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Sex differences have been observed in acute COVID-19 and Long COVID (LC) outcomes, with greater disease severity and mortality during acute infection in males and a greater proportion of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to the pathogenesis of LC. To investigate the immunologic underpinnings of LC development and persistence, we used single-cell transcriptomics, single-cell proteomics, and plasma proteomics on blood samples obtained during acute SARS-CoV-2 infection and at 3 and 12 months post-infection in a cohort of 45 patients who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Specifically, males who would develop LC at 3 months had widespread increases in TGF-β signaling during acute infection in proliferating NK cells. Females who would develop LC demonstrated increased expression of XIST , an RNA gene implicated in autoimmunity, and increased IL1 signaling in monocytes at 12 months post infection. Several immune features of LC were also conserved across sexes. Both males and females with LC had reduced co-stimulatory signaling from monocytes and broad upregulation of NF-κB transcription factors. In both sexes, those with persistent LC demonstrated increased LAG3, a marker of T cell exhaustion, reduced ETS1 transcription factor expression across lymphocyte subsets, and elevated intracellular IL-4 levels in T cell subsets, suggesting that ETS1 alterations may drive an aberrantly elevated Th2-like response in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics., Competing Interests: Competing interests: Upinder Singh reports research support from National Institutes of Health, Agency for Healthcare Research and Quality, and Pfizer, Inc.; she is an advisor to Regeneron and Gilead. Catherine Blish is an advisor to Immunebridge and DeepCell on topics unrelated to this research. All other authors declare no competing interests.

While preventing vertical HIV transmission has been very successful, HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations in T cell memory differentiation between iHEU and iHUU being significant from week 15 of life. The altered memory T cell differentiation in iHEU was preceded by lower TCR Vβ clonotypic diversity and linked to TCR clonal depletion within the naïve T cell compartment. Compared to iHUU, iHEU had elevated CD56 lo CD16 lo Perforin + CD38 + CD45RA + FcεRIγ + NK cells at 1 month postpartum and whose abundance pre-vaccination were predictive of vaccine-induced pertussis and rotavirus antibody responses post 3 months of life. Collectively, HIV/ARV exposure disrupted the trajectory of innate and adaptive immunity from birth which may underlie relative vulnerability to infections in iHEU., (© 2024. The Author(s).)

Infants who are exposed to HIV but uninfected (iHEU) have higher risk of infectious morbidity than infants who are HIV-unexposed and uninfected (iHUU), possibly due to altered immunity. As infant gut microbiota may influence immune development, we evaluated the effects of HIV exposure on infant gut microbiota and its association with tetanus toxoid vaccine responses. We evaluated the gut microbiota of 82 South African (61 iHEU and 21 iHUU) and 196 Nigerian (141 iHEU and 55 iHUU) infants at <1 and 15 weeks of life by 16S rRNA gene sequencing. Anti-tetanus antibodies were measured by enzyme-linked immunosorbent assay at matched time points. Gut microbiota in the 278 included infants and its succession were more strongly influenced by geographical location and age than by HIV exposure. Microbiota of Nigerian infants, who were exclusively breastfed, drastically changed over 15 weeks, becoming dominated by Bifidobacterium longum subspecies infantis . This change was not observed among South African infants, even when limiting the analysis to exclusively breastfed infants. The Least Absolute Shrinkage and Selection Operator regression suggested that HIV exposure and gut microbiota were independently associated with tetanus titers at week 15, and that high passively transferred antibody levels, as seen in the Nigerian cohort, may mitigate these effects. In conclusion, in two African cohorts, HIV exposure minimally altered the infant gut microbiota compared to age and setting, but both specific gut microbes and HIV exposure independently predicted humoral tetanus vaccine responses.IMPORTANCEGut microbiota plays an essential role in immune system development. Since infants HIV-exposed and uninfected (iHEU) are more vulnerable to infectious diseases than unexposed infants, we explored the impact of HIV exposure on gut microbiota and its association with vaccine responses. This study was conducted in two African countries with rapidly increasing numbers of iHEU. Infant HIV exposure did not substantially affect gut microbial succession, but geographic location had a strong effect. However, both the relative abundance of specific gut microbes and HIV exposure were independently associated with tetanus titers, which were also influenced by baseline tetanus titers (maternal transfer). Our findings provide insight into the effect of HIV exposure, passive maternal antibody, and gut microbiota on infant humoral vaccine responses., Competing Interests: The authors declare no conflict of interest.

Background: Atlanto-axial (AA) subluxation can be a complex syndrome in dogs. Accurate identification and assessment of this condition are key to providing treatment and resolution., Aim: The purpose of this retrospective study was to describe the magnetic resonance imaging (MRI) characteristics of AA subluxation and associated neurologic deficits., Methods: A multicenter review of dogs with a diagnosis of AA subluxation was conducted, evaluating signalment, neurologic grade, duration of signs, and MRI characteristics. MRI characteristics included degree of spinal cord compression and joint subluxation, integrity of odontoid ligaments, presence of a dens, spinal cord signal intensity, and presence of syringohydromyelia, hydrocephalus, and Chiari-like malformation. A control population with normal AA joints was also evaluated. MR images of 42 dogs with AA subluxation were compared to 26 age and breed-matched control dogs., Results: Affected dogs had a median age of 27 months and a median weight of 2.7 kg, and the most commonly affected breed was the Yorkshire terrier (47.5%). Spinal cord signal hyperintensity, increased AA joint size, and cross-sectional cord compression at the level of the dens and mid-body C2 were associated with AA subluxation. No associations were found between cord compression, the appearance of the dens, or cord signal intensity and neurologic grade. Affected dogs did not have a higher incidence of Chiari-like malformation, syringohydromyelia, or hydrocephalus than control dogs, and their neurologic grade was not associated with MRI findings. Lack of dens and/or odontoid ligaments was associated with larger subluxations., Conclusion: Dogs with clinical signs of AA subluxation were significantly more likely to have intramedullary hyperintensity at the level of compression ( p = 0.0004), an increased AA joint cavity size ( p = 0.0005), and increased spinal cord compression at the level of dens and mid-body C2 ( p ≤ 0.05). The authors suggest an AA joint cavity size >1.4 mm and a subluxation distance >2.5 mm as cutoffs for MRI diagnosis of AA subluxation in dogs. No differences were noted between dogs with AA subluxation and control dogs regarding syringohydromyelia, hydrocephalus, and Chiari-like malformation., Competing Interests: The authors declare that there are no known conflicts of interest associated with this publication and there was no financial support necessary for this work that could have influenced the outcome.

