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Historical and ethnographic sources depict use of portable braced shaft weapons, or pikes, in megafauna hunting and defense during Late Holocene millennia in North and South America, Africa, Eurasia and Southeast Asia. Given the predominance of megafauna in Late Pleistocene North America during the centuries when Clovis points appeared and spread across much of the continent (13,050-12,650 cal BP), braced weapons may have been used in hunting of megaherbivores and defense against megacarnivores. Drawing from historical examples of pike use against lions, jaguars, boars, grizzlies, carabao and warhorses we consider the possibility of a fluted lithic pike. Associated osseous rods have been problematic as Clovis foreshafts due to the bevel angle and the apparent weakness of the splint haft when great strength is needed for deep penetration in megafauna hunting. However our review of Late Holocene pike use in megafauna encounters indicates the sharp tip becomes less important after hide or armor has been pierced because compression is sustained. Thus, foreshaft collapse after hide entry may not limit but rather increase the efficacy of the braced weapon. We conduct preliminary static experiments to model a fluted pike that adjusts during compression such that haft collapse and point detachment (when point jams on impact with bone) preserve the fluted biface, beveled rod and wooden mainshaft tip. In addition to Clovis point attributes and association with osseous rods, potential archaeological correlates of Clovis pike use include the high frequency of Clovis point isolates and concentrations of complete points with unbutchered mammoth remains at sites such as Naco in Arizona., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Byram et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Quitting smoking may lead to improvement in substance use, psychiatric symptoms, and pain, especially among high-risk populations who are more likely to experience comorbid conditions. However, causal inferences regarding smoking cessation and its subsequent benefits have been limited., Methods: We emulated a hypothetical open-label randomized control trial of smoking cessation using longitudinal observational data of HIV-positive and HIV-negative US veterans from 2003-2015 in the Veterans Aging Cohort Study. We followed individuals from the first time they self-reported current cigarette smoking (baseline). We categorized participants as quitters or non-quitters at the first follow-up visit (approximately 1 year after baseline). Using inverse probability weighting to adjust for confounding and selection bias, we estimated odds ratios for improvement of co-occurring conditions (unhealthy alcohol use, cannabis use, illicit opioid use, cocaine use, depressive symptoms, anxiety symptoms, and pain symptoms) at second follow-up (approximately 2 years after baseline) for those who quit smoking compared to those who did not, among individuals who had the condition at baseline., Results: Of 4,165 eligible individuals (i.e., current smokers at baseline), 419 reported no current smoking and 2,330 reported current smoking at the first follow-up. Adjusted odds ratios (95% confidence intervals) for associations between quitting smoking and improvement of each condition at second follow-up were: 2.10 (1.01, 4.35) for unhealthy alcohol use, 1.75 (1.00, 3.06) for cannabis use, 1.10 (0.58, 2.08) for illicit opioid use, and 2.25 (1.20, 4.24) for cocaine use, 0.78 (0.44, 1.38) for depressive symptoms, 0.93 (0.58, 1.49) for anxiety symptoms, and 1.31 (0.84, 2.06) for pain symptoms., Conclusions: While a causal interpretation of our findings may not be warranted, we found evidence for decreased substance use among veterans who quit cigarette smoking but none for the resolution of psychiatric conditions or pain symptoms. Findings suggest the need for additional resources combined with smoking cessation to reduce psychiatric and pain symptoms for high-risk populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ban et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ward et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The COVID-19 pandemic caused disruption in healthcare delivery due to reductions in both health facility capacity and care-seeking behavior. For women experiencing obstetric complications, access to comprehensive emergency obstetric care is critical for maternal and child health. In Kenya, pandemic-related restrictions began in March 2020 and were compounded by a healthcare worker strike in December 2020. We examined medical record data at Coast General Teaching and Referral Hospital, a large public hospital, and conducted staff interviews to understand how healthcare disruptions impacted care delivery and perinatal outcomes. Routinely collected data from all mother-baby dyads admitted to the Labor and Delivery Ward from January 2019 through March 2021 were included in interrupted time-series analyses. Outcomes included number of admissions and proportion of deliveries that resulted in caesarean sections and adverse birth outcomes. Interviews were conducted with nurses and medical officers to understand how the pandemic impacted clinical care. Pre-pandemic, the ward averaged 810 admissions/month, compared to 492 admissions/month post-pandemic (average monthly decrease: 24.9 admissions; 95% CI: -48.0, -1.8). The proportion of stillbirths increased 0.3% per month during the pandemic compared to the pre-pandemic period (95% CI: 0.1, 0.4). No significant differences were seen in the proportion of other adverse obstetrical outcomes. Interview results suggested that pandemic-related disruptions included reduced access to surgical theaters and protective equipment, and absence of COVID-19 guidelines. While these disruptions were perceived as impacting care for high-risk pregnancies, providers believed that overall quality of care did not diminish during the pandemic. However, they expressed concern about a likely increase in at-home births. In conclusion, while the pandemic had minimal adverse impact on hospital-based obstetrical outcomes, it reduced the number of patients able to access care. Emergency preparedness guidelines and public health messaging promoting timely obstetrical care are needed to ensure continuation of services during future healthcare disruptions., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RSM has received research funding, paid to the University of Washington from Hologic Corporation. All other authors (JEL, GW, NA, DO, BAR, NWK, KS, AA, and PO) declare that they have no competing interests., (Copyright: © 2023 Long et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. We show that this local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. We further show how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Miller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Intimate partner violence (IPV) is a major public health problem and is the most common form of violence against women. Women with HIV in serodifferent relationships may be at an increased risk of IPV compared to women without HIV, hindering their ability to adhere to antiretroviral therapy, clinic appointments, and condom use during sex. This study assessed the prevalence and correlates of IPV in the past year among women with HIV in serodifferent relationships in Nairobi, Kenya., Methods: This cross-sectional study included women with HIV in serodifferent relationships who were at least 18 years old and provided written informed consent. Their experience of physical, sexual, or emotional violence in the past year by the current partner was assessed using 13 questions adapted from the World Health Organization survey on violence against women. Standardized instruments were used to assess sociodemographic and behavioral factors. Associations between intimate partner violence and other variables were evaluated using log binomial regression models., Results: Of the 159 women enrolled, 47 (29.6%, 95% CI 22.9-37.2%) reported IPV in the past year. Of these, 32 (68.1%) reported emotional, 27 (57.4%) physical, and 27 (57.4%) sexual violence. In the multivariate model, pregnancy (adjusted prevalence ratio [aPR] 2.14, 95% CI 1.09-4.20), alcohol use (minimal drinking aPR 1.91, 95% CI 1.10-3.33; moderate/severe drinking aPR 1.17, 95% CI 0.53-2.59), male partner controlling behavior (aPR 2.09, 95% CI 1.24-3.51), and past physical violence (aPR 1.93, 95% CI 1.22-3.05) remained significantly associated with a higher prevalence of IPV in the past year., Conclusion: This study identified a high prevalence of IPV in the past year among women with HIV in serodifferent relationships. Pregnant women and women who had experienced prior violence had a higher prevalence of IPV. These data highlight the need to screen for IPV during clinic visits, and to offer evidence based interventions to support women in serodifferent relationships who have experienced IPV., Competing Interests: Dr. Richardson received compensation from Gilead for Data Safety Monitoring Board membership and Mock FDA Panel membership and Dr. McClelland currently receives research funding, paid to the University of Washington from Hologic Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Children who have been discharged from hospital in sub-Saharan Africa remain at substantial risk of mortality in the post-discharge period. Antimicrobial resistance (AMR) may be an important factor. We sought to determine the prevalence and risk factors associated with AMR in commensal Escherichia coli (E. coli) from Kenyan children at the time of discharge., Methodology/principle Findings: Fecal samples were collected from 406 children aged 1-59 months in western Kenya at the time of discharge from hospital and cultured for E. coli. Susceptibility to ampicillin, ceftriaxone, cefotaxime, ceftazidime, cefoxitin, imipenem, ciprofloxacin, gentamicin, combined amoxicillin/clavulanic acid, trimethoprim-sulfamethoxazole, azithromycin, and chloramphenicol was determined by disc diffusion according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Poisson regression was used to determine associations between participant characteristics and the presence of extended-spectrum beta-lactamases (ESBL) producing E. coli. Non-susceptibility to ampicillin (95%), gentamicin (44%), ceftriaxone (46%), and the presence of ESBL (44%) was high. Receipt of antibiotics during the hospitalization was associated with the presence of ESBL (aPR = 2.23; 95% CI: 1.29-3.83) as was being hospitalized within the prior year (aPR = 1.32 [1.07-1.69]). Open defecation (aPR = 2.02; 95% CI: 1.39-2.94), having a toilet shared with other households (aPR = 1.49; 95% CI: 1.17-1.89), and being female (aPR = 1.42; 95% CI: 1.15-1.76) were associated with carriage of ESBL E. coli., Conclusions/significance: AMR is common among isolates of E. coli from children at hospital discharge in Kenya, including nearly half having detectable ESBL., Competing Interests: The authors have declared that no competing interests exist.

