Functional annotation of a divergent genome using sequence and structure-based similarity.

BACKGROUND: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. RESULTS: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. CONCLUSION: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

The Association of Childbirth with Medical Debt in the USA, 2019-2020.

BACKGROUND: Medical debt affects one in five adults in the USA and may disproportionately burden postpartum women due to pregnancy-related medical costs. OBJECTIVE: To evaluate the association between childbirth and medical debt, and the correlates of medical debt among postpartum women, in the USA. DESIGN: Cross-sectional. PARTICIPANTS: We analyzed female "sample adults" 18-49 years old in the 2019-2020 National Health Interview Survey, a nationally representative household survey. MAIN MEASURES: Our primary exposure was whether the subject gave birth in the past year. We had two family-level debt outcomes: problems paying medical bills and inability to pay medical bills. We examined the association between live birth and medical debt outcomes, unadjusted and adjusted for potential confounders in multivariable logistic regressions. Among postpartum women, we also examined the association between medical debt with maternal asthma, hypertension, and gestational diabetes and several sociodemographic factors. KEY RESULTS: Our sample included n = 12,163 women, n = 645 with a live birth in the past year. Postpartum women were younger, more likely to have Medicaid, and lived in larger families than those not postpartum. 19.8% of postpartum women faced difficulty with medical bills versus 15.1% who were not; in multivariable regression, postpartum women had 48% higher adjusted odds of medical debt problems (95% CI 1.13, 1.92). Results were similar when examining inability to pay medical bills, and similar differences were seen for privately insured women. Among postpartum women, those with lower incomes and with asthma or gestational diabetes, but not hypertension, had significantly higher adjusted odds of medical debt problems. CONCLUSIONS: Postpartum women experience higher levels of medical debt than other women; poorer women and those with common chronic diseases may have an even higher burden. Policies to expand and improve health coverage for this population are needed to improve maternal health and the welfare of young families.

The associations of plant-based food and metabolic syndrome using NHANES 2015-16 data.

BACKGROUND: Consuming a diet rich in plant-based foods (PBFs) may be protective for risk of metabolic syndrome (MetS) and chronic disease. However, the impact of consuming healthy versus all types of PBF on MetS is unknown. METHODS: The relationship between consumption of PBF (both healthy and all) was examined using data from the 2015 to 2016 National Health and Nutrition Examination Survey (NHANES). The amount of PBFs consumed was calculated as average daily servings, whereas dichotomous MetS variables were based on the National Cholesterol Education Adult Program Treatment Panel III (2005). After weighting and multiple imputation, adjusted associations were examined using logistic regression and marginal probabilities. RESULTS: Consumption of healthy PBF was significantly associated with reduced risk for elevated waist circumference (P = 0.017; odds ratio, OR 0.96, 95% confidence interval, CI 0.94-0.98) and MetS (P = 0.006; OR 0.96, 95% CI 0.93-0.99). Interactions revealed significant protective effects for females who were $\ge$ 60 years. CONCLUSIONS: In the adjusted model, a 1-unit increase in daily servings of healthy PBF was associated with a 4% lower risk for prevalence of elevated waist circumference and a 4% decrease in risk for prevalence of MetS. Increasing consumption of PBF may offer a viable strategy for reducing risk of MetS.

Effect of height versus height/weight-based spinal bupivacaine on maternal hemodynamics for elective cesarean in short stature patients: a randomized clinical trial.