The vaginal ecosystem is closely tied to human health and reproductive outcomes, yet its dynamics in the wake of childbirth remain poorly characterized. Here, we profile the vaginal microbiota and cytokine milieu of participants sampled longitudinally throughout pregnancy and for at least one year postpartum. We show that delivery, regardless of mode, is associated with a vaginal pro-inflammatory cytokine response and the loss of Lactobacillus dominance. By contrast, neither the progression of gestation nor the approach of labor strongly altered the vaginal ecosystem. At 9.5-months postpartum-the latest timepoint at which cytokines were assessed-elevated inflammation coincided with vaginal bacterial communities that had remained perturbed (highly diverse) from the time of delivery. Time-to-event analysis indicated a one-year postpartum probability of transitioning to Lactobacillus dominance of 49.4%. As diversity and inflammation declined during the postpartum period, dominance by L. crispatus, the quintessential health-associated commensal, failed to return: its prevalence before, immediately after, and one year after delivery was 41%, 4%, and 9%, respectively. Revisiting our pre-delivery data, we found that a prior live birth was associated with a lower odds of L. crispatus dominance in pregnant participants-an outcome modestly tempered by a longer ( > 18-month) interpregnancy interval. Our results suggest that reproductive history and childbirth in particular remodel the vaginal ecosystem and that the timing and degree of recovery from delivery may help determine the subsequent health of the woman and of future pregnancies., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The spatiotemporal structure of the human microbiome 1,2 , proteome 3 and metabolome 4,5 reflects and determines regional intestinal physiology and may have implications for disease 6 . Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals 7 . To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease., (© 2023. The Author(s).)

Background: With the approval of the HIV-1 capsid inhibitor, lenacapavir, capsid sequencing will be required for managing lenacapavir-experienced individuals with detectable viremia. Successful sequence interpretation will require examining new capsid sequences in the context of previously published sequence data., Methods: We analyzed published HIV-1 group M capsid sequences from 21,012 capsid-inhibitor naïve individuals to characterize amino acid variability at each position and influence of subtype and cytotoxic T lymphocyte (CTL) selection pressure. We determined the distributions of usual mutations, defined as amino acid differences from the group M consensus, with a prevalence ≥ 0.1%. Co-evolving mutations were identified using a phylogenetically-informed Bayesian graphical model method., Results: 162 (70.1%) positions had no usual mutations (45.9%) or only conservative usual mutations with a positive BLOSUM62 score (24.2%). Variability correlated independently with subtype-specific amino acid occurrence (Spearman rho = 0.83; p < 1 × 10 -9 ) and the number of times positions were reported to contain an HLA-associated polymorphism, an indicator of CTL pressure (rho = 0.43; p = 0.0002)., Conclusions: Knowing the distribution of usual capsid mutations is essential for sequence quality control. Comparing capsid sequences from lenacapavir-treated and lenacapavir-naïve individuals will enable the identification of additional mutations potentially associated with lenacapavir therapy.

HIV-1 pol nucleotide ambiguities encoding amino acid mixtures occur commonly during population-based genotypic drug resistance testing. However, few studies have addressed the validity of sequences with fully ambiguous codons (FACs) containing codons translatable to more than four amino acids. We identified 839 published HIV-1 pol sequences with 846 FACs at 131 positions and determined their distribution relative to 215 HLA-associated pol positions (HAPs) and 84 drug-resistance positions. Among HIV-1 reverse transcriptase (RT) and protease sequences from antiretroviral therapy (ART)-naive and -experienced persons, there was a strong correlation between the likelihood a position was a FAC and that it was an HAP (Spearman's correlation coefficient rho >0.40; p  < 1e-6). Among HIV-1 RT sequences from ART-experienced persons, there was a correlation between the likelihood that a position was a FAC and that it was a drug-resistance position (rho = 0.2; p  = 8e-4). In the context of population-based genotypic resistance testing, FACs usually result from antiviral or immune selection pressure.