Background: The United States (US) Expanded Access Program (EAP) to coronavirus disease 2019 (COVID-19) convalescent plasma was initiated in response to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. While randomized clinical trials were in various stages of development and enrollment, there was an urgent need for widespread access to potential therapeutic agents. The objective of this study is to report on the demographic, geographical, and chronological characteristics of patients in the EAP, and key safety metrics following transfusion of COVID-19 convalescent plasma., Methods and Findings: Mayo Clinic served as the central institutional review board for all participating facilities, and any US physician could participate as a local physician-principal investigator. Eligible patients were hospitalized, were aged 18 years or older, and had-or were at risk of progression to-severe or life-threatening COVID-19; eligible patients were enrolled through the EAP central website. Blood collection facilities rapidly implemented programs to collect convalescent plasma for hospitalized patients with COVID-19. Demographic and clinical characteristics of all enrolled patients in the EAP were summarized. Temporal patterns in access to COVID-19 convalescent plasma were investigated by comparing daily and weekly changes in EAP enrollment in response to changes in infection rate at the state level. Geographical analyses on access to convalescent plasma included assessing EAP enrollment in all national hospital referral regions, as well as assessing enrollment in metropolitan areas and less populated areas that did not have access to COVID-19 clinical trials. From April 3 to August 23, 2020, 105,717 hospitalized patients with severe or life-threatening COVID-19 were enrolled in the EAP. The majority of patients were 60 years of age or older (57.8%), were male (58.4%), and had overweight or obesity (83.8%). There was substantial inclusion of minorities and underserved populations: 46.4% of patients were of a race other than white, and 37.2% of patients were of Hispanic ethnicity. Chronologically and geographically, increases in the number of both enrollments and transfusions in the EAP closely followed confirmed infections across all 50 states. Nearly all national hospital referral regions enrolled and transfused patients in the EAP, including both in metropolitan and in less populated areas. The incidence of serious adverse events was objectively low (<1%), and the overall crude 30-day mortality rate was 25.2% (95% CI, 25.0% to 25.5%). This registry study was limited by the observational and pragmatic study design that did not include a control or comparator group; thus, the data should not be used to infer definitive treatment effects., Conclusions: These results suggest that the EAP provided widespread access to COVID-19 convalescent plasma in all 50 states, including for underserved racial and ethnic minority populations. The study design of the EAP may serve as a model for future efforts when broad access to a treatment is needed in response to an emerging infectious disease., Trial Registration: ClinicalTrials.gov NCT#: NCT04338360., Competing Interests: The authors have declared that no competing interests exist.

Ultrasound can be used to assess injury and structural changes to the soft-tissue structure of the foot. It may be useful to assess the feet of people with diabetes who are at increased risk of plantar soft-tissue pathological changes. The aim of this study was to determine if ultrasound measurements of plantar soft-tissue thickness and assessments of tissue acoustic characteristics are reliable in people with and without diabetes mellitus. A repeated measures design was used to determine intra-observer reliability for ultrasound measurements of plantar skin and fat pad thickness and intra- and inter-observer reliability of plantar skin and fat pad tissue characterisation assessments made at foot sites which are at risk of tissue injury in people with diabetes. Thickness measurements and tissue characterisation assessments were obtained at the heel and forefoot in both the unloaded and compressed states and included discrete layers of the plantar tissues: skin, microchamber, horizontal fibrous band, macrochamber and total soft-tissue depth. At each site, relative intra-observer reliability was achieved for the measurement of at least one plantar tissue layer. The total soft-tissue thickness measured in the unloaded state (ICC 0.925-0.976) demonstrated intra-observer reliability and is the most sensitive for detecting small change on repeated measures. Intra-observer agreement was demonstrated for tissue characteristic assessments of the skin at the heel (k = 0.70), fat pad at the lateral sesamoid region (k = 0.70) and both skin and fat pad at the second (k = 0.80, k = 0.70 respectively) and third metatarsal heads (k = 0.90, k = 0.79 respectively). However, acceptable inter-observer agreement was not demonstrated for any tissue characteristic assessment, therefore the use of multiple observers should be avoided when making these assessments., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The postpartum period can be challenging for women living with HIV. Understanding how the postpartum period impacts ART adherence and condomless sex could inform the development of comprehensive sexual and reproductive health and HIV services tailored to the needs of women living with HIV during this critical interval., Methods: In a longitudinal cohort study of HIV-seropositive Kenyan women, late ART refills and self-reported condomless sex were compared between the woman's pregnancy and the postpartum period. Analyses were conducted using generalized estimating equations and adjusted for alcohol use, depressive symptoms, intimate partner violence (IPV), and having a recent regular partner. Effect modification was explored for selected variables., Results and Discussion: 151 women contributed visits. Late ART refills occurred at 7% (32/439) of pregnancy visits compared to 18% (178/1016) during the postpartum period (adjusted relative risk [aRR] 2.44, 95% confidence interval [CI] 1.62-3.67). This association differed by women's education level. Women with ≥8 years of education had late ART refills more during the postpartum period than pregnancy (aRR 3.00, 95%CI 1.95-4.62). In contrast, in women with <8 years of education, late ART refills occurred similarly during pregnancy and the postpartum period (aRR 0.88, 95%CI 0.18-4.35). Women reported condomless sex at 10% (60/600) of pregnancy visits compared to 7% (72/1081) of postpartum visits (aRR 0.76, 95%CI 0.45-1.27). This association differed by whether women had experienced recent IPV. Women without recent IPV had a significant decline in condomless sex from pregnancy to postpartum (aRR 0.53, 95%CI 0.30-0.95) while women with recent IPV had no significant change in condomless sex from pregnancy to postpartum (aRR 1.76, 95%CI 0.87-3.55)., Conclusion: Improved support for ART adherence during the postpartum period and addressing IPV to limit condomless sex could improve HIV treatment and prevention outcomes for HIV-seropositive women as well as their infants and sexual partners., Competing Interests: BAR received compensation from Gilead for Data Safety Monitoring Board membership and Mock FDA Panel membership. RSM currently receives research funding, paid to the University of Washington from Hologic Corporation and has received honoraria for consulting from Lupin Pharmaceuticals Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

How organisms control when to transition between different stages of development is a key question in biology. In plants, epigenetic silencing by Polycomb repressive complex 1 (PRC1) and PRC2 plays a crucial role in promoting developmental transitions, including from juvenile-to-adult phases of vegetative growth. PRC1/2 are known to repress the master regulator of vegetative phase change, miR156, leading to the transition to adult growth, but how this process is regulated temporally is unknown. Here we investigate whether transcription factors in the VIVIPAROUS/ABI3-LIKE (VAL) gene family provide the temporal signal for the epigenetic repression of miR156. Exploiting a novel val1 allele, we found that VAL1 and VAL2 redundantly regulate vegetative phase change by controlling the overall level, rather than temporal dynamics, of miR156 expression. Furthermore, we discovered that VAL1 and VAL2 also act independently of miR156 to control this important developmental transition. In combination, our results highlight the complexity of temporal regulation in plants., Competing Interests: The authors have declared that no competing interests exist.

