Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH.

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. Through whole genome sequencing we have uncovered conserved V483M Ptprh mutations in PyMT induced tumors. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while rescue with catalytically dead PTPRH did not. A dose response curve illustrated that two human NSCLC lines with naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Osimertinib treatment of these tumors resulted in a reduction of tumor volume relative to vehicle controls. PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Together these data suggest mutations in PTPRH in NSCLC is inhibitory to PTPRH function, resulting in aberrant EGFR activity and ultimately may result in clinically actionable alterations using existing therapies.

A Multifaceted Evaluation of a COVID-19 Contact Tracing Program in King County, Washington.

CONTEXT: Despite the massive scale of COVID-19 case investigation and contact tracing (CI/CT) programs operating worldwide, the evidence supporting the intervention's public health impact is limited. OBJECTIVE: To evaluate the Public Health-Seattle & King County (PHSKC) CI/CT program, including its reach, timeliness, effect on isolation and quarantine (I&Q) adherence, and potential to mitigate pandemic-related hardships. DESIGN: This program evaluation used descriptive statistics to analyze surveillance records, case and contact interviews, referral records, and survey data provided by a sample of cases who had recently ended isolation. SETTING: The PHSKC is one of the largest governmental local health departments in the United States. It serves more than 2.2 million people who reside in Seattle and 38 other municipalities. PARTICIPANTS: King County residents who were diagnosed with COVID-19 between July 2020 and June 2021. INTERVENTION: The PHSKC integrated COVID-19 CI/CT with prevention education and service provision. RESULTS: The PHSKC CI/CT team interviewed 42 900 cases (82% of cases eligible for CI/CT), a mean of 6.1 days after symptom onset and 3.4 days after SARS-CoV-2 testing. Cases disclosed the names and addresses of 10 817 unique worksites (mean = 0.8/interview) and 11 432 other recently visited locations (mean = 0.5/interview) and provided contact information for 62 987 household members (mean = 2.7/interview) and 14 398 nonhousehold contacts (mean = 0.3/interview). The CI/CT team helped arrange COVID-19 testing for 5650 contacts, facilitated grocery delivery for 7253 households, and referred 9127 households for financial assistance. End of I&Q Survey participants (n = 304, 54% of sampled) reported self-notifying an average of 4 nonhousehold contacts and 69% agreed that the information and referrals provided by the CI/CT team helped them stay in isolation. CONCLUSIONS: In the 12-month evaluation period, CI/CT reached 42 611 households and identified thousands of exposure venues. The timing of CI/CT relative to infectiousness and difficulty eliciting nonhousehold contacts may have attenuated the intervention's effect. Through promotion of I&Q guidance and services, CI/CT can help mitigate pandemic-related hardships.

Isoniazid or rifampicin preventive therapy with and without screening for subclinical TB: a modeling analysis.

BACKGROUND: Short-course, rifamycin-based regimens could facilitate scale-up of tuberculosis preventive therapy (TPT), but it is unclear how stringently tuberculosis (TB) disease should be ruled out before TPT use. METHODS: We developed a state-transition model of a TPT intervention among two TPT-eligible cohorts: adults newly diagnosed with HIV in South Africa (PWH) and TB household contacts in Pakistan (HHCs). We modeled two TPT regimens-4 months of rifampicin [4R] or 6 months of isoniazid [6H]-comparing each to a reference of no intervention. Before initiating TPT, TB disease was excluded either through symptom-only screening or with additional radiographic screening that could detect subclinical TB but might limit access to the TPT intervention. TPT's potential curative effects on both latent and subclinical TB were modeled, as were both acquisitions of resistance and prevention of drug-resistant disease. Although all eligible individuals received the screening and/or TPT interventions, the modeled TB outcomes comprised only those with latent or subclinical TB that would have progressed to symptomatic disease if untreated. RESULTS: When prescribed after only symptom-based TB screening (such that individuals with subclinical TB were included among TPT recipients), 4R averted 45 active (i.e., symptomatic) TB cases (95% uncertainty range 24-79 cases or 40-89% of progressions to active TB) per 1000 PWH [17 (9-29, 43-94%) per 1000 HHCs]; 6H averted 37 (19-66, 52-73%) active TB cases among PWH [13 (7-23, 53-75%) among HHCs]. With this symptom-only screening, for each net rifampicin resistance case added by 4R, 12 (3-102) active TB cases were averted among PWH (37 [9-580] among HHCs); isoniazid-resistant TB was also reduced. Similarly, 6H after symptom-only screening increased isoniazid resistance while reducing overall and rifampicin-resistant active TB. Screening for subclinical TB before TPT eliminated this net increase in resistance to the TPT drug; however, if the screening requirement reduced TPT access by more than 10% (the estimated threshold for 4R among HHCs) to 30% (for 6H among PWH), it was likely to reduce the intervention's overall TB prevention impact. CONCLUSIONS: All modeled TPT strategies prevent TB relative to no intervention, and differences between TPT regimens or between screening approaches are small relative to uncertainty in the outcomes of any given strategy. If most TPT-eligible individuals can be screened for subclinical TB, then pairing such screening with rifamycin-based TPT maximizes active TB prevention and does not increase rifampicin resistance. Where subclinical TB cannot be routinely excluded without substantially reducing TPT access, the choice of TPT regimen requires weighing 4R's efficacy advantages (as well as its greater safety and shorter duration that we did not directly model) against the consequences of rifampicin resistance in a small fraction of recipients.