For marine ectotherms, larval success, planktonic larval duration and dispersal trajectories are strongly influenced by temperature, and therefore, ocean warming and heatwaves have profound impacts on these sensitive stages. Warming, through increased poleward flow in regions with western boundary currents, such as the East Australia Current (EAC), provides opportunities for range extension as propagules track preferred conditions. Two sea urchin species, Centrostephanus rodgersii and Heliocidaris tuberculata, sympatric in the EAC warming hotspot, exhibit contrasting responses to warming. Over half a century, C. rodgersii has undergone marked poleward range extension, but the range of H. tuberculata has not changed. We constructed thermal performance curves (TPC) to determine if contrasting developmental thermal tolerance can explain this difference. The temperatures tested encompassed present-day distribution and forecast ocean warming/heatwave conditions. The broad and narrow thermal optimum (Topt) ranges for C. rodgersii and H. tuberculata larvae (7.2 and 4.7°C range, respectively) matched their realized (adult distribution) thermal niches. The cool and warm temperatures for 50% development to the feeding larva approximated temperatures at adult poleward range limits. Larval cool tolerances with respect to mean local temperature differed, 6.0 and 3.8°C respectively. Larval warm tolerances were similar for both species as are the adult warm range edges. The larvae of both species would be sensitive to heatwaves. Centrostephanus rodgersii has stayed in place and shifted in space, likely due to its broad cold-warm larval thermal tolerance and large thermal safety margins. Phenotypic plasticity of the planktonic stage of C. rodgersii facilitated its range extension. In contrast, larval cold intolerance of H. tuberculata explains its restricted range and will delay poleward extension as the region warms. In a warming ocean, we show that intrinsic thermal biology traits of the pelagic stage provide an integrative tool to explain species-specific variation in range shift patterns., (© 2022 John Wiley & Sons Ltd.)

Few studies have investigated immune cell ontogeny throughout the neonatal and early pediatric period, when there is often increased vulnerability to infections. In this study, we evaluated the dynamics of two critical T cell populations, T regulatory (Treg) cells and Th17 cells, over the first 36 wk of human life. First, we observed distinct CD4 + T cells phenotypes between cord blood and peripheral blood, collected within 12 h of birth, showing that cord blood is not a surrogate for newborn blood. Second, both Treg and Th17 cells expanded in a synchronous fashion over 36 wk of life. However, comparing infants exposed to HIV in utero, but remaining uninfected, with HIV-unexposed uninfected control infants, there was a lower frequency of peripheral blood Treg cells at birth, resulting in a delayed expansion, and then declining again at 36 wk. Focusing on birth events, we found that Treg cells coexpressing CCR4 and α4β7 inversely correlated with plasma concentrations of CCL17 (the ligand for CCR4) and intestinal fatty acid binding protein, IL-7, and CCL20. This was in contrast with Th17 cells, which showed a positive association with these plasma analytes. Thus, despite the stereotypic expansion of both cell subsets over the first few months of life, there was a disruption in the balance of Th17 to Treg cells at birth likely being a result of gut damage and homing of newborn Treg cells from the blood circulation to the gut., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: Hydrocolloid dressings (HCD) are helpful in chronic wound care, but research is limited in acute postoperative wounds. HCD can potentially be incorporated into a simplified wound care regimen after excisional surgeries., Objective: To examine whether a one-time HCD application after dermatologic surgery results in greater patient satisfaction and improved postoperative outcomes compared with conventional daily dressings (CDD)., Methods: We examined patients who underwent Mohs or standard surgical excision with linear closure followed by HCD. The patients additionally had a history of excisional surgery with CDD in the past 5 years. A modified version of the validated Bluebelle Wound Healing Questionnaire was administered., Results: The survey response rate was 74.4% (64/86). Compared with CDD, HCD rated higher in comfort, convenience, scar appearance, and simplicity of wound care instructions ( P  < .0001). Nearly all patients (96.8%) preferred HCD over CDD., Limitations: Variability in time from prior dermatologic surgery may introduce recall bias. Prior surgeries involving CDD were sometimes performed by a different surgeon, which could introduce other confounding factors., Conclusions: A simplified wound care regimen involving HCD can potentially lead to increased comfort, convenience, simplicity, and a subjective improvement in scar appearance, though additional studies are needed., Competing Interests: None disclosed.

High throughput sequencing (HTS)-based technology enables identifying and quantifying non-culturable microbial organisms in all environments. Microbial sequences have enhanced our understanding of the human microbiome, the soil and plant environment, and the marine environment. All molecular microbial data pose statistical challenges due to contamination sequences from reagents, batch effects, unequal sampling, and undetected taxa. Technical biases and heteroscedasticity have the strongest effects, but different strains across subjects and environments also make direct differential abundance testing unwieldy. We provide an introduction to a few statistical tools that can overcome some of these difficulties and demonstrate those tools on an example. We show how standard statistical methods, such as simple hierarchical mixture and topic models, can facilitate inferences on latent microbial communities. We also review some nonparametric Bayesian approaches that combine visualization and uncertainty quantification. The intersection of molecular microbial biology and statistics is an exciting new venue. Finally, we list some of the important open problems that would benefit from more careful statistical method development.

For short development species, like the sea urchin Heliocidaris erythrogramma, the entire planktonic duration can be impacted by marine heatwaves (MHW). Developmental thermal tolerance of this species through metamorphosis was investigated over a broad range (7.6-28.0 °C), including temperatures across its distribution and MHW conditions. In controls (19.5-21.0 °C), 80% of individuals developed to metamorphosis at day 5, doubling to 10 days at 14.0 °C. The thermal range (14.4-21.2 °C) of metamorphosis on day 7 reflected the realised thermal niche with 25.9 °C the upper temperature for success (T 40 ). By day 10, juvenile tolerance narrowed to the local range (16.2-19.0 °C), similar to levels tolerated by adults, indicating negative carryover effects across the metamorphic transition. Without phenotypic adjustment or adaptation, regional warming will be detrimental, although populations may be sustained by thermotolerant offspring. Our results show the importance of the metamorphic transition in understanding the cumulative sensitivity of species to MHW., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

BACKGROUNDElevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare with those observed in critically ill patients with acute respiratory distress syndrome (ARDS) or sepsis due to other causes.METHODSWe used a Luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1β, IL-1RA, IL-6, IL-8, IL-18, and TNF-α) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTSFifteen hospitalized COVID-19 patients, 9 of whom were critically ill, were compared with critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1β, IL-1RA, IL-6, IL-8, IL-18, and TNF-α between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSIONLevels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 may be unwarranted.FUNDINGFunding was received from NHLBI K23 HL125663 (AJR); The Bill and Melinda Gates Foundation OPP1113682 (AJR and CAB); Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687 NIH/NIAID U19AI057229-16; Stanford Maternal Child Health Research Institute; and Chan Zuckerberg Biohub (CAB).