Spatial inhibition of return (IOR) refers to the phenomenon by which individuals are slower to respond to stimuli appearing at a previously cued location compared to un-cued locations. Here with a group of older adults (n = 56, 58-80 (67.9±5.2) year old, 31 females, 18.7±3.6 years of education), we provide evidence supporting the notion that spatial IOR is mildly impaired in individuals with mild cognitive impairment (MCI) or mild Alzheimer's disease (AD), and the impairment is detectable using a double cue paradigm. Furthermore, reduced spatial IOR in high-risk healthy older individuals is associated with reduced memory and other neurocognitive task performance, suggesting that the double cue spatial IOR paradigm may be useful in detecting MCI and early AD., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Oral potentially malignant disorders (OPMDs) encompass histologically benign, dysplastic, and cancerous lesions that are often indistinguishable by appearance and inconsistently managed. We assessed the potential impact of test-and-treat pathways enabled by a point-of-care test for OPMD characterization., Materials and Methods: We constructed a decision-analytic model to compare life expectancy of test-treat strategies for 60-year-old patients with OPMDs in the primary dental setting, based on a trial for a point-of-care cytopathology tool (POCOCT). Eight strategies of OPMD detection and evaluation were compared, involving deferred evaluation (no further characterization), prompt OPMD characterization using POCOCT measurements, or the commonly recommended usual care strategy of routine referral for scalpel biopsy. POCOCT pathways differed in threshold for additional intervention, including surgery for any dysplasia or malignancy, or for only moderate or severe dysplasia or cancer. Strategies with initial referral for biopsy also reflected varied treatment thresholds in current practice between surgery and surveillance of mild dysplasia. Sensitivity analysis was performed to assess the impact of variation in parameter values on model results., Results: Requisite referral for scalpel biopsy offered the highest life expectancy of 20.92 life-years compared with deferred evaluation (+0.30 life-years), though this outcome was driven by baseline assumptions of limited patient adherence to surveillance using POCOCT. POCOCT characterization and surveillance offered only 0.02 life-years less than the most biopsy-intensive strategy, while resulting in 27% fewer biopsies. When the probability of adherence to surveillance and confirmatory biopsy was ≥ 0.88, or when metastasis rates were lower than reported, POCOCT characterization extended life-years (+0.04 life-years) than prompt specialist referral., Conclusion: Risk-based OPMD management through point-of-care cytology may offer a reasonable alternative to routine referral for specialist evaluation and scalpel biopsy, with far fewer biopsies. In patients who adhere to surveillance protocols, POCOCT surveillance may extend life expectancy beyond biopsy and follow up visual-tactile inspection., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: SKK reports royalties from Wolters Kluwer and honorarium from the American Journal of Roentgenology for work unrelated to this study. JTM has a provisional patent pending for Screening and Assessment of Potentially Malignant Oral Lesions. (McDevitt J.T., McRae M.P. Provisional Patent, 31K5390-206256-0028-Corporation P1US). In addition, he has ownership positions and an equity interest in both SensoDx II LLC and OraLiva, INC. and serves on their Scientific Advisory Boards. All other authors declare no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Excessive alcohol intake has been associated with poor adherence to antiretroviral therapy (ART). The impact of alcohol on viral suppression is particularly important among groups at high risk of HIV transmission, such as female sex workers (FSWs). Few studies have directly evaluated the association between alcohol use and HIV viral load. We hypothesized that hazardous or harmful alcohol use is associated with detectable plasma viral load among HIV-positive FSWs., Methods: A prospective cohort study was conducted among HIV-positive FSWs in Mombasa, Kenya. Hazardous or harmful alcohol use was assessed yearly and defined as an Alcohol Use Disorders Identification Test (AUDIT) score ≥7. Detectable viral load was assessed every six months and defined as ≥180 c/mL. Adherence measures were collected monthly and included late ART refill (>48 hours) and self-reported adherence, using both a validated self-rating scale of ability to take medication and visual analog scale (VAS) of ART use in the last month. Generalized estimating equations were used to estimate adjusted relative risks (aRR) and 95% confidence intervals (CI)., Results: This analysis included 366 participants followed monthly between October 2012 and March 2018. At baseline, AUDIT scores indicated hazardous alcohol use (AUDIT 7-15) in 14.3%, harmful alcohol use (AUDIT 16-19) in 1.4%, and alcohol dependency (AUDIT ≥20) in 1.4% of participants. After adjusting for potential confounders, a combined exposure including hazardous, harmful, and dependent alcohol use was not associated with detectable viral load (aRR 1.10, 95%CI 0.63-1.92) or late ART refill (aRR 1.13, 95%CI 0.82-1.56), but was associated with lower self-rated ability to take medication (aRR 2.38, 95%CI 1.42-3.99) and a lower rate of self-reported perfect ART adherence by VAS (aRR 2.62, 95%CI 1.84-3.71)., Conclusions: In this FSW cohort, while participants reporting hazardous, harmful, or dependent alcohol use were not more likely to have a detectable viral load, they were more likely to report lower ART adherence. These results suggest that interventions targeting alcohol use among this population of FSWs may not have a large impact on viral suppression., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RSM receives research funding for a study of high-risk HPV collection methods, paid to the University of Washington, from Hologic Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors declared that no competing interests exist.

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo., Competing Interests: The authors have declared that no competing interests exist.

Trehalose metabolism in yeast has been linked to a variety of phenotypes, including heat resistance, desiccation tolerance, carbon-source utilization, and sporulation. The relationships among the several phenotypes of mutants unable to synthesize trehalose are not understood, even though the pathway is highly conserved. One of these phenotypes is that tps1Δ strains cannot reportedly grow on media containing glucose or fructose, even when another carbon source they can use (e.g. galactose) is present. Here we corroborate the recent observation that a small fraction of yeast tps1Δ cells do grow on glucose, unlike the majority of the population. This is not due to a genetic alteration, but instead resembles the persister phenotype documented in many microorganisms and cancer cells undergoing lethal stress. We extend these observations to show that this phenomenon is glucose-specific, as it does not occur on another highly fermented carbon source, fructose. We further demonstrate that this phenomenon appears to be related to mitochondrial complex III function, but unrelated to inorganic phosphate levels in the cell, as had previously been suggested. Finally, we found that this phenomenon is specific to S288C-derived strains, and is the consequence of a variant in the MKT1 gene., Competing Interests: The authors have read the journal’s policy and have the following conflicts: authors PAG, DGH, RSM, and DB are affiliated with Calico Life Sciences LLC. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-562271) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further support the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for the discovery of new chemotherapeutic agents for a range of diseases., Competing Interests: The authors have declared that no competing interests exist.

Significant population declines in Acropora cervicornis and A. palmata began in the 1970s and now exceed over 90%. The losses were caused by a combination of coral disease and bleaching, with possible contributions from other stressors, including pollution and predation. Reproduction in the wild by fragment regeneration and sexual recruitment is inadequate to offset population declines. Starting in 2007, the Coral Restoration Foundation™ evaluated the feasibility of outplanting A. cervicornis colonies to reefs in the Florida Keys to restore populations at sites where the species was previously abundant. Reported here are the results of 20 coral outplanting projects with each project defined as a cohort of colonies outplanted at the same time and location. Photogrammetric analysis and in situ monitoring (2007 to 2015) measured survivorship, growth, and condition of 2419 colonies. Survivorship was initially high but generally decreased after two years. Survivorship among projects based on colony counts ranged from 4% to 89% for seven cohorts monitored at least five years. Weibull survival models were used to estimate survivorship beyond the duration of the projects and ranged from approximately 0% to over 35% after five years and 0% to 10% after seven years. Growth rate averaged 10 cm/year during the first two years then plateaued in subsequent years. After four years, approximately one-third of surviving colonies were ≥ 50 cm in maximum diameter. Projects used three to sixteen different genotypes and significant differences did not occur in survivorship, condition, or growth. Restoration times for three reefs were calculated based on NOAA Recovery Plan (NRP) metrics (colony abundance and size) and the findings from projects reported here. Results support NRP conclusions that reducing stressors is required before significant population growth and recovery will occur. Until then, outplanting protects against local extinction and helps to maintain genetic diversity in the wild., Competing Interests: We have read the journal's policy and Steven Miller may have a competing interest: He is on the board of the Coral Restoration Foundation.