Clinic-based diabetes screening at the time of HIV testing and associations with poor clinical outcomes in South Africa: a cohort study.

BACKGROUND: HIV clinical care programs in high burden settings are uniquely positioned to facilitate diabetes diagnosis, which is a major challenge. However, in sub-Saharan Africa, data on the burden of diabetes among people living with HIV (PLHIV) and its impact on HIV outcomes is sparse. METHODS: We enrolled adults presenting for HIV testing at an outpatient clinic in Durban. Those who tested positive for HIV-infection were screened for diabetes using a point-of-care hemoglobin A1c (HbA1c) test. We used log-binomial, Poisson, and Cox proportional hazard models adjusting for confounders to estimate the relationship of diabetes (HbA1c ≥ 6.5%) with the outcomes of HIV viral suppression (< 50 copies/mL) 4-8 months after antiretroviral therapy initiation, retention in care, hospitalization, tuberculosis, and death over 12 months. RESULTS: Among 1369 PLHIV, 0.5% (n = 7) reported a prior diabetes diagnosis, 20.6% (95% CI 18.5-22.8%, n = 282) screened positive for pre-diabetes (HbA1c 5.7-6.4%) and 3.5% (95% CI 2.7-4.6%, n = 48) for diabetes. The number needed to screen to identify one new PLHIV with diabetes was 46.5 persons overall and 36.5 restricting to those with BMI ≥ 25 kg/m2. Compared to PLHIV without diabetes, the risk of study outcomes among those with diabetes was not statistically significant, although the adjusted hazard of death was 1.79 (95% CI 0.41-7.87). CONCLUSIONS: Diabetes and pre-diabetes were common among adults testing positive for HIV and associated with death. Clinic-based diabetes screening could be targeted to higher risk groups and may improve HIV treatment outcomes.

Urine Tenofovir Concentrations Correlate With Plasma and Relate to Tenofovir Disoproxil Fumarate Adherence: A Randomized, Directly Observed Pharmacokinetic Trial (TARGET Study).

BACKGROUND: Direct measurement of tenofovir (TFV) in urine could be an objective measure to monitor adherence to preexposure prophylaxis (PrEP) or TFV-based antiretroviral therapy (ART). METHODS: We conducted a 3-arm randomized, pharmacokinetic study of tenofovir disoproxil fumarate (TDF) 300 mg/emtricitabine (FTC) 200 mg among adults living with human immunodeficiency virus. Participants were randomized to receive controlled TDF/FTC dosing as (1) "perfect" adherence (daily); (2) "moderate" adherence (4 doses/week); or (3) "low" adherence (2 doses/week). We obtained trough spot urine and plasma samples during a 6-week directly observed therapy period and a 4-week washout period. TFV concentrations were compared between adherence arms using 1-way analysis of variance. RESULTS: Among 28 participants, the median age was 33 years and 16 (57%) were male. Correlation between TFV plasma and urine concentrations was strong (ρ = 0.78; P < .0001). Median (interquartile range) steady-state trough TFV concentrations (ng/mL) for perfect, moderate, and low TDF adherence were 41 (26-52), 16 (14-19), and 4 (3-5) in plasma; and 6480 (3940-14 300), 3405 (2210-5020), and 448 (228-675) in urine. Trough TFV concentrations at steady state were significantly different between the 3 adherence arms for plasma (P < .0001) and urine (P = .0002). Following drug cessation, TFV concentrations persisted longer in urine than plasma samples. Washout urine TFV concentrations and time to undetectable concentrations did not differ between the 3 randomized adherence groups. CONCLUSIONS: Urine TFV concentrations can inform interpretation of novel point-of-care urine-based TFV assays to assess recent TDF adherence. CLINICAL TRIALS REGISTRATION: NCT03012607

Tuberculosis-related stigma among adults presenting for HIV testing in KwaZulu-Natal, South Africa.