PURPOSE: Doses of spinal bupivacaine adjusted to patient height or height/weight have been shown to provide hemodynamic stability during cesarean section. However, their effects in short stature parturients are unknown. METHODS: In this double-blind, randomized clinical trial, we randomly assigned short parturients (height < 150 cm) undergoing elective cesarean section, to receive doses of intrathecal hyperbaric bupivacaine either height or height/weight-adjusted, in a 1:1 ratio. The primary outcome was post-spinal hypotension (defined as systolic blood pressure [SBP] < 90% of baseline between spinal administration and delivery of the baby). Secondary outcomes included severe post-spinal hypotension (SBP < 80% of baseline), post-delivery hypotension (SBP < 90% and < 80% of baseline), intraoperative bradycardia, nausea and vomiting, shivering, rescue analgesic needed, and spinal block characteristics. RESULTS: A total of 112 patients underwent randomization. Post-spinal hypotension (SBP < 90% of baseline) occurred in 52% of the patients in the height/weight group and in 55% in the height group (difference - 3.5%: 95% confidence interval [CI] - 22 to 14.8, P = 0.705). There was no significant difference between the two groups in the occurrences of post-spinal severe hypotension (SBP < 80% of baseline), post-delivery hypotension, and spinal block characteristics. Six patients (11%) in the height/weight group needed intraoperative rescue analgesic compared to none in the height group (P = 0.027). CONCLUSION: We found that height-based dosing in short parturients provides the optimal trade-off between intraoperative hemodynamic instability and provision of pain-free anesthesia. TRIAL REGISTRATION: clinicaltrial.gov-NCT04082676. https://clinicaltrials.gov/ct2/show/NCT04082676 .

Intrathecal morphine does not prevent chronic postsurgical pain after elective Caesarean delivery: a randomised controlled trial.

BACKGROUND: Morphine is frequently added to spinal anaesthesia for Caesarean delivery. We aimed to determine whether intrathecal morphine for spinal anaesthesia decreases the risk of chronic postsurgical pain (CPSP). METHODS: In this randomised, double-blind, placebo-controlled trial, 290 healthy parturients undergoing elective Caesarean delivery were randomly assigned in a 1:1 ratio to receive either intrathecal morphine 100 µg (n=145) or normal saline (control; n=145) as a part of spinal anaesthesia. Anaesthetic care and postoperative pain management were standardised in all patients. The primary outcome was the incidence of CPSP at 3 months. Secondary outcomes included CPSP at 6 months, pain severity, and pain interference, measured by the Brief Pain Inventory questionnaire using an 11-point numeric rating scale, at 3 and 6 months after the surgery. RESULTS: Two hundred and seventy-six patients completed the 3-month follow-up, 139 in the morphine group and 137 in the placebo group. The incidences of CPSP at 3 months were 19% (27 of 139) in the morphine group and 18% (25 of 137) in the placebo group (odds ratio, 1.08; 95% confidence interval, 0.59-1.97; P=0.803). At 6 months, CPSP was present in 23 of 139 (16%) morphine group patients compared with 19 of 137 (14%) in the placebo group (odds ratio, 1.23; 95% confidence interval, 0.63-2.38; P=0.536). Brief Pain Inventory questionnaire scores for pain severity and pain interference at 3 and 6 months were similar between groups. CONCLUSIONS: Administration of morphine 100 µg as a component of spinal anaesthesia for elective Caesarean delivery failed to reduce the incidence of chronic pain at 3 and 6 months after surgery. CLINICAL TRIAL REGISTRATION: NCT03451695.

Factors associated with failed epidural blood patch after accidental dural puncture in obstetrics: a prospective, multicentre, international cohort study.

BACKGROUND: Epidural blood patch is commonly used for management of post-dural puncture headache after accidental dural puncture. The primary aim was to determine factors associated with failed epidural blood patch. METHODS: In this prospective, multicentre, international cohort study, parturients ≥18 yr receiving an epidural blood patch for treatment of post-dural puncture headache were included. Failed epidural blood patch was defined as headache intensity numeric rating scale (NRS) score ≥7 in the upright position at 4, 24, or 48 h, or the need for a second epidural blood patch, and complete success by NRS=0 at 0-48 h after epidural blood patch. All others were considered partial success. Multinominal logistic regression was used for statistical analyses with P<0.01 considered statistically significant. RESULTS: In all, 643 women received an epidural blood patch. Complete data to classify failure were available in 591 (91.9%) women. Failed epidural blood patch occurred in 167 (28.3%) patients; 195 (33.0%) were completely successful and 229 (38.7%) partially successful. A total of 126 women (19.8%) received a second epidural blood patch. A statistically significant association with failure was observed in patients with a history of migraine, when the accidental dural puncture occurred between lumbar levels L1/L3 compared with L3/L5 and when epidural blood patch was performed <48 h compared with ≥48 h after accidental dural puncture. In patients having radiological investigations, three intracranial bleeds were diagnosed. CONCLUSIONS: Failed epidural blood patch occurred in 28.3% of women. Independent modifiable factors associated with failure were higher lumbar level of accidental dural puncture and short interval between accidental dural puncture and epidural blood patch. A history of migraine was associated with a higher risk of second epidural blood patch. CLINICAL TRIAL REGISTRATION: NCT02362828.