Background: The ECHO trial has relieved apprehension about intramuscular depot medroxyprogesterone acetate (DMPA-IM), however it is still important to understand how DMPA-IM affects the vaginal environment. We sought to describe how DMPA-IM initiation influences vaginal bacteria associated with HIV acquisition in postpartum women., Methods: Vaginal swabs were collected for Nugent score determination and taxon-specific quantitative PCR of eight bacteria. Enrollment occurred at contraceptive initiation (DMPA-IM or non-hormonal contraception (non-HC)) and repeat vaginal swabs were collected after three months. Generalized estimating equations were used to estimate changes in Nugent score, total bacterial load, and taxa concentrations among contraceptive groups., Results: Women who chose DMPA-IM (n = 33) were more likely to be married (97%vs.67%) and have resumed intercourse since delivery (52%vs.29%) compared to women who chose non-HC (n = 21). After three months, significant decreases in the concentrations of Sneathia species, Mycoplasma hominis, and Parvimonas species Type 1 were seen among non-HC users, however concentrations remained stable among DMPA-IM users; contraceptive method was associated with significantly different changes in M. hominis concentration between groups (p = 0.010)., Conclusions: Our findings suggest that postpartum use of DMPA-IM and non-HC may have differential impacts on the vaginal concentrations of some bacteria that have previously been associated with HIV acquisition., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: David N. Fredricks has a financial relationship with BD for licensure of molecular diagnosis of BV. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A reservoir of HIV-infected cells that persists despite suppressive antiretroviral therapy (ART) is the source of viral rebound upon ART cessation and the major barrier to a cure. Understanding reservoir seeding dynamics will help identify the best timing for HIV cure strategies. Here we characterize reservoir seeding using longitudinal samples from before and after ART initiation in individuals who sequentially became infected with genetically distinct HIV variants (superinfected). We previously identified cases of superinfection in a cohort of Kenyan women, and the dates of both initial infection and superinfection were determined. Six women, superinfected 0.2-5.2 years after initial infection, were subsequently treated with ART 5.4-18.0 years after initial infection. We performed next-generation sequencing of HIV gag and env RNA from plasma collected during acute infection as well as every ~2 years thereafter until ART initiation, and of HIV DNA from PBMCs collected 0.9-4.8 years after viral suppression on ART. We assessed phylogenetic relationships between HIV DNA reservoir sequences and longitudinal plasma RNA sequences prior to ART, to determine proportions of initial and superinfecting variants in the reservoir. The proportions of initial and superinfection lineage variants present in the HIV DNA reservoir were most similar to the proportions present in HIV RNA immediately prior to ART initiation. Phylogenetic analysis confirmed that the majority of HIV DNA reservoir sequences had the smallest pairwise distance to RNA sequences from timepoints closest to ART initiation. Our data suggest that while reservoir cells are created throughout pre-ART infection, the majority of HIV-infected cells that persist during ART entered the reservoir near the time of ART initiation. We estimate the half-life of pre-ART DNA reservoir sequences to be ~25 months, which is shorter than estimated reservoir decay rates during suppressive ART, implying continual decay and reseeding of the reservoir up to the point of ART initiation., Competing Interests: The authors have declared that no competing interests exist.

Background: Many regions of sub-Saharan Africa experience a high prevalence of both schistosomiasis and HIV-1, leading to frequent coinfection. Higher plasma HIV-1 viral loads are associated with faster disease progression and genital HIV-1 loads are a primary determinant of HIV-1 transmission risk. We hypothesized that schistosome infection would be associated with higher HIV-1 viral loads in plasma and genital samples., Methods/principal Findings: We utilized data from individuals who HIV-1 seroconverted while enrolled in one of four prospective cohort studies. Plasma and genital viral loads collected 4-24 months after the estimated date of HIV-1 acquisition, but prior to antiretroviral therapy initiation, were included. Detection of circulating anodic antigen in archived blood samples, collected prior to HIV-1 seroconversion, identified participants with active schistosomiasis; immunoblots determined the schistosome species causing infection. Our analysis included 370 HIV-1 seroconverters with plasma viral load results, of whom 82 (22%) had schistosomiasis. We did not find a statistically significant association between schistosomiasis and higher HIV-1 set point plasma viral loads (-0.17 log10 copies/ml, 95% CI -0.38 to 0.03); S. mansoni infection was associated with a lower set point (-0.34 log10 copies/ml, 95% CI -0.58 to -0.09). We found no association between schistosomiasis and cervical (+0.07 log10 copies/swab, 95% CI -0.20 to 0.34) or vaginal (+0.11 log10 copies/swab, 95% CI -0.17 to 0.39) set point viral loads; S. haematobium infection was associated with lower cervical viral loads (-0.59 log10 copies/swab, 95% CI -1.11 to -0.06)., Conclusions/significance: These results do not support the hypotheses that schistosome coinfection increases plasma or genital HIV-1 viral loads., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RSM has received funding for research, paid to the University of Washington, from Hologic Corporation. All other authors have declared that no competing interests exist.

Balancer chromosomes are multiply inverted and rearranged chromosomes that are widely used in Drosophila genetics. First described nearly 100 years ago, balancers are used extensively in stock maintenance and complex crosses. Recently, the complete molecular structures of several commonly used balancers were determined by whole-genome sequencing. This revealed a surprising amount of variation among balancers derived from a common progenitor, identified genes directly affected by inversion breakpoints, and cataloged mutations shared by balancers. These studies emphasized that it is important to choose the optimal balancer, because different inversions suppress meiotic recombination in different chromosomal regions. In this review, we provide a brief history of balancers in Drosophila, discuss how they are used today, and provide examples of unexpected recombination events involving balancers that can lead to stock breakdown., Competing Interests: The authors have declared that no competing interests exist.

Background: Timely diagnosis of infant HIV infection is essential for antiretroviral therapy (ART) initiation. In a randomized controlled trial, we found the Texting Improves Testing (TextIT) intervention (a theory-based text messaging system) to be efficacious for improving infant HIV testing rates and maternal retention in prevention of mother-to-child HIV transmission (PMTCT) programs. Using an implementation science approach, we aimed to evaluate real-world effectiveness of the intervention., Methods and Findings: In a pragmatic, cluster-randomized, stepped-wedge trial with 2 time periods of observation, we randomly allocated 10 clinics to begin implementing the intervention immediately and 10 clinics to begin implementing 6 months later. To approximate real-world conditions, inclusion criteria were broad. Women at clinics implementing the intervention received up to 14 text messages during pregnancy and after delivery and had the option to respond to text messages, call, or send inquiry text messages to a designated clinic phone. The primary outcomes were infant HIV testing and maternal retention in care during the first 8 weeks after delivery. We used modified Poisson regression with robust variance estimation to estimate the relative risk and 95% confidence intervals (CIs). Generalized estimating equations were applied on individual-level data to account for clustering by site. Between February 2015 and December 2016, 4,681 women were assessed for study participation, and 2,515 were included. Participant characteristics at enrollment did not differ by study arm. Overall median age was 27 years (interquartile range [IQR] 23-30), median gestational age was 30 weeks (IQR 28-34), 99% were receiving ART, and 87% who enrolled during intervention phases owned a phone. Of 2,326 infants analyzed, 1,466 of 1,613 (90.9%) in the intervention group and 609 of 713 (85.4%) in the control group met the primary outcome of HIV virologic testing performed before 8 weeks after birth (adjusted relative risk [aRR] 1.03; 95% CI 0.97-1.10; P = 0.3). Of 2,472 women analyzed, 1,548 of 1,725 (90%) in the intervention group and 571 of 747 (76%) in the control group met the primary outcome of retention in care during the first 8 weeks after delivery (aRR 1.12; 95% CI 0.97-1.30; P = 0.1). This study had two main limitations. Staff at all facilities were aware of ongoing observation, which may have contributed to increased rates of infant HIV testing and maternal retention in care at both intervention and control facilities, and programmatic initiatives to improve maternal and infant retention in care were ongoing at all facilities at the time of this study, which likely limited the ability to demonstrate effectiveness of the trial intervention., Conclusions: In this study, a larger proportion of infants in the intervention group received HIV testing compared with the control group, but the difference was small and not statistically significant. There was also a nonsignificant increase in maternal postpartum retention in the intervention periods. Despite the lack of a significant effect of the intervention, key lessons emerged, both for strengthening PMTCT and for implementation research in general. Perhaps most important, improving the implementation of usual care may have been sufficient to substantially improve infant HIV testing rates., Trial Registration: ClinicalTrials.gov Trial Number NCT02350140., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RSM receives research funding, paid to the University of Washington, from Hologic Corporation. EHG is a member of the Editorial Board of PLOS Medicine.