Background: We evaluated the impact of low-cost water, sanitation, handwashing (WSH) and child nutrition interventions on enteropathogen carriage in the WASH Benefits cluster-randomized controlled trial in rural Bangladesh., Methods: We analyzed 1411 routine fecal samples from children 14±2 months old in the WSH (n = 369), nutrition counseling plus lipid-based nutrient supplement (n = 353), nutrition plus WSH (n = 360), and control (n = 329) arms for 34 enteropathogens using quantitative PCR. Outcomes included the number of co-occurring pathogens; cumulative quantity of four stunting-associated pathogens; and prevalence and quantity of individual pathogens. Masked analysis was by intention-to-treat., Results: 326 (99.1%) control children had one or more enteropathogens detected (mean 3.8±1.8). Children receiving WSH interventions had lower prevalence and quantity of individual viruses than controls (prevalence difference for norovirus: -11% [95% confidence interval [CI], -5 to -17%]; sapovirus: -9% [95%CI, -3 to -15%]; and adenovirus 40/41: -9% [95%CI, -2 to - 15%]). There was no difference in bacteria, parasites, or cumulative quantity of stunting-associated pathogens between controls and any intervention arm., Conclusions: WSH interventions were associated with fewer enteric viruses in children aged 14 months. Different strategies are needed to reduce enteric bacteria and parasites at this critical young age., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Selective and neutral forces shape human microbiota assembly in early life. The Tsimane are an indigenous Bolivian population with infant care-associated behaviors predicted to increase mother-infant microbial dispersal. Here, we characterize microbial community assembly in 47 infant-mother pairs from six Tsimane villages, using 16S rRNA gene amplicon sequencing of longitudinal stool and tongue swab samples. We find that infant consumption of dairy products, vegetables, and chicha (a fermented drink inoculated with oral microbes) is associated with stool microbiota composition. In stool and tongue samples, microbes shared between mothers and infants are more abundant than non-shared microbes. Using a neutral model of community assembly, we find that neutral processes alone explain the prevalence of 79% of infant-colonizing microbes, but explain microbial prevalence less well in adults from river villages with more regular access to markets. Our results underscore the importance of neutral forces during microbiota assembly. Changing lifestyle factors may alter traditional modes of microbiota assembly by decreasing the role of neutral processes.

Background: HIV-1 and HIV-2 differ in their antiretroviral (ARV) susceptibilities and drug resistance mutations (DRMs)., Methods: We analyzed published HIV-2 pol sequences to identify HIV-2 treatment-selected mutations (TSMs). Mutation prevalences were determined by HIV-2 group and ARV status. Nonpolymorphic mutations were those in <1% of ARV-naive persons. TSMs were those associated with ARV therapy after multiple comparisons adjustment., Results: We analyzed protease (PR) sequences from 483 PR inhibitor (PI)-naive and 232 PI-treated persons; RT sequences from 333 nucleoside RT inhibitor (NRTI)-naive and 252 NRTI-treated persons; and integrase (IN) sequences from 236 IN inhibitor (INSTI)-naive and 60 INSTI-treated persons. In PR, 12 nonpolymorphic TSMs occurred in ≥11 persons: V33I, K45R, V47A, I50V, I54M, T56V, V62A, A73G, I82F, I84V, F85L, L90M. In RT, 9 nonpolymorphic TSMs occurred in ≥10 persons: K40R, A62V, K70R, Y115F, Q151M, M184VI, S215Y. In IN, 11 nonpolymorphic TSMs occurred in ≥4 persons: Q91R, E92AQ, T97A, G140S, Y143G, Q148R, A153G, N155H, H156R, R231 5-amino acid insertions. Nine of 32 nonpolymorphic TSMs were previously unreported., Conclusions: This meta-analysis confirmed the ARV association of previously reported HIV-2 DRMs and identified novel TSMs. Genotypic and phenotypic studies of HIV-2 TSMs will improve approaches to predicting HIV-2 ARV susceptibility and treating HIV-2-infected persons., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: At low mutation-detection thresholds, next generation sequencing (NGS) for HIV-1 genotypic resistance testing is susceptible to artifactual detection of mutations arising from PCR error and APOBEC-mediated G-to-A hypermutation., Methods: We analyzed published HIV-1 pol Illumina NGS data to characterize the distribution of mutations at eight NGS mutation detection thresholds: 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1%. At each threshold, we determined proportions of amino acid mutations that were unusual (defined as having a prevalence <0.01% in HIV-1 group M sequences) or signature APOBEC mutations., Results: Eight studies, containing 855 samples, in the NCBI Sequence Read Archive were analyzed. As detection thresholds were lowered, there was a progressive increase in the proportion of positions with usual and unusual mutations and in the proportion of all mutations that were unusual. The median proportion of positions with an unusual mutation increased gradually from 0% at the 20% threshold to 0.3% at the 1% threshold and then exponentially to 1.3% (0.5% threshold), 6.9% (0.2% threshold), and 23.2% (0.1% threshold). In two of three studies with available plasma HIV-1 RNA levels, the proportion of positions with unusual mutations was negatively associated with virus levels. Although the complete set of signature APOBEC mutations was much smaller than that of unusual mutations, the former outnumbered the latter in one-sixth of samples at the 0.5%, 1%, and 2% thresholds., Conclusions: The marked increase in the proportion of positions with unusual mutations at thresholds below 1% and in samples with lower virus loads suggests that, at low thresholds, many unusual mutations are artifactual, reflecting PCR error or G-to-A hypermutation. Profiling the numbers of unusual and signature APOBEC pol mutations at different NGS mutation detection thresholds may be useful to avoid selecting a threshold that is too low and poses an unacceptable risk of identifying artifactual mutations., Competing Interests: The authors have declared that no competing interests exist.