BACKGROUND: Tuberculosis (TB)-related stigma presents a major barrier to care of persons with TB through its impact on treatment initiation and retention in care. This is particularly challenging in settings with high prevalence of both TB and HIV where fear of HIV/AIDS can amplify stigma surrounding TB. The purpose of this study was to validate a TB stigma scale for use among persons presenting for outpatient HIV screening in the Umlazi township of South Africa and evaluate factors associated with TB-related stigma in this high HIV burden setting. METHODS: In this cross-sectional study, we measured TB-related stigma in adults prior to HIV testing using a 12-item scale designed to assess experienced and felt TB-related stigma. RESULTS: Among 848 adults, mean age was 32 years, 54% were male, and the median TB stigma score was 19 of 36 (interquartile range 15-23). We identified two factors in the stigma scale which had excellent reliability (Cronbach's alpha 0.85, 0.89). Persons with high TB stigma were more likely to be male (adjusted relative risk ratio [aRRR] 1.56, 95% confidence interval [CI] 1.11-2.28) and have accurate knowledge of TB transmission (aRRR 1.90, 95% CI 1.16-3.10) as compared to those with low stigma. Variables not significantly associated with stigma in the multivariate model included education, income, prior TB or HIV diagnoses, and depression. CONCLUSIONS: Male sex and TB knowledge were associated with higher TB stigma in an outpatient HIV clinic in a South African township. Identifying risk factors associated with stigma will be important to guide stigma reduction interventions.

Mineral Metabolism Disturbances and Arteriovenous Fistula Maturation.

BACKGROUND: The arteriovenous fistula (AVF) is central to haemodialysis treatment, but up to half of surgically created AVF fail to mature. Chronic kidney disease often leads to mineral metabolism disturbances that may interfere with AVF maturation through adverse vascular effects. This study tested associations between mineral metabolism markers and vein histology at AVF creation and unassisted and overall clinical AVF maturation. METHODS: Concentrations of fibroblast growth factor 23, parathyroid hormone, calcium, phosphate, and vitamin D metabolites: 1,25(OH)2D, 24,25(OH)2D, 25(OH)D, and bioavailable 25(OH)D were measured in pre-operative serum samples from 562 of 602 participants in the Haemodialysis Fistula Maturation Study, a multicentre, prospective cohort study of patients undergoing surgical creation of an autologous upper extremity AVF. Unassisted and overall AVF maturation were ascertained for 540 and 527 participants, respectively, within nine months of surgery or four weeks of dialysis initiation. Study personnel obtained vein segments adjacent to the portion of the vein used for anastomosis, which were processed, embedded, and stained for measurement of neointimal hyperplasia, calcification, and collagen deposition in the medial wall. RESULTS: Participants in this substudy were 71% male, 43% black, and had a mean age of 55 years. Failure to achieve AVF maturation without assistance occurred in 288 (53%) participants for whom this outcome was determined. In demographic and further adjusted models, mineral metabolism markers were not significantly associated with vein histology characteristics, unassisted AVF maturation failure, or overall maturation failure, other than a biologically unexplained association of higher 24,25(OH)2D with overall failure. This exception aside, associations were non-significant for continuous and categorical analyses and relevant subgroups. CONCLUSIONS: Serum concentrations of measured mineral metabolites were not substantially associated with major histological characteristics of veins in patients undergoing AVF creation surgery, or with AVF maturation failure, suggesting that efforts to improve AVF maturation rates should increase attention to other processes such as vein mechanics, anatomy, and cellular metabolism among end stage renal disease patients.

Interaction of nutritional status and diabetes on active and latent tuberculosis: a cross-sectional analysis.