Background: Hepatocelluar cancer (HCC) is the leading cause of death among people with hepatitis C virus (HCV)-related cirrhosis. Our aim was to determine the optimal surveillance frequency for patients with HCV-related compensated cirrhosis., Methods: We developed a decision analytic Markov model and validated it against data from the Veterans Outcomes and Costs Associated with Liver Disease (VOCAL) study group and published epidemiologic studies. Four strategies of different surveillance intervals were compared: no surveillance and ultrasound surveillance every 12, 6, and 3 months. We estimated lifetime survival, life expectancy, quality adjusted life years (QALY), total costs associated with each strategy, and incremental cost effectiveness ratios. We applied a willingness to pay threshold of $100,000. Analysis was conducted for two scenarios: a scenario reflecting current HCV and HCC surveillance compliance rates and treatment use and an aspirational scenario., Results: In the current scenario the preferred strategy was 3-month surveillance with an incremental cost-effectiveness ratio (ICER) of $7,159/QALY. In the aspirational scenario, 6-month surveillance was preferred with an ICER of $82,807/QALY because treating more people with HCV led to a lower incidence of HCC. Sensitivity analyses suggested that surveillance every 12 months would suffice in the particular circumstance when patients are very likely to return regularly for testing and when appropriate HCV and HCC treatment is readily available. Compared with the current scenario, the aspirational scenario resulted in a 1.87 year gain in life expectancy for the cohort because of large reductions in decompensated cirrhosis and HCC incidence., Conclusions: HCC surveillance has good value for money for patients with HCV-related compensated cirrhosis. Investments to improve adherence to surveillance should be made when rates are suboptimal. Surveillance every 12 months will suffice when patients are very likely to return regularly for testing and when appropriate HCV and HCC treatment is readily available., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Female sex workers (FSWs) in sub-Saharan Africa are a key population for HIV prevention and treatment interventions, but less attention is given to their family planning needs. We evaluated the prevalence and predictors of unmet contraceptive need in HIV-positive FSWs., Study Design: This cross-sectional analysis used data from an existing longitudinal study of FSWs in Mombasa, Kenya. This analysis included women who were HIV positive, age ≥18 years, pre-menopausal, not currently pregnant or desiring pregnancy, and reported exchanging sex for cash or in-kind payment at the time of enrollment. Unmet contraceptive need was defined as non-use of modern non-barrier contraceptives and not currently trying to become pregnant. Poisson regression was used to identify factors independently associated with unmet contraceptive need., Results: Among 346 HIV-positive FSWs, 125 (36.1%) reported modern non-barrier contraceptive use, leaving 221 (63.9%, 95%CI 58.8-68.9%) with unmet contraceptive need. Condom use was the only form of contraception for 129 (37.3%) participants. In unadjusted analyses, unmet contraceptive need was associated with physical abuse in the past year by someone other than a regular partner (PR 1.2, 95%CI 1.0-1.5), desire for (more) children (PR 1.3, 95%CI 1.1-1.5), and having 2-3 previous pregnancies compared to 0-1 prior pregnancies (PR 0.8, 95%CI 0.6-0.9). In adjusted analyses, lower number of previous pregnancies and having desire for future children remained significantly associated with a higher prevalence of unmet contraceptive need., Conclusions: Unmet need for modern non-barrier contraception was found in two-thirds of HIV-positive FSWs who reported that they were not currently trying to become pregnant, and was higher in women with the lowest number of prior pregnancies (0-1 prior pregnancies) and in those reporting desire for (more) children in the future. These findings highlight the need for concerted efforts to identify and eliminate barriers to contraceptive use in FSWs living with HIV., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RSM receives research funding for a study of high-risk HPV collection methods, paid to the University of Washington, from Hologic Corporation. All other authors declared that no competing interests exist. We confirm that the listed competing interests do not alter our adherence to PLOS ONE data sharing policies.

During early meiotic prophase, homologous chromosomes are connected along their entire lengths by a proteinaceous tripartite structure known as the synaptonemal complex (SC). Although the components that comprise the SC are predominantly studied in this canonical ribbon-like structure, they can also polymerize into repeated structures known as polycomplexes. We find that in Drosophila oocytes, the ability of SC components to assemble into canonical tripartite SC requires the E3 ubiquitin ligase Seven in absentia (Sina). In sina mutant oocytes, SC components assemble into large rod-like polycomplexes instead of proper SC. Thus, the wild-type Sina protein inhibits the polymerization of SC components, including those of the lateral element, into polycomplexes. These polycomplexes persist into meiotic stages when canonical SC has been disassembled, indicating that Sina also plays a role in controlling SC disassembly. Polycomplexes induced by loss-of-function sina mutations associate with centromeres, sites of double-strand breaks, and cohesins. Perhaps as a consequence of these associations, centromere clustering is defective and crossing over is reduced. These results suggest that while features of the polycomplexes can be recognized as SC by other components of the meiotic nucleus, polycomplexes nonetheless fail to execute core functions of canonical SC., Competing Interests: The authors have declared that no competing interests exist.

Background: A study of a comprehensive HIV Care Coordination Program (CCP) showed effectiveness in increasing viral load suppression (VLS) among PLWH in New York City (NYC). We evaluated the cost-effectiveness of a scale-up of the CCP in NYC., Methods: We incorporated observed effects and costs of the CCP into a computer simulation of HIV in NYC, comparing strategy scale-up with no implementation. The simulation combined a deterministic compartmental model of HIV transmission with a stochastic microsimulation of HIV progression, and was calibrated to NYC HIV epidemiological data from 1997 to 2009. We assessed incremental cost-effectiveness from a health sector perspective using 2017 $US, a 20-year time horizon, and a 3% annual discount rate. We explored two scenarios: (1) two-year average enrollment and (2) continuous enrollment., Results: In scenario 1, scale-up resulted in a cost-per-infection-averted of $898,104 and a cost-per-QALY-gained of $423,721. In sensitivity analyses, scale-up achieved cost-effectiveness if effectiveness increased from RR1.11 to RR1.37 or costs decreased by 41.7%. Limiting the intervention to persons with unsuppressed viral load prior to enrollment (RR1.32) attenuated the cost reduction necessary to 11.5%. In scenario 2, scale-up resulted in a cost-per-infection-averted of $705,171 and cost-per-QALY-gained of $720,970. In sensitivity analyses, scale-up achieved cost-effectiveness if effectiveness increased from RR1.11 to RR1.46 or program costs decreased by 71.3%. Limiting the intervention to persons with unsuppressed viral load attenuated the cost reduction necessary to 38.7%., Conclusion: Cost-effective CCP scale-up would require reduced costs and/or focused enrollment within NYC, but may be more readily achieved in cities with lower background VLS levels., Competing Interests: The authors have declared that no competing interests exist.

Mussels of the genus Bathymodiolus are among the most widespread colonizers of hydrothermal vent and cold seep environments, sustained by endosymbiosis with chemosynthetic bacteria. Presumed species of Bathymodiolus are abundant at newly discovered cold seeps on the Mid-Atlantic continental slope, however morphological taxonomy is challenging, and their phylogenetic affinities remain unestablished. Here we used mitochondrial sequence to classify species found at three seep sites (Baltimore Canyon seep (BCS; ~400m); Norfolk Canyon seep (NCS; ~1520m); and Chincoteague Island seep (CTS; ~1000m)). Mitochondrial COI (N = 162) and ND4 (N = 39) data suggest that Bathymodiolus childressi predominates at these sites, although single B. mauritanicus and B. heckerae individuals were detected. As previous work had suggested that methanotrophic and thiotrophic interactions can both occur at a site, and within an individual mussel, we investigated the symbiont communities in gill tissues of a subset of mussels from BCS and NCS. We constructed metabarcode libraries with four different primer sets spanning the 16S gene. A methanotrophic phylotype dominated all gill microbial samples from BCS, but sulfur-oxidizing Campylobacterota were represented by a notable minority of sequences from NCS. The methanotroph phylotype shared a clade with globally distributed Bathymodiolus spp. symbionts from methane seeps and hydrothermal vents. Two distinct Campylobacterota phylotypes were prevalent in NCS samples, one of which shares a clade with Campylobacterota associated with B. childressi from the Gulf of Mexico and the other with Campylobacterota associated with other deep-sea fauna. Variation in chemosynthetic symbiont communities among sites and individuals has important ecological and geochemical implications and suggests shifting reliance on methanotrophy. Continued characterization of symbionts from cold seeps will provide a greater understanding of the ecology of these unique environments as well and their geochemical footprint in elemental cycling and energy flux., Competing Interests: CSA Ocean Sciences, Inc was the management entity, and our data sharing policies were dictated by the funders – which were federal, and therefore had open data sharing. This does not alter our adherence to PLOS ONE policies on sharing data and material.