In humans, the composition of microbial communities differs among body sites and between habitats within a single site. Patterns of variation in the distribution of organisms across time and space are referred to as "biogeography." The human oral cavity is a critical observatory for exploring microbial biogeography because it is spatially structured, easily accessible, and its microbiota has been linked to the promotion of both health and disease. The biogeographic features of microbial communities residing in spatially distinct, but ecologically similar, environments on the human body, including the subgingival crevice, have not yet been adequately explored. The purpose of this paper is twofold. First, we seek to provide the dental community with a primer on biogeographic theory, highlighting its relevance to the study of the human oral cavity. We summarize what is known about the biogeographic variation of dental caries and periodontitis and postulate that disease occurrence reflects spatial patterning in the composition and structure of oral microbial communities. Second, we present a number of methods that investigators can use to test specific hypotheses using biogeographic theory. To anchor our discussion, we apply each method to a case study and examine the spatial variation of the human subgingival microbiota in 2 individuals. Our case study suggests that the composition of subgingival communities may conform to an anterior-to-posterior gradient within the oral cavity. The gradient appears to be structured by both deterministic and nondeterministic processes, although additional work is needed to confirm these findings. A better understanding of biogeographic patterns and processes will lead to improved efficacy of dental interventions targeting the oral microbiota., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

While low-carbohydrate and low-fat diets can both lead to weight-loss, a substantial variability in achieved long-term outcomes exists among obese but otherwise healthy adults. We examined the hypothesis that structural differences in the gut microbiota explain a portion of variability in weight-loss using two cohorts of obese adults enrolled in the Diet Intervention Examining The Factors Interacting with Treatment Success (DIETFITS) study. A total of 161 pre-diet fecal samples were sequenced from a discovery cohort (n = 66) and 106 from a validation cohort (n = 56). An additional 157 fecal samples were sequenced from the discovery cohort after 10 weeks of dietary intervention. We found no specific bacterial signatures associated with weight loss that were consistent across both cohorts. However, the gut microbiota plasticity (i.e. variability), was correlated with long-term (12-month) weight loss in a diet-dependent manner; on the low-fat diet subjects with higher pre-diet daily plasticity had higher sustained weight loss, whereas on the low-carbohydrate diet those with higher plasticity over 10 weeks of dieting had higher 12-month weight loss. Our findings suggest the potential importance of gut microbiota plasticity for sustained weight-loss. We highlight the advantages of evaluating kinetic trends and assessing reproducibility in studies of the gut microbiota.

The human microbiome is a complex ecological system, and describing its structure and function under different environmental conditions is important from both basic scientific and medical perspectives. Viewed through a biostatistical lens, many microbiome analysis goals can be formulated as latent variable modeling problems. However, although probabilistic latent variable models are a cornerstone of modern unsupervised learning, they are rarely applied in the context of microbiome data analysis, in spite of the evolutionary, temporal, and count structure that could be directly incorporated through such models. We explore the application of probabilistic latent variable models to microbiome data, with a focus on Latent Dirichlet allocation, Non-negative matrix factorization, and Dynamic Unigram models. To develop guidelines for when different methods are appropriate, we perform a simulation study. We further illustrate and compare these techniques using the data of Dethlefsen and Relman (2011, Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proceedings of the National Academy of Sciences108, 4554-4561), a study on the effects of antibiotics on bacterial community composition. Code and data for all simulations and case studies are available publicly., (© The Author 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The simultaneous study of multiple measurement types is a frequently encountered problem in practical data analysis. It is especially common in microbiome research, where several sources of data-for example, 16s-rRNA, metagenomic, metabolomic, or transcriptomic data-can be collected on the same physical samples. There has been a proliferation of proposals for analyzing such multitable microbiome data, as is often the case when new data sources become more readily available, facilitating inquiry into new types of scientific questions. However, stepping back from the rush for new methods for multitable analysis in the microbiome literature, it is worthwhile to recognize the broader landscape of multitable methods, as they have been relevant in problem domains ranging across economics, robotics, genomics, chemometrics, and neuroscience. In different contexts, these techniques are called data integration, multi-omic, and multitask methods, for example. Of course, there is no unique optimal algorithm to use across domains-different instances of the multitable problem possess specific structure or variation that are worth incorporating in methodology. Our purpose here is not to develop new algorithms, but rather to 1) distill relevant themes across different analysis approaches and 2) provide concrete workflows for approaching analysis, as a function of ultimate analysis goals and data characteristics (heterogeneity, dimensionality, sparsity). Towards the second goal, we have made code for all analysis and figures available online at https://github.com/krisrs1128/multitable&#95;review.