BACKGROUND: Malnutrition and diabetes are risk factors for active tuberculosis (TB), possible risk factors for latent TB infection (LTBI), and may interact to alter their effect on these outcomes. Studies to date have not investigated this interaction. METHODS: We enrolled 919 newly diagnosed active TB patients and 1113 household contacts at Primary Health Centres in Puducherry and Tamil Nadu, India from 2014 to 2018. In cross-sectional analyses, we used generalized estimating equations to measure additive and multiplicative interaction of body mass index (BMI) and diabetes on two outcomes, active TB and LTBI. RESULTS: Among overweight or obese adults, active TB prevalence was 12-times higher in diabetic compared to non-diabetic participants, 2.5-times higher among normal weight adults, and no different among underweight adults (P for interaction < 0.0001). Diabetes was associated with 50 additional active TB cases per 100 overweight or obese participants, 56 per 100 normal weight participants, and 17 per 100 underweight participants (P for interaction < 0.0001). Across BMI categories, screening 2.3-3.8 active TB patients yielded one hyperglycemic patient. LTBI prevalence did not differ by diabetes and BMI*diabetes interaction was not significant. CONCLUSIONS: BMI and diabetes are associated with newly diagnosed active TB, but not LTBI. Diabetes conferred the greatest risk of active TB in overweight and obese adults whereas the burden of active TB associated with diabetes was similar for normal and overweight or obese adults. Hyperglycemia was common among all active TB patients. These findings highlight the importance of bi-directional diabetes-active TB screening in India.

A randomized clinical pharmacokinetic trial of Tenofovir in blood, plasma and urine in adults with perfect, moderate and low PrEP adherence: the TARGET study.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is key component of pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) for HIV, but existing tools to monitor drug adherence are often inaccurate. Detection of tenofovir (TFV) in accessible biological samples, such as fingerprick blood, urine or oral fluid samples could be a novel objective measure of recent TDF adherence. To measure TFV concentrations associated with different levels of TDF adherence, we designed a randomized clinical trial to assess the blood, urine and oral fluid concentrations of TFV in adults with perfect, moderate and low drug adherence. METHODS/DESIGN: A randomized, open-label, clinical pharmacokinetic study of tenofovir in healthy adult volunteers without HIV or Hepatitis B infection in Thailand. Consenting, eligible participants are randomized (1:1:1) among three groups to receive a controlled number of TDF (300 mg) doses in a combination pill with emtricitabine (FTC, 200 mg) for six weeks. Participants in Group 1 receive a single TDF/FTC tablet once daily (Perfect adherence); Group 2 receive a single TDF/FTC tablet 4 times/week (Moderate adherence); and Group 3 receive a single TDF/FTC tablet 2 times/week (Low adherence). Blood, plasma, urine and oral fluid samples are collected for drug measurement during three study phases: (i) initial 6-week treatment phase; (ii) intensive 24-h blood sampling phase after 6 weeks; (iii) 4-week washout phase. Thirty adults with evaluable pharmacokinetic samples (10 per group) will be enrolled [based on ensuring 25% precision in pharmacokinetic parameter estimates]. Pre-dose drug concentrations during the treatment phase will be descriptive and comparisons between groups performed using a Kruskal-Wallis test. A non-compartmental pharmacokinetic analysis will be performed on the intensive sampling data at Week 7 and the time course of TFV washout in the difference biological matrices will be reported based on the detected concentrations following drug cessation. DISCUSSION: The results of this randomized trial will define the target concentration thresholds of TFV in blood, urine and oral fluid that can distinguish between different levels of TDF adherence. Such adherence 'benchmarks' can be applied to real-time drug testing and novel point-of-care tests to identify individuals with poor PrEP or ART adherence. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03012607 .

Cough Aerosols of Mycobacterium tuberculosis in the Prediction of Incident Tuberculosis Disease in Household Contacts.

BACKGROUND: Tuberculosis disease develops in only 5%-10% of humans infected with Mycobacterium tuberculosis The mechanisms underlying this variability remain poorly understood. We recently demonstrated that colony-forming units of M. tuberculosis in cough-generated aerosols are a better predictor of infection than the standard sputum acid-fast bacilli smear. We hypothesized that cough aerosol cultures may also predict progression to tuberculosis disease in contacts. METHODS: We conducted a retrospective cohort study of 85 patients with smear-positive tuberculosis and their 369 household contacts in Kampala, Uganda. Index case patients underwent a standard evaluation, and we cultured M. tuberculosis from cough aerosols. Contacts underwent a standard evaluation at enrollment, and they were later traced to determine their tuberculosis status. RESULTS: During a median follow-up of 3.9 years, 8 (2%) of the contacts developed tuberculosis disease. In unadjusted and adjusted analyses, incident tuberculosis disease in contacts was associated with sputum Mycobacterial Growth Indicator Tube culture (odds ratio, 8.2; 95% confidence interval, 1.1-59.2; P = .04), exposure to a high-aerosol tuberculosis case patient (6.0, 1.4-25.2; P = .01), and marginally, human immunodeficiency virus in the contact (6.11; 0.89-41.7; P = .07). We present data demonstrating that sputum and aerosol specimens measure 2 related but different phenomena. CONCLUSIONS: We found an increased risk of tuberculosis progression among contacts of high-aerosol case patients. The hypothesis that a larger infectious inoculum, represented by high aerosol production, determines the risk of disease progression deserves evaluation in future prospective studies.