Vaginal washing is a common practice associated with adverse outcomes including bacterial vaginosis (BV) and HIV infection. Prior studies have not examined the associations between vaginal washing and individual vaginal bacteria, or whether these associations are independent of the effect of vaginal washing on BV. The purpose of this study was to characterize the association between vaginal washing and the presence and concentrations of vaginal bacteria associated with optimal and sub-optimal vaginal states. The analysis utilized data from participants in the placebo arm of the Preventing Vaginal Infections trial, which enrolled HIV-uninfected women from the United States and Kenya. Detection of bacterial taxa associated with BV was compared between visits with versus without reported vaginal washing. The effect of vaginal washing on a number of vaginal bacteria differed substantially (p<0.05) between the US and Kenya, so results were stratified by country. In US women, vaginal washing was associated with a significantly higher likelihood of detection of BV associated bacterium 1 (BVAB1) (relative risk [RR] 1.55, 95% confidence interval [CI] 1.15-2.09, p = 0.004), BVAB2 (RR 1.99, 95%CI 1.46-2.71, p<0.001), Mageeibacillus indolicus (RR 2.08, 95%CI 1.46-2.96, p<0.001), Atopobium vaginae (RR 1.34, 95%CI 1.13-1.59, p = 0.001), Leptotrichia/Sneathia species (RR 1.66, 95% CI 1.33-2.09, p<0.001), Megasphaera species (RR 1.78, 95%CI 1.34-2.37, p<0.001) and Gardnerella vaginalis (RR 1.08, 95%CI 1.01-1.16, p = 0.02). No significant association between vaginal washing and bacterial detection was found in Kenyan women. Adjustment for bacterial vaginosis diagnosed by Gram stain did not alter these results. This study provides evidence that the association between vaginal washing and detection of individual bacterial taxa can vary regionally. For some vaginal bacteria, the association with vaginal washing may be independent of the effect on Gram stain detection of BV. Larger prospective studies in diverse geographic settings should explore whether eliminating vaginal washing impacts the presence and concentrations of key vaginal bacteria., Competing Interests: RSM receives research funding, paid to the University of Washington, from Hologic Corporation. All other authors reported no conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over., Competing Interests: The authors have declared that no competing interests exist.

Objective: Text messages significantly improve uptake of infant HIV testing in clinical trial contexts. Women who were excluded from a randomized trial in Kenya were followed to create a comparison between women who were enrolled and did not receive the study SMS intervention and women who were screened but not enrolled., Design: Parallel-cohort randomized controlled trial analysis., Methods: We compared time to infant HIV testing between women in three groups: the Trial SMS group, the Trial Control group, and the Comparison Cohort comprised of women who were screened but not enrolled., Results: Of the 1,115 women screened, 388 (35%) were eligible for trial enrollment, and were randomized to receive either intervention text messages (Trial SMS; N = 195) or continue usual care (Trial Control; N = 193). Among 727 women not enrolled in the study (Comparison Cohort), we obtained infant HIV testing data from clinic records for 510 (70%). The cumulative probability of infant HIV testing was highest in the Trial SMS group (92.0%; 95% CI 87.5-95.3), followed by the Trial Control group (85.1%; 95% CI 79.5-89.8), and lowest among women in the Comparison Cohort (43.4%; 95% CI 39.2-47.8)., Conclusions: Both the Trial SMS group and the Trial Control group were significantly more likely to have their infants tested for HIV compared to the Comparison Cohort, providing evidence of a "clinical trial effect." This analysis suggests that SMS interventions should be implemented as an adjunct to consistent and engaged delivery of basic health services., Competing Interests: Dr. McClelland receives research funding, paid to the University of Washington, from Hologic Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: HIV-1-discordant couples that remain discordant despite repeated exposure may differ from the general population in their distribution of transmission risk factors, including low plasma viral load (PVL) in the infected partner even in the absence of antiretroviral therapy (ART)., Methods: We followed two cohorts of HIV-1-infected Kenyan women: females in discordant couples (FDC) and female sex workers (FSW). We compared the distribution of undetectable (<150 copies/mL) and low PVL (<1,000 copies/mL) between the cohorts using bootstrap methods and exact Poisson regression., Results: We evaluated 296 FDC and 220 FSW. At baseline, FDC were more likely to have undetectable (RR = 6.94, bootstrap 95% CI: 3.47, 20.81) and low PVL (RR = 3.53, bootstrap 95% CI: 2.57, 5.65) than FSW. Similarly, both repeat undetectable PVL (RR = 9.36, bootstrap 95% CI: 6.04, 10.97) and repeat low (RR = 4.99, bootstrap 95% CI: 2.33, 14.04) PVL were more likely among FDC than FSW during follow-up., Discussion: We observed higher prevalence of viral control in FDC compared to FSW in the absence of ART, suggesting potentially higher prevalence of biological HIV resistance factors among discordant couples., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Link4Health, a cluster-RCT, demonstrated the effectiveness of a combination strategy targeting barriers at various HIV continuum steps on linkage to and retention in care; showing effectiveness in achieving linkage to HIV care within 1 month plus retention in care at 12 months after HIV testing for people living with HIV (RR 1.48, 95% CI 1.19-1.96, p = 0.002). In addition to standard of care, Link4Health included: 1) Point-of-care CD4+ count testing; 2) Accelerated ART initiation; 3) Mobile phone appointment reminders; 4) Care and prevention package including commodities and informational materials; and 5) Non-cash financial incentive. Our objective was to evaluate the cost-effectiveness of a scale-up of the Link4Health strategy in Swaziland., Methods and Findings: We incorporated the effects and costs of the Link4Health strategy into a computer simulation of the HIV epidemic in Swaziland, comparing a scenario where the strategy was scaled up to a scenario with no implementation. The simulation combined a deterministic compartmental model of HIV transmission with a stochastic microsimulation of HIV progression calibrated to Swaziland epidemiological data. It incorporated downstream health costs potentially saved and infections potentially prevented by improved linkage and treatment adherence. We assessed the incremental cost-effectiveness ratio of Link4Health compared to standard care from a health sector perspective reported in US$2015, a time horizon of 20 years, and a discount rate of 3% in accordance with WHO guidelines.[1] Our results suggest that scale-up of the Link4Health strategy would reduce new HIV infections over 20 years by 11,059 infections, a 7% reduction from the projected 169,019 cases and prevent 5,313 deaths, an 11% reduction from the projected 49,582 deaths. Link4Health resulted in an incremental cost per infection prevented of $13,310 and an incremental cost per QALY gained of $3,560/QALY from the health sector perspective., Conclusions: Using a threshold of <3 x per capita GDP, the Link4Health strategy is likely to be a cost-effective strategy for responding to the HIV epidemic in Swaziland., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

Background: As access to antiretroviral therapy in sub-Saharan Africa continues to expand, more women with HIV can expect to survive through their reproductive years. Modern contraceptives can help women choose the timing and spacing of childbearing. However, concerns remain that women with HIV who use non-barrier forms of modern contraception may engage in more condomless sex because of their decreased risk of unintended pregnancy. We examined whether non-barrier modern contraceptive use by HIV-positive female sex workers was associated with increased frequency of recent condomless sex, measured by detection of prostate-specific antigen (PSA) in vaginal secretions., Methods: Women who were HIV-positive and reported transactional sex were included in this analysis. Pregnant and post-menopausal follow-up time was excluded, as were visits at which women reported trying to get pregnant. At enrollment and quarterly follow-up visits, a pelvic speculum examination with collection of vaginal secretions was conducted for detection of PSA. In addition, women completed a structured face-to-face interview about their current contraceptive methods and sexual risk behavior at enrollment and monthly follow-up visits. Log-binomial generalized estimating equations regression was used to test for associations between non-barrier modern contraceptive use and detection of PSA in vaginal secretions and self-reported condomless sex. Data from October 2012 through September 2014 were included in this analysis., Results: Overall, 314 women contributed 1,583 quarterly examination visits. There was minimal difference in PSA detection at contraceptive-exposed versus contraceptive-unexposed visits (adjusted relative risk [aRR] 1.28, 95% confidence interval [95% CI] 0.93-1.76). There was a higher rate of self-reported condomless sex at visits where women reported using modern contraceptives, but this difference was not statistically significant after adjustment for potential confounding factors (aRR 1.59, 95% CI 0.98-2.58)., Conclusion: Non-barrier methods of modern contraception were not associated with increased risk of objective evidence of condomless sex.