Objectives/hypothesis: Head and neck squamous cell carcinoma represents the sixth most common cancer. As a result of field cancerization, second primaries and recurrences are high. Hence, research has focused on chemoprevention. Curcumin, a polyphenol compound with anticarcinogenic properties, is one such promising nutraceutical. As poor bioavailability limits curcumin's use, a novel gum formulation was tested allowing for direct mucosal absorption into the bloodstream. This preliminary study validates curcumin gum efficacy by assessing release and transmucosal absorption, along with measuring its effects on serum cytokine levels., Study Design: Clinical trial., Methods: Protocols consisting of initial chew (chewing gum for 30 minutes) and revised chew (alternating chewing and parking gum against buccal mucosa for 30 minutes) were tested in healthy volunteers. High-performance liquid chromatography measured remnant curcumin in chewed gum, serum, and saliva. Serum levels were assayed for 15 proinflammatory cytokines via multiplex analysis., Results: Revised chew samples demonstrated significantly higher curcumin release and absorption (P = .0078). Curcumin serum levels were significantly higher at 4 hours in samples > 2.0 g of curcumin release (P = .01). As saliva levels decreased, a concurrent increase in serum levels was observed, with no significance in the inverse relationship (P = .1423). When evaluating differences between gender, race, and age, the Asian population showed significantly lower curcumin release and serum levels (P = .009). CXCL1 (GRO-α) and TNF-α were significantly decreased in serum after chewing the gum (P = .036, P < .001, respectively)., Conclusions: Enhanced mucosal contact appears critical in improving curcumin release and absorption. CXCL1 and TNF-α both represent potential biomarkers for the future study of curcumin chemoprevention., Level of Evidence: 2b Laryngoscope, 129:1597-1603, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: The accuracy of microbial community surveys based on marker-gene and metagenomic sequencing (MGS) suffers from the presence of contaminants-DNA sequences not truly present in the sample. Contaminants come from various sources, including reagents. Appropriate laboratory practices can reduce contamination, but do not eliminate it. Here we introduce decontam ( https://github.com/benjjneb/decontam ), an open-source R package that implements a statistical classification procedure that identifies contaminants in MGS data based on two widely reproduced patterns: contaminants appear at higher frequencies in low-concentration samples and are often found in negative controls., Results: Decontam classified amplicon sequence variants (ASVs) in a human oral dataset consistently with prior microscopic observations of the microbial taxa inhabiting that environment and previous reports of contaminant taxa. In metagenomics and marker-gene measurements of a dilution series, decontam substantially reduced technical variation arising from different sequencing protocols. The application of decontam to two recently published datasets corroborated and extended their conclusions that little evidence existed for an indigenous placenta microbiome and that some low-frequency taxa seemingly associated with preterm birth were contaminants., Conclusions: Decontam improves the quality of metagenomic and marker-gene sequencing by identifying and removing contaminant DNA sequences. Decontam integrates easily with existing MGS workflows and allows researchers to generate more accurate profiles of microbial communities at little to no additional cost.

Recent studies suggest that the microbiome has an impact on gestational health and outcome. However, characterization of the pregnancy-associated microbiome has largely relied on 16S rRNA gene amplicon-based surveys. Here, we describe an assembly-driven, metagenomics-based, longitudinal study of the vaginal, gut, and oral microbiomes in 292 samples from 10 subjects sampled every three weeks throughout pregnancy. Nonhuman sequences in the amount of 1.53 Gb were assembled into scaffolds, and functional genes were predicted for gene- and pathway-based analyses. Vaginal assemblies were binned into 97 draft quality genomes. Redundancy analysis (RDA) of microbial community composition at all three body sites revealed gestational age to be a significant source of variation in patterns of gene abundance. In addition, health complications were associated with variation in community functional gene composition in the mouth and gut. The diversity of Lactobacillus iners -dominated communities in the vagina, unlike most other vaginal community types, significantly increased with gestational age. The genomes of co-occurring Gardnerella vaginalis strains with predicted distinct functions were recovered in samples from two subjects. In seven subjects, gut samples contained strains of the same Lactobacillus species that dominated the vaginal community of that same subject and not other Lactobacillus species; however, these within-host strains were divergent. CRISPR spacer analysis suggested shared phage and plasmid populations across body sites and individuals. This work underscores the dynamic behavior of the microbiome during pregnancy and suggests the potential importance of understanding the sources of this behavior for fetal development and gestational outcome., (© 2018 Goltsman et al.; Published by Cold Spring Harbor Laboratory Press.)

In human and murine studies, IFN-γ is a critical mediator immunity to influenza. IFN-γ production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-γ and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-γ early in infection, we set out to identify the drivers of the human NK cell IFN-γ response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-γ in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-γ response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-γ response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-γ response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-γ and identifies a previously unrecognized influenza innate immune evasion strategy., (Copyright © 2018 by The American Association of Immunologists, Inc.)

A dog with lameness, stifle effusion, and osteophytes, but in which the stifle retained stability for 10 months after the onset of lameness, was evaluated with 3T magnetic resonance imaging (MRI). Imaging revealed a tear in the caudal meniscotibial ligament of the medial meniscus. Arthroscopy findings correlated well with MRI and a partial meniscectomy was subsequently carried out, without any surgery to stabilize the stifle, and lameness resolved.

Spatial and temporal patterns in microbial communities provide insights into the forces that shape them, their functions and roles in health and disease. Here, we used spatial and ecological statistics to analyze the role that saliva plays in structuring bacterial communities of the human mouth using >9000 dental and mucosal samples. We show that regardless of tissue type (teeth, alveolar mucosa, keratinized gingiva, or buccal mucosa), surface-associated bacterial communities vary along an ecological gradient from the front to the back of the mouth, and that on exposed tooth surfaces, the gradient is pronounced on lingual compared to buccal surfaces. Furthermore, our data suggest that this gradient is attenuated in individuals with low salivary flow due to Sjögren's syndrome. Taken together, our findings imply that salivary flow influences the spatial organization of microbial communities and that biogeographical patterns may be useful for understanding host physiological processes and for predicting disease.