Associations between variety of fruits and vegetables consumed, diet quality, and sociodemographic factors among 8th and 11th grade adolescents in Texas.

OBJECTIVE: To examine demographic and dietary correlates of consumption of a variety of fruits and vegetables (FV) among Texas adolescents. Different types of FV are needed for adequate dietary intake of vitamins and phytochemicals for proper development and functioning throughout the lifespan. DESIGN: Cross-sectional analysis from the Texas Surveillance of Physical Activity and Nutrition (Texas SPAN) data comparing consumption of a variety of fruit and vegetables by gender, race/ethnicity, and region (Texas-Mexico border/non-border). SETTING: Middle, high schools in Texas. PARTICIPANTS: 8th, 11th grade Texas adolescents (n=9,056 representing N=659,288) mean age 14.8 years. RESULTS: Within this sample, mean fruit and vegetable variety scores (0-7) ranged from 2.47-2.65. Boys consumed a significantly greater variety of fruit than girls (m=1.12 compared to 1.04). Adolescents in non-border regions consumed a greater variety of vegetables and FV compared to those in border regions. FV variety was associated with healthier eating in the full sample, particularly in the highest Socioeconomic Status (SES) tertile. Within the highest SES tertile, a one-unit increase in variety of fruit, vegetable, and FV was associated with significant increases (p<0.001) in a healthy eating measure, the SPAN Healthy Eating Index: Fruit variety (ß=1.33, SE=.29), Vegetable variety (ß=0.90, SE=0.28), and FV Variety (ß=.81, SE=0.19). CONCLUSIONS: Consumption of a greater variety of FV appears to be associated with a healthier overall diet. Associations of FV variety with healthy eating were most significant in the highest SES tertile. These findings support the need to further examine consuming a variety of FV within healthy eating behavior.

Validation of the FRESH Austin food frequency questionnaire using multiple 24-h dietary recalls.

OBJECTIVE: The purpose of the current study was to examine the validity of an FFQ utilised in the Food Retail: Evaluating Strategies for a Healthy Austin (FRESH Austin) study, designed to evaluate changes in the consumption of fruits and vegetables (FV) in diverse low-income communities in Austin, TX. DESIGN: The FRESH Austin FFQ was validated against three 24-h dietary recalls (24hDR). All dietary assessments were administered (in-person or by telephone) by trained investigators. SETTING: Recruitment was conducted at sites within the geographic areas targeted in the FRESH Austin recruitment. People at a community health clinic, a local health centre and a YMCA within the intervention area were approached by trained and certified data collectors, and invited to participate. PARTICIPANTS: Among fifty-six participants, 83 % were female, 46 % were non-White, 24 % had income < $25 K/year and 30 % spoke only/mostly Spanish at home. RESULTS: The FFQ and average of three 24hDR produce similar estimates of average total servings/d across FV (6·68 and 6·40 servings/d, respectively). Correlations produced measures from 0·01 for 'Potatoes' and 0·59 for 'Other Vegetables'. Mean absolute percentage errors values were small for all FV, suggesting the variance of the error estimates was also small. Bland-Altman plots indicate acceptable levels of agreement between the two methods. CONCLUSION: These outcomes indicate that the FRESH FFQ is a valid instrument for assessing FV consumption. The validation of the FRESH Austin FFQ provides important insights for evaluating community-based efforts to increase FV consumption in diverse populations.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

Objective and perceived barriers and facilitators of daily fruit and vegetable consumption among under-resourced communities in Central Texas.