Improving the sensitivity of the Xpert MTB/RIF assay on sputum pellets by decreasing the amount of added sample reagent: a laboratory and clinical evaluation.

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosis in sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of M. tuberculosis into sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosis culture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which M. tuberculosis was detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%; P=0.03) and for smear-negative specimens (67% versus 58%; P=0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosis detection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

Prospective cross-sectional evaluation of the small membrane filtration method for diagnosis of pulmonary tuberculosis.

Smear microscopy has suboptimal sensitivity, and there is a need to improve its performance since it is commonly used to diagnose tuberculosis (TB). We prospectively evaluated the diagnostic accuracy of the small membrane filtration (SMF) method, an approach that uses a vacuum manifold and is designed to concentrate bacilli onto a filter that can be examined microscopically. We enrolled hospitalized adults suspected to have pulmonary TB in Kampala, Uganda. We obtained a clinical history and three spontaneously expectorated sputum specimens for smear microscopy (direct, concentrated, and SMF), MGIT (mycobacterial growth indicator tube) 960 and Lowenstein-Jensen (LJ) cultures, and Xpert MTB/RIF testing. We performed per-specimen (primary) and per-patient analyses. From October 2012 to June 2013, we enrolled 212 patients (579 sputum specimens). The participants were mostly female (63.2%), and 81.6% were HIV infected; their median CD4 cell count was 47 cells/µl. Overall, 19.0%, 20.4%, 27.1%, 25.2%, and 25.9% of specimens tested positive by direct smear, concentrated smear, MGIT culture, LJ culture, and Xpert test, respectively. In the per-specimen analysis, the sensitivity of the SMF method (48.5%; 95% confidence interval [CI], 37.4 to 59.6) was lower than those of direct smear (60.9%; 51.4 to 70.5 [P = 0.0001]) and concentrated smear (63.3%; 53.6 to 73.1 [P < 0.0001]). Subgroup analyses showed that SMF performed poorly in specimens having a low volume or low bacterial load. The SMF method performed poorly compared to standard smear techniques and was sensitive to sample preparation techniques. The optimal laboratory SMF protocol may require striking a fine balance between sample dilution and filtration failure rate.

Impact of cryoablation on operative outcomes in thoracotomy patients.

OBJECTIVES: Cryoablation is increasingly being utilized as an alternative to epidurals for patients undergoing thoracotomies. Current evidence suggests cryoablation may decrease postoperative analgesia utilization, but could increase operative times. We hypothesized that the adoption of intraoperative cryoablation to manage post-thoracotomy pain would result in reduced length of stay and reduced perioperative analgesia compared to routine epidural use. METHODS: A retrospective analysis was performed from a single, quaternary referral centre, prospective database on patients receiving thoracotomies between January 2020 and March 2022. Patients undergoing transthoracic hiatal hernia repair, lung resection or double-lung transplant were divided between epidural and cryoablation cohorts. Primary outcomes were length of stay, intraoperative procedure time, crossover pain management and oral narcotic usage the day before discharge. RESULTS: During the study period, 186 patients underwent a transthoracic hiatal hernia repair, lung resection or double-lung transplant with 94 receiving a preoperative epidural and 92 undergoing cryoablation. Subgroup analysis demonstrated no significant differences in demographics, operative length, length of stay or perioperative narcotic use. Notably, over a third of patients in each cryoablation subgroup received a postoperative epidural (45.5% transthoracic hiatal hernia repair, 38.5% lung resection and 45.0% double-lung transplant) for further pain management during their admission. CONCLUSIONS: Cryoablation use was not associated with an increase in procedure time, a decrease in narcotic use or length of stay. Surprisingly, many cryoablation patients received epidurals in the postoperative period for further pain control. Additional analysis is needed to fully understand the benefits and costs of epidural versus cryoablation strategies.