Background: Tafenoquine is an investigational 8-aminoquinoline for the prevention of Plasmodium vivax relapse. Tafenoquine has a long half-life and the potential for more convenient dosing, compared with the currently recommended 14-day primaquine regimen., Methods: This randomized, active-control, double-blind trial was conducted in Bangkok, Thailand. Seventy patients with microscopically confirmed P. vivax were randomized (2:1) to tafenoquine 400 mg once daily for 3 days or 2500 mg total dose chloroquine phosphate (1500 mg chloroquine base) given over 3 days plus primaquine 15 mg daily for 14 days. Patients were followed to day 120., Results: Day 28 adequate clinical response rate in the per-protocol population was 93% (40/43) (90%CI 83-98%) with tafenoquine, and 100% (22/22) (90%CI 87-100%) with chloroquine/primaquine. Day 120 relapse prevention was 100% (35/35) with tafenoquine (90%CI 92-100%), and 95% (19/20) (90%CI 78-100%) with chloroquine/primaquine. Mean (SD) parasite, gametocyte and fever clearance times with tafenoquine were 82.5 h (32.3), 49.1 h (33.0), and 41.1 h (31.4) versus 40.0 h (15.7), 22.7 h (16.4), and 24.7 h (17.7) with chloroquine/primaquine, respectively. Peak methemoglobin was 1.4-25.6% (median 7.4%, mean 9.1%) in the tafenoquine arm, and 0.5-5.9% (median 1.5%, mean 1.9%) in the chloroquine/primaquine arm. There were no clinical symptoms of methemoglobinemia in any patient., Discussion: Although there was no difference in efficacy in this study, the slow rate of parasite, gametocyte and fever clearance indicates that tafenoquine should not be used as monotherapy for radical cure of P. vivax malaria. Also, monotherapy increases the potential risk of resistance developing to this long-acting agent. Clinical trials of single-dose tafenoquine 300 mg combined with standard 3-day chloroquine or artemisinin-based combination therapy are ongoing., Trial Registration: Clinicaltrials.gov NCT01290601.

Tropical wetlands are thought to be the most important source of interannual variability in atmospheric methane (CH4) concentrations, yet sparse data prevents them from being incorporated into Earth system models. This problem is particularly pronounced in the neotropics where bottom-up models based on water table depth are incongruent with top-down inversion models suggesting unaccounted sinks or sources of CH4. The newly documented vast areas of peatlands in the Amazon basin may account for an important unrecognized CH4 source, but the hydrologic and biogeochemical controls of CH4 dynamics from these systems remain poorly understood. We studied three zones of a peatland in Madre de Dios, Peru, to test whether CH4 emissions and pore water concentrations varied with vegetation community, soil chemistry and proximity to groundwater sources. We found that the open-canopy herbaceous zone emitted roughly one-third as much CH4 as the Mauritia flexuosa palm-dominated areas (4.7 ± 0.9 and 14.0 ± 2.4 mg CH4 m-2 h-1, respectively). Emissions decreased with distance from groundwater discharge across the three sampling sites, and tracked changes in soil carbon chemistry, especially increased soil phenolics. Based on all available data, we calculate that neotropical peatlands contribute emissions of 43 ± 11.9 Tg CH4 y-1, however this estimate is subject to geographic bias and will need revision once additional studies are published.

Objective: To inform the design of a combination intervention strategy targeting HIV-infected unhealthy alcohol users in Maharashtra, India, that could be tested in future randomized control trials., Methods: Using probabilistic compartmental simulation modeling we compared intervention strategies targeting HIV-infected unhealthy alcohol users on antiretroviral therapy (ART) in Maharashtra, India. We tested interventions targeting four behaviors (unhealthy alcohol consumption, risky sexual behavior, depression and antiretroviral adherence), in three formats (individual, group based, community) and two durations (shorter versus longer). A total of 5,386 possible intervention combinations were tested across the population for a 20-year time horizon and intervention bundles were narrowed down based on incremental cost-effectiveness analysis using a two-step probabilistic uncertainty analysis approach., Results: Taking into account uncertainty in transmission variables and intervention cost and effectiveness values, we were able to reduce the number of possible intervention combinations to be used in a randomized control trial from over 5,000 to less than 5. The most robust intervention bundle identified was a combination of three interventions: long individual alcohol counseling; weekly Short Message Service (SMS) adherence counseling; and brief sex risk group counseling., Conclusions: In addition to guiding policy design, simulation modeling of HIV transmission can be used as a preparatory step to trial design, offering a method for intervention pre-selection at a reduced cost.

The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.

Feline dysautonomia (FD) is a multiple system neuropathy of unknown aetiology. An apparently identical disease occurs in horses (equine grass sickness, EGS), dogs, rabbits, hares, sheep, alpacas and llamas. Horses with acute EGS have a marked reduction in plasma concentrations of the sulphur amino acids (SAA) cyst(e)ine and methionine, which may reflect exposure to a neurotoxic xenobiotic. The aim of this study was to determine whether FD cats have alterations in amino acid profiles similar to those of EGS horses. Amino acids were quantified in plasma/serum from 14 FD cats, 5 healthy in-contact cats which shared housing and diet with the FD cats, and 6 healthy control cats which were housed separately from FD cats and which received a different diet. The adequacy of amino acids in the cats' diet was assessed by determining the amino acid content of tinned and dry pelleted foods collected immediately after occurrences of FD. Compared with controls, FD cats had increased concentrations of many essential amino acids, with the exception of methionine which was significantly reduced, and reductions in most non-essential amino acids. In-contact cats also had inadequate methionine status. Artefactual loss of cysteine during analysis precluded assessment of the cyst(e)ine status. Food analysis indicated that the low methionine status was unlikely to be attributable to dietary inadequacy of methionine or cystine. Multi-mycotoxin screening identified low concentrations of several mycotoxins in dry food from all 3 premises. While this indicates fungal contamination of the food, none of these mycotoxins appears to induce the specific clinico-pathologic features which characterise FD and equivalent multiple system neuropathies in other species. Instead, we hypothesise that ingestion of another, as yet unidentified, dietary neurotoxic mycotoxin or xenobiotic, may cause both the characteristic disease pathology and the plasma SAA depletion.

Objective: Young women bear the greatest burden of sexually transmitted infections (STIs), so it is important to identify and address barriers to STI screening in this population. We conducted a qualitative study to explore the feasibility of STI screening among adolescent girls and young women in Mombasa, Kenya., Methods: We conducted 17 in-depth interviews (IDIs) (8 with adolescent girls and 9 with young women) and 6 focus group discussions (FGDs) (4 with adolescent girls and 2 with young women, total 55 participants). The audio recordings for the IDIs and FGDs were translated and transcribed into English. Transcripts were independently reviewed by two researchers, and a set of codes was designed to help analyze the data using the content analysis approach. Data content was then analyzed manually and digitally using ATLAS.ti, and consensus was reached on central and specific emergent themes discussed by the research team., Results: Adolescent girls and young women in Mombasa, Kenya expressed willingness to participate in STI screening. A major incentive for screening was participants' desire to know their STI status, especially following perceived high-risk sexual behavior. Lack of symptoms and fear of positive test results were identified as barriers to STI screening at the individual level, while parental notification and stigmatization from parents, family members and the community were identified as barriers at the community level. Uncomfortable or embarrassing methods of specimen collection were an additional barrier. Thus, urine-based screening was felt to be the most acceptable., Conclusion: Kenyan adolescent girls and young women seem willing to participate in screening for STIs using urine testing. Addressing stigmatization by parents, health care workers and the community could further facilitate STI screening in this population., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Genital ulcer disease (GUD) prevalence increases in the first month of antiretroviral treatment (ART), followed by a return to baseline prevalence by month 3. Since most GUD is caused by herpes simplex virus type 2 (HSV-2), we hypothesized that genital HSV detection would follow a similar pattern after treatment initiation., Methods: We conducted a prospective cohort study of 122 HSV-2 and HIV-1 co-infected women with advanced HIV disease who initiated ART and were followed closely with collection of genital swab specimens for the first three months of treatment., Results: At baseline, the HSV detection rate was 32%, without significant increase in genital HSV detection noted during the first month or the third month of ART. HIV-1 shedding declined during this period; no association was also noted between HSV and HIV-1 shedding during this period., Conclusion: Because other studies have reported increased HSV detection in women initiating ART and we have previously reported an increase in GUD during early ART, it may be prudent to counsel HIV-1 infected women initiating ART that HSV shedding in the genital tract may continue after ART initiation., Competing Interests: The authors have declared that no competing interests exist.