Recent advances have made it possible to analyze high-throughput marker-gene sequencing data without resorting to the customary construction of molecular operational taxonomic units (OTUs): clusters of sequencing reads that differ by less than a fixed dissimilarity threshold. New methods control errors sufficiently such that amplicon sequence variants (ASVs) can be resolved exactly, down to the level of single-nucleotide differences over the sequenced gene region. The benefits of finer resolution are immediately apparent, and arguments for ASV methods have focused on their improved resolution. Less obvious, but we believe more important, are the broad benefits that derive from the status of ASVs as consistent labels with intrinsic biological meaning identified independently from a reference database. Here we discuss how these features grant ASVs the combined advantages of closed-reference OTUs-including computational costs that scale linearly with study size, simple merging between independently processed data sets, and forward prediction-and of de novo OTUs-including accurate measurement of diversity and applicability to communities lacking deep coverage in reference databases. We argue that the improvements in reusability, reproducibility and comprehensiveness are sufficiently great that ASVs should replace OTUs as the standard unit of marker-gene analysis and reporting.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. Previous studies have suggested that the maternal vaginal microbiota contributes to the pathophysiology of PTB, but conflicting results in recent years have raised doubts. We conducted a study of PTB compared with term birth in two cohorts of pregnant women: one predominantly Caucasian ( n = 39) at low risk for PTB, the second predominantly African American and at high-risk ( n = 96). We profiled the taxonomic composition of 2,179 vaginal swabs collected prospectively and weekly during gestation using 16S rRNA gene sequencing. Previously proposed associations between PTB and lower Lactobacillus and higher Gardnerella abundances replicated in the low-risk cohort, but not in the high-risk cohort. High-resolution bioinformatics enabled taxonomic assignment to the species and subspecies levels, revealing that Lactobacillus crispatus was associated with low risk of PTB in both cohorts, while Lactobacillus iners was not, and that a subspecies clade of Gardnerella vaginalis explained the genus association with PTB. Patterns of cooccurrence between L. crispatus and Gardnerella were highly exclusive, while Gardnerella and L. iners often coexisted at high frequencies. We argue that the vaginal microbiota is better represented by the quantitative frequencies of these key taxa than by classifying communities into five community state types. Our findings extend and corroborate the association between the vaginal microbiota and PTB, demonstrate the benefits of high-resolution statistical bioinformatics in clinical microbiome studies, and suggest that previous conflicting results may reflect the different risk profile of women of black race., Competing Interests: The authors declare no conflict of interest.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.

Our work focuses on the stability, resilience, and response to perturbation of the bacterial communities in the human gut. Informative flash flood-like disturbances that eliminate most gastrointestinal biomass can be induced using a clinically-relevant iso-osmotic agent. We designed and executed such a disturbance in human volunteers using a dense longitudinal sampling scheme extending before and after induced diarrhea. This experiment has enabled a careful multidomain analysis of a controlled perturbation of the human gut microbiota with a new level of resolution. These new longitudinal multidomain data were analyzed using recently developed statistical methods that demonstrate improvements over current practices. By imposing sparsity constraints we have enhanced the interpretability of the analyses and by employing a new adaptive generalized principal components analysis, incorporated modulated phylogenetic information and enhanced interpretation through scoring of the portions of the tree most influenced by the perturbation. Our analyses leverage the taxa-sample duality in the data to show how the gut microbiota recovers following this perturbation. Through a holistic approach that integrates phylogenetic, metagenomic and abundance information, we elucidate patterns of taxonomic and functional change that characterize the community recovery process across individuals. We provide complete code and illustrations of new sparse statistical methods for high-dimensional, longitudinal multidomain data that provide greater interpretability than existing methods.

Minority variant human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are associated with an increased risk of virological failure during treatment with NNRTI-containing regimens. To determine whether individuals to whom variants with isolated NNRTI-associated drug resistance were transmitted are at increased risk of virological failure during treatment with a non-NNRTI-containing regimen, we identified minority variant resistance mutations in 33 individuals with isolated NNRTI-associated transmitted drug resistance and 49 matched controls. We found similar proportions of overall and nucleoside reverse transcriptase inhibitor-associated minority variant resistance mutations in both groups, suggesting that isolated NNRTI-associated transmitted drug resistance may not be a risk factor for virological failure during treatment with a non-NNRTI-containing regimen., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

OBJECTIVE To compare the accuracy of ultrasonography and MRI for diagnosing medial meniscal lesions in dogs with cranial cruciate ligament (CCL) deficiency. DESIGN Diagnostic test evaluation. ANIMALS 26 dogs (31 stifle joints) with CCL deficiency. PROCEDURES A single surgeon physically examined each dog and performed ultrasonography and arthroscopy of affected stifle joints to identify medial meniscal lesions. Video recordings of the arthroscopic procedure were saved and subsequently reviewed by the same surgeon and by a second surgeon working independently and blinded to results of all examinations. A radiologist blinded to results of all examinations evaluated MRI scans of the affected joints. Correct classification rate (CCR), sensitivity, and specificity of ultrasonography and MRI were calculated twice, with each of the 2 surgeons' arthroscopic assessments used as the reference standard. RESULTS Compared with arthroscopic examination by the unblinded surgeon, ultrasonography had a CCR of 90%, sensitivity of 95% (95% confidence interval [CI], 73% to 100%), and specificity of 82% (95% CI, 48% to 97%). For MRI, these values were 84%, 75% (51% to 90%), and 100% (68% to 100%), respectively. Compared with arthroscopic assessment by the blinded surgeon, ultrasonography had a CCR of 84%, sensitivity of 86% (95% CI, 64% to 96%), and specificity of 78% (95% CI, 40% to 96%). For MRI, these values were 77%, 68% (45% to 82%), and 100% (63% to 100%), respectively. CONCLUSIONS AND CLINICAL RELEVANCE These data suggested imperfect performance but clinical usefulness of both ultrasonography and MRI for diagnosing medial meniscal lesions in dogs.