INTRODUCTION: Fruit and vegetable consumption (FVC) continues to be low, particularly among people living in under-resourced communities. Identifying barriers and facilitators of FVC and whether those barriers and facilitators differ for racially and ethnically minoritized people is imperative for developing effective and equitable public health policies and interventions. METHODS: A baseline cohort of 390 participants from Central Texas communities historically lacking healthy food retailers completed a survey including FVC, 7 psychosocial barriers and facilitators of FVC, distance to a grocery retailer, participation in government assistance programs, and race/ethnicity. RESULTS: Not having time to prepare fruits and vegetables was the only significant psychosocial barrier identified (B = -.11, t(390) = 2.04, P = .04), but was not significant after accounting for sociodemographic variables. Significant facilitators of daily FVC were liking F&V (B=.31, t(390) = 6.40, P<.001), participating in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) (B=.14, t(390) = 2.81, P = .005), and Hispanic/Latino(a) ethnicity (B = -.21, t(390) = 4.30, P<.001). The final model accounted for 17% of the variance in daily FVC (R2=.17, F(4, 375) = 7.69, P < .001). Black, white and Other race participants were more likely to report having difficulty using F&V before spoiling than Hispanic/Latino(a) participants (P = .003). White and Other race participants were more likely to report that F&V were hard to prepare (P = .006) and that they didn't have time to prepare F&V (P = .005). DISCUSSION: When designing public health policy and interventions to increase FVC, researchers could prioritize identifying ways to alleviate time constraints, increase F&V liking, and help eligible participants to enroll in WIC. Strategies that reduce the risk of F&V spoiling and make F&V easier to prepare may also benefit some groups.

Outcomes of a Community-Wide Health Intervention in a Low-Income, Primarily Hispanic Community: The Go! Austin/Vamos! Austin (GAVA) Initiative.

OBJECTIVES: To describe outcomes of a 4-year physical activity (PA) and nutrition intervention (2013-2017) in Dove Springs, a low-income urban community in Texas. METHOD: Go! Austin/Vamos! Austin is a place-based intervention targeting the built and social environments of PA and nutrition. Baseline and follow-up measures related to PA and nutrition were obtained from 357 parent-child dyads (final n = 236) in the intervention community and a control community. A three-level dose of exposure measure was created to indicate the amount of exposure to intervention activities across the 4 years. Pre-post changes in key outcomes by level of exposure and contrasts across "high exposure" and "no exposure" categories were obtained using repeated-measures regression, adjusting for important confounders. RESULTS: "High exposure" adult participants showed consistently more favorable changes than "no exposure" participants across a variety of indicators, including positive perceptions and utilization of community PA resources, amount of moderate PA, utilization of retail outlets offering fresh produce, and measures of healthy eating. Few improvements were seen in child-level outcomes. CONCLUSIONS: Community interventions can successfully improve health-promoting behaviors provided they ensure sufficient dose of exposure.

School-based gardening, cooking and nutrition intervention increased vegetable intake but did not reduce BMI: Texas sprouts - a cluster randomized controlled trial.