Structure of EvaA: a paradigm for sugar 2,3-dehydratases.

Unusual deoxysugars found appended to natural products often provide or enhance the pharmacokinetic activities of the parent compound. The preferred carbohydrate donors for the biosynthesis of such glycosylated natural products are the dTDP-linked sugars. Many of the biologically relevant dTDP-deoxysugars are constructed around the 2,6-dideoxyhexoses or the 2,3(4),6-trideoxyhexoses. A key step in the biosynthesis of these sugars is the removal of the hexose C-2' hydroxyl group and the oxidation of the C-3' hydroxyl group to a carbonyl moiety. Enzymes that catalyze these reactions are referred to as 2,3-dehydratases and have been, for the most part, largely uncharacterized. Here we report the first structural analysis of a sugar 2,3-dehydratase. For this investigation, the enzyme, EvaA, was cloned from Amycolatopsis orientalis, and the structure was solved and refined to a nominal resolution of 1.7 Å. On the basis of the resulting model, it is clear that EvaA belongs to the large Nudix hydrolase superfamily and is most similar to GDP-mannose hydrolase. Each subunit of the EvaA dimer folds into two domains that clearly arose via gene duplication. Two dTDP-sugar binding pockets, A and B, are present in each EvaA subunit. On the basis of site-directed mutagenesis experiments and activity assays, it appears that pocket A functions as the active site and pocket B is simply a remnant left behind from the gene duplication event. As 2,3-dehydration is crucial for the biosynthesis of many unusual deoxysugars, this investigation provides key structural insight into this widely conserved reaction.

Viral suppression after 12 months of antiretroviral therapy in low- and middle-income countries: a systematic review.

OBJECTIVE: To establish estimates of viral suppression in low- and middle-income countries (LMICs) in patients who received antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection. METHODS: Data on viral suppression after 12 months of ART in LMICs were collected from articles published in 2003 to 2011 and from abstracts of conferences held between 2009 and 2011. Pooled proportions for on-treatment and intention-to-treat populations were used as summary estimates. Random-effects models were used for heterogeneous groups of studies (I (2) > 75%). FINDINGS: Overall, 49 studies covering 48 cohorts and 30 016 individuals met the inclusion criteria. With thresholds for suppression between 300 and 500 copies of viral ribonucleic acid (RNA) per ml of plasma, 84.3% (95% confidence interval, CI: 80.4-87.9) of the pooled on-treatment population and 70.5% (95% CI: 65.2-75.6) of the intention-to-treat population showed suppression. Use of different viral RNA thresholds changed the proportions showing suppression: to 84% and 76% of the on-treatment population with thresholds set above 300 and at or below 200 RNA copies per ml, respectively, and to 78%, 71% and 63% of the intention-to-treat population at thresholds set at 1000, 300 to 500, and 200 or fewer copies per ml, respectively. CONCLUSION: The pooled estimates of viral suppression recorded after 12 months of ART in LMICs provide benchmarks that other ART programmes can use to set realistic goals and perform predictive modelling. Evidence from this review suggests that the current international target - i.e. viral suppression in > 70% of the intention-to-treat population, with a threshold of 1000 copies per ml - should be revised upwards.

Evaluation of mortality risk after COVID-19 vaccination, Utah 2021.

INTRODUCTION: In order to evaluate trends in death after COVID-19 vaccination we analyzed the timing of death relative to vaccination date and the causes of death in vaccinated Utahns in 2021. METHODS: We matched people in the Utah immunization registry with documented COVID-19 vaccinations between December 18, 2020 and December 31, 2021 to Utah's 2021 vital statistics death records. Vaccinated people were categorized as having one, two, or ≥ three COVID-19 vaccine doses in a time-updated metric. We examined crude mortality rates by dosing groups in two-week intervals for all deaths, and by COVID-19 versus non-COVID-19 causes, within the 44 weeks following receipt of the most recent vaccine. RESULTS: We identified 2,072,908 individuals who received at least one dose of COVID-19 vaccine of whom 10,997 died in 2021. Only 17.5 % of the total vaccinated population was age 65+, while 80.9 % of those who died were over 65. In the four weeks following the first or second vaccination, all-cause mortality was low and then stabilized for the remainder of the evaluation period at a bi-weekly average of 33.0 and 39.0 deaths/100,000 people for one and two doses, respectively. Typical seasonal variation in death was observed among those with two doses. Small sample size precluded analysis of those with ≥ three doses, but trends were similar. CONCLUSIONS: Mortality rates in the 44 weeks following the COVID-19 vaccination did not show trends suggesting an increase in mortality related to COVID-19 vaccination, reinforcing the safety of COVID-19 vaccines. This represents an accessible approach for local evaluation.