Magnetic resonance imaging (MRI) is a common test for dogs with suspected intradural spinal cord lesions, however studies on diagnostic performance for this test are lacking. Objectives of this multi-institutional, retrospective, case-control study were to estimate sensitivity and specificity of MRI for (1) distinguishing between histopathologically confirmed intradural spinal cord disease versus degenerative myelopathy in dogs, (2) categorizing intradural spinal cord diseases as neoplastic, inflammatory, or vascular; and (3) determining tumor type within the etiologic category of neoplasia. Additional aims were to (1) determine whether knowledge of clinical data affects sensitivity and specificity of MRI diagnoses; and (2) report interrater agreement for MRI classification of intradural spinal lesions. Cases were recruited from participating hospital databases over a 7-year period. Three reviewers independently evaluated each MRI study prior to and after provision of clinical information. A total of 87 cases were sampled (17 degenerative myelopathy, 53 neoplasia, nine inflammatory, and eight vascular). Magnetic resonance imaging had excellent (>97.6%) sensitivity for diagnosis of intradural spinal cord lesions but specificity varied before and after provision of clinical data (68.6% vs. 82.4%, P = 0.023). Magnetic resonance imaging had good sensitivity (86.8%) and moderate specificity (64.7-72.5%) for diagnosing neoplasia. Sensitivity was lower for classifying inflammatory lesions but improved with provision of clinical data (48.1% vs. 81.5%, P = 0.015). Magnetic resonance imaging was insensitive for diagnosing vascular lesions (25.0%). Interrater agreement was very good for correctly diagnosing dogs with intradural lesions (ĸ = 0.882-0.833), and good (ĸ = 0.726-0.671) for diagnosing dogs with neoplasia., (© 2017 American College of Veterinary Radiology.)

Objectives: The purpose of this study was to investigate whether platelet-rich plasma (PRP) enhances osseous healing in conjunction with a high tibial osteotomy in dogs., Study Design: Randomized controlled trial., Methods: Sixty-four client-owned pet dogs with naturally occurring rupture of the anterior cruciate ligament and that were to be treated with a high tibial osteotomy (tibial plateau leveling osteotomy) were randomized into the treatment or control group. Dogs in the treatment group received autologous platelet-rich plasma activated with calcium chloride and bovine thrombin to produce a well-formed PRP gel that was placed into the osteotomy at the time of surgery. Dogs in the control group received saline lavage of the osteotomy. All dogs had the osteotomy stabilized with identical titanium alloy implants and all aspects of the surgical procedure and post-operative care were identical among dogs of the two groups. Bone healing was assessed at exactly 28, 49, and 70 days after surgery with radiography and ultrasonography and with MRI at day 28. The effect of PRP on bone healing was assessed using a repeated measures analysis of covariance with radiographic and ultrasonographic data and using a t-test with the MRI data., Results: Sixty dogs completed the study. There were no significant differences in age, weight, or gender distribution between the treatment and control groups. Twenty-seven dogs were treated with PRP and 33 were in the control group. The average platelet concentration of the PRP was 1.37x106 platelets/μL (±489x103) with a leukocyte concentration of 5.45x103/μL (±3.5x103). All dogs demonstrated progressive healing over time and achieved clinically successful outcomes. Time since surgery and patient age were significant predictors of radiographic healing and time since surgery was a significant predictor of ultrasonographic assessment of healing. There was no significant effect of PRP treatment as assessed radiographically, ultrasonographically, or with MRI., Conclusion: The PRP used in this study did not hasten osseous union in dogs treated with a high tibial osteotomy.

Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 - A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F - were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To determine whether assessment of morphological MRI sequences or delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) would have strong correlations with arthroscopic assessment of cartilage pathology in dogs with naturally occurring medial compartment pathology of the elbow., Methods: Dogs tentatively diagnosed with medial coronoid disease had evaluation of their affected elbows using radiography, morphological MRI sequences, and dGEMRIC MRI evaluation prior to arthroscopy. Elbow radiographs were graded 0-6 for severity of changes. Cartilage of the medial coronoid process (MCP) and humeral trochlea (HT) were scored on a 0-3 scale using anatomical MRI sequences. The T1 relaxation times for the MCP and trochlea were quantified using dGEMRIC. Cartilage pathology was graded arthroscopically using a modified Outerbridge score (MOS) by a surgeon blinded to MRI assessment. Correlations between radiography and MOS, and between MRI and MOS, were quantified., Results: Twenty-six elbows in 14 dogs were evaluated. There were statistically significant ( p  < 0.05) moderate correlations between radiographic scores and MOS for the MCP ( r  = 0.71) and HT (0.57). There was a statistically significant moderate correlation between morphological MRI scoring and MOS for the HT ( r  = 0.54; p  < 0.05), but not for the MCP ( p  > 0.05). There was a weak, but significant correlation, between the dGEMRIC value and MOS of the MCP ( r  = 0.41; p  < 0.05), but no correlation between the dGEMRIC values and MOS for the HT ( p  > 0.05)., Clinical Relevance: Statistically significant correlations to MOS were identified for both radiography and MRI but neither diagnostic modality provided sufficiently strong correlations to serve as a substitute for arthroscopic evaluation of the articular cartilage.