Correct developmental timing is essential for plant fitness and reproductive success. Two important transitions in shoot development-the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition-are mediated by a group of genes targeted by miR156, SQUAMOSA PROMOTER BINDING PROTEIN (SBP) genes. To determine the developmental functions of these genes in Arabidopsis thaliana, we characterized their expression patterns, and their gain-of-function and loss-of-function phenotypes. Our results reveal that SBP-LIKE (SPL) genes in Arabidopsis can be divided into three functionally distinct groups: 1) SPL2, SPL9, SPL10, SPL11, SPL13 and SPL15 contribute to both the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition, with SPL9, SP13 and SPL15 being more important for these processes than SPL2, SPL10 and SPL11; 2) SPL3, SPL4 and SPL5 do not play a major role in vegetative phase change or floral induction, but promote the floral meristem identity transition; 3) SPL6 does not have a major function in shoot morphogenesis, but may be important for certain physiological processes. We also found that miR156-regulated SPL genes repress adventitious root development, providing an explanation for the observation that the capacity for adventitious root production declines as the shoot ages. miR156 is expressed at very high levels in young seedlings, and declines in abundance as the shoot develops. It completely blocks the expression of its SPL targets in the first two leaves of the rosette, and represses these genes to different degrees at later stages of development, primarily by promoting their translational repression. These results provide a framework for future studies of this multifunctional family of transcription factors, and offer new insights into the role of miR156 in Arabidopsis development.

Losses to follow-up (LTFU) remain an important programmatic challenge. While numerous patient-level factors have been associated with LTFU, less is known about facility-level factors. Data from the East African International epidemiologic Databases to Evaluate AIDS (EA-IeDEA) Consortium was used to identify facility-level factors associated with LTFU in Kenya, Tanzania and Uganda. Patients were defined as LTFU if they had no visit within 12 months of the study endpoint for pre-ART patients or 6 months for patients on ART. Adjusting for patient factors, shared frailty proportional hazard models were used to identify the facility-level factors associated with LTFU for the pre- and post-ART periods. Data from 77,362 patients and 29 facilities were analyzed. Median age at enrolment was 36.0 years (Interquartile Range: 30.1, 43.1), 63.9% were women and 58.3% initiated ART. Rates (95% Confidence Interval) of LTFU were 25.1 (24.7-25.6) and 16.7 (16.3-17.2) per 100 person-years in the pre-ART and post-ART periods, respectively. Facility-level factors associated with increased LTFU included secondary-level care, HIV RNA PCR turnaround time >14 days, and no onsite availability of CD4 testing. Increased LTFU was also observed when no nutritional supplements were provided (pre-ART only), when TB patients were treated within the HIV program (pre-ART only), and when the facility was open ≤4 mornings per week (ART only). Our findings suggest that facility-based strategies such as point of care laboratory testing and separate clinic spaces for TB patients may improve retention.

Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5' of ITS1 and the 3' of ITS2, while skipping the uninformative 5.8S region. Operational taxonomic units (OTUs) were picked from scaffolds clustered at 97% identity, searched by BLAST against the nt database, and given taxonomic assignments using the paired-read lowest common ancestor approach. Taxonomic assignments and quantitative patterns were consistent with known plant distributions, phenology, and observational reports of pollen foraging, but revealed an unexpected contribution from non-crop graminoids and wetland plants. The mean number of plant species assignments per sample was 23.0 (+/- 5.5) and the mean species diversity (effective number of equally abundant species) was 3.3 (+/- 1.2). Bray-Curtis similarities showed good agreement among samples from the same apiary and sampling date. Rarefaction plots indicated that fewer than 50,000 reads are typically needed to characterize pollen samples of this complexity. Our results show that a pre-compiled, curated reference database is not essential for genus-level assignments, but species-level assignments are hindered by database gaps, reference length variation, and probable errors in the taxonomic assignment, requiring post-hoc evaluation. Although the effective per-sample yield achieved using custom MiSeq amplicon primers was less than the machine maximum, primarily due to lower "read2" quality, further protocol optimization and/or a modest reduction in multiplex scale should offset this difficulty. As small quantities of pollen are sufficient for amplification, our approach might be extendable to other questions or species for which large pollen samples are not available.

Background: Allergic non-asthmatic (ANA) adults experience upper airway symptoms of allergic disease such as rhinorrhea, congestion and sneezing without symptoms of asthma. The aim of this study was to utilize PET-CT functional imaging to determine whether allergen challenge elicits a pulmonary response in ANA subjects or whether their allergic disease is truly isolated to the upper airways., Methods: In 6 ANA subjects, bronchoalveolar lavages (BAL) were performed at baseline and 24h after instillation of an allergen and a diluent in separate lung lobes. After instillation (10h), functional imaging was performed to quantify and compare regional perfusion, ventilation, fractional gas content (Fgas), and glucose uptake rate (Ki) between the baseline, diluent and allergen lobes. BAL cell counts were also compared., Results: In ANA subjects, compared to the baseline and diluent lobes, perfusion and ventilation were significantly lower in the allergen lobe (median [inter-quartile range], baseline vs. diluent vs. allergen: Mean-normalized perfusion; 0.87 [0.85-0.97] vs. 0.90 [0.86-0.98] vs. 0.59 [0.55-0.67]; p<0.05. Mean-normalized ventilation 0.89 [0.88-0.98] vs. 0.95 [0.89-1.02] vs. 0.63 [0.52-0.67], p<0.05). In contrast, no significant differences were found in Fgas between baseline, diluent and allergen lobes or in Ki. Total cell counts, eosinophil and neutrophil cell counts (cells/ml BAL) were significantly greater in the allergen lobe compared to the baseline lobe (all P<0.05)., Conclusions: Despite having no clinical symptoms of a lower airway allergic response (cough and wheeze) allergic non-asthmatic subjects have a pulmonary response to allergen exposure which manifests as reduced ventilation and perfusion.

HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women's Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.

Variance is a statistical parameter used to characterize heterogeneity or variability in data sets. However, measurements commonly include noise, as random errors superimposed to the actual value, which may substantially increase the variance compared to a noise-free data set. Our aim was to develop and validate a method to estimate noise-free spatial heterogeneity of pulmonary perfusion using dynamic positron emission tomography (PET) scans. On theoretical grounds, we demonstrate a linear relationship between the total variance of a data set derived from averages of n multiple measurements, and the reciprocal of n. Using multiple measurements with varying n yields estimates of the linear relationship including the noise-free variance as the constant parameter. In PET images, n is proportional to the number of registered decay events, and the variance of the image is typically normalized by the square of its mean value yielding a coefficient of variation squared (CV(2)). The method was evaluated with a Jaszczak phantom as reference spatial heterogeneity (CV(r)(2)) for comparison with our estimate of noise-free or 'true' heterogeneity (CV(t)(2)). We found that CV(t)(2) was only 5.4% higher than CV(r)2. Additional evaluations were conducted on 38 PET scans of pulmonary perfusion using (13)NN-saline injection. The mean CV(t)(2) was 0.10 (range: 0.03-0.30), while the mean CV(2) including noise was 0.24 (range: 0.10-0.59). CV(t)(2) was in average 41.5% of the CV(2) measured including noise (range: 17.8-71.2%). The reproducibility of CV(t)(2) was evaluated using three repeated PET scans from five subjects. Individual CV(t)(2) were within 16% of each subject's mean and paired t-tests revealed no difference among the results from the three consecutive PET scans. In conclusion, our method provides reliable noise-free estimates of CV(t)(2) in PET scans, and may be useful for similar statistical problems in experimental data.