Structural studies of AntD: an N-Acyltransferase involved in the biosynthesis of D-Anthrose.

The unusual dideoxy sugar d-anthrose has been identified as an important component in the endospores of infectious agents such as Bacillus anthracis and Bacillus cereus. Specifically, it is the terminal sugar on the bacterium's exosporium, and it provides a point of interaction between the spore and the host. The biosynthesis of d-anthrose involves numerous steps starting from α-d-glucose 1-phosphate. Here we present a combined structural and functional investigation of AntD from B. cereus. This enzyme plays a key role in d-anthrose biosynthesis by catalyzing the acylation of the C-4″ amino group of dTDP-4-amino-4,6-dideoxyglucose using 3-hydroxy-3-methylbutyryl-CoA as the acyl donor. For this investigation, two ternary complexes of AntD were determined to 1.8 Å resolution: one in which the protein contained bound ß-hydroxybutyryl-CoA and dTDP and the second with CoA and dTDP-4-amino-4,6-dideoxyglucose. On the basis of these high-resolution structures, it was shown that the side chain of Asp 94 lies within hydrogen bonding distance of the sugar C-4″ amino group, and the side chain of Ser 84 resides near the carbonyl oxygen of ß-hydroxybutyryl-CoA. To test the roles of these residues in the catalytic mechanism of AntD, various site-directed mutant proteins were prepared and subjected to kinetic and structural analyses. The D94A and D94N mutant proteins demonstrated enzymatic activity, albeit with significantly reduced catalytic efficiencies. The S84A mutant protein showed an approximate 10-fold decrease in activity. Interestingly, the S84C and S84T mutant proteins were both active but demonstrated substrate inhibition. The three-dimensional structures of all of the mutant proteins were nearly identical to that of the wild-type enzyme, indicating that the changes in their kinetic parameters were not due to major conformational changes. Taken together, these data suggest that Asp 94 is important for substrate binding, but probably does not function as an enzymatic base, and that Ser 84 most likely plays a role in the formation of an oxyanion hole.

Structural and functional studies on a 3'-epimerase involved in the biosynthesis of dTDP-6-deoxy-D-allose.

Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft.

Tenofovir-Diphosphate in Dried Blood Spots vs Tenofovir in Urine/Plasma for Oral Preexposure Prophylaxis Adherence Monitoring.

Background: Tenofovir-diphosphate (TFV-DP) measured in dried blood spots (DBS) and tenofovir (TFV) measured in urine/plasma have been used to measure TFV-based oral pre-exposure prophylaxis (PrEP) adherence. However, there are limited data comparing these 3 metrics and their appropriate use for PrEP adherence monitoring. Methods: We collected DBS, urine, and plasma samples from HIV-negative adults randomized to a low (2 doses/week), moderate (4 doses/week), or perfect (7 doses/week) adherence group (via directly observed therapy) of tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) for 6 weeks, followed by a 4-week washout phase. Drug concentrations were measured using liquid chromatography tandem mass spectrometry. Linear mixed-effects modeling was used to examine associations between drug concentrations and dosing time. Results: Among 28 participants, the median age was 33 years, and 12 (43%) were female. At steady state, 25th percentile TFV-DP concentrations were 466, 779, and 1375 fmol/3 mm punch in the low, moderate, and perfect adherence group, respectively. Correlation was stronger between quantifiable TFV-DP and plasma TFV (r = 0.65; P < .01) than between TFV-DP and urine TFV (r = 0.50; P < .01). Among all participants, each additional week of cumulative dosing on average led to a mean increase of 158 fmol/3 mm punch (P < .001) in TFV-DP during the dosing phase. Each additional day after the last dose was associated with 43 fmol/3 mm punch lower TFV-DP (P = .07). Conclusions: TFV-DP levels in DBS provide valuable insight into both dosing recency and cumulative doses from variable adherence patterns. Our observed benchmark TFV-DP concentrations were slightly higher than prior predicted estimates based on convenience samples.