBF4-dependent kinase (DDK), is required for the firing of DNA replication origins. CDC7 is also involved in replication stress responses and its depletion sensitises cells to drugs that affect fork progression, including Topoisomerase 2 poisons. Although CDC7 is an important regulator of cell division, relatively few substrates and bona-fide CDC7 phosphorylation sites have been identified to date in human cells. In this study, we have generated an active recombinant CDC7/DBF4 kinase that can utilize bulky ATP analogues. By performing in vitro kinase assays using benzyl-thio-ATP, we have identified TOP2A as a primary CDC7 substrate in nuclear extracts, and serine 1213 and serine 1525 as in vitro phosphorylation sites. We show that CDC7/DBF4 and TOP2A interact in cells, that this interaction mainly occurs early in S-phase, and that it is compromised after treatment with CDC7 inhibitors. We further provide evidence that human DBF4 localises at centromeres, to which TOP2A is progressively recruited during S-phase. Importantly, we found that CDC7/DBF4 down-regulation, as well S1213A/S1525A TOP2A mutations can advance the timing of centromeric TOP2A recruitment in S-phase. Our results indicate that TOP2A is a novel DDK target and have important implications for centromere biology

Mitotic stress is an integral part of the oncogene-induced senescence program that promotes multinucleation and cell cycle arrest

Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80°C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method