BACKGROUND: Although school garden programs have been shown to improve dietary behaviors, there has not been a cluster-randomized controlled trial (RCT) conducted to examine the effects of school garden programs on obesity or other health outcomes. The goal of this study was to evaluate the effects of a one-year school-based gardening, nutrition, and cooking intervention (called Texas Sprouts) on dietary intake, obesity outcomes, and blood pressure in elementary school children. METHODS: This study was a school-based cluster RCT with 16 elementary schools that were randomly assigned to either the Texas Sprouts intervention (n = 8 schools) or to control (delayed intervention, n = 8 schools). The intervention was one school year long (9 months) and consisted of: a) Garden Leadership Committee formation; b) a 0.25-acre outdoor teaching garden; c) 18 student gardening, nutrition, and cooking lessons taught by trained educators throughout the school-year; and d) nine monthly parent lessons. The delayed intervention was implemented the following academic year and received the same protocol as the intervention arm. Child outcomes measured were anthropometrics (i.e., BMI parameters, waist circumference, and body fat percentage via bioelectrical impedance), blood pressure, and dietary intake (i.e., vegetable, fruit, and sugar sweetened beverages) via survey. Data were analyzed with complete cases and with imputations at random. Generalized weighted linear mixed models were used to test the intervention effects and to account for clustering effect of sampling by school. RESULTS: A total of 3135 children were enrolled in the study (intervention n = 1412, 45%). Average age was 9.2 years, 64% Hispanic, 47% male, and 69% eligible for free and reduced lunch. The intervention compared to control resulted in increased vegetable intake (+ 0.48 vs. + 0.04 frequency/day, p = 0.02). There were no effects of the intervention compared to control on fruit intake, sugar sweetened beverages, any of the obesity measures or blood pressure. CONCLUSION: While this school-based gardening, nutrition, and cooking program did not reduce obesity markers or blood pressure, it did result in increased vegetable intake. It is possible that a longer and more sustained effect of increased vegetable intake is needed to lead to reductions in obesity markers and blood pressure. CLINICAL TRIALS NUMBER: NCT02668744 .

Mapping Food Insecurity-Related 2-1-1 Calls in a 10-County Area of Central Texas by Zip Code: Exploring the Role of Geographic Food Access, Urbanicity and Demographic Indicators.

Food insecurity is a public health issue that affects 12% of Americans. Individuals living in food insecure households are more likely to suffer from conditions such as undernutrition, obesity and chronic diseases. Food insecurity has been linked to limited geographic access to food; however, past studies have used limited measures of access which do not fully capture the nuances of community context. The purpose of this study was to explore the association between food insecurity and geographic food access by level of urbanicity. 2-1-1 calls made in 2018 in Central Texas were classified as food needs versus non-food needs. Supermarket and convenience stores were mapped using ArcGIS. Geographic food access was operationalized as the presence of supermarkets and convenience stores: within the zip code; only in neighboring zip codes; and not located within or in neighboring zip codes. Descriptive statistics and binomial logistic regression were used to examine associations between geographic access and 2-1-1 food calls, stratified by level of urbanicity. 11% of the 2-1-1 calls made in 2018 (N = 55,405) were regarding food needs. Results showed that peri-urban and rural callers living in zip codes that only had supermarkets in neighboring zip codes had greater odds of calling about food needs compared to those that had supermarkets within the zip code. These findings indicate that geographic food access is associated with food insecurity, but this relationship varies by urbanicity. Thus, the development of food insecurity mitigation programs in peri-urban and rural areas is needed.

Management practices for postdural puncture headache in obstetrics: a prospective, international, cohort study.

BACKGROUND: Accidental dural puncture is an uncommon complication of epidural analgesia and can cause postdural puncture headache (PDPH). We aimed to describe management practices and outcomes after PDPH treated by epidural blood patch (EBP) or no EBP. METHODS: Following ethics committee approval, patients who developed PDPH after accidental dural puncture were recruited from participating countries and divided into two groups, those receiving EBP or no EBP. Data registered included patient and procedure characteristics, headache symptoms and intensity, management practices, and complications. Follow-up was at 3 months. RESULTS: A total of 1001 patients from 24 countries were included, of which 647 (64.6%) received an EBP and 354 (35.4%) did not receive an EBP (no-EBP). Higher initial headache intensity was associated with greater use of EBP, odds ratio 1.29 (95% confidence interval 1.19-1.41) per pain intensity unit increase. Headache intensity declined sharply at 4 h after EBP and 127 (19.3%) patients received a second EBP. On average, no or mild headache (numeric rating score≤3) was observed 7 days after diagnosis. Intracranial bleeding was diagnosed in three patients (0.46%), and backache, headache, and analgesic use were more common at 3 months in the EBP group. CONCLUSIONS: Management practices vary between countries, but EBP was more often used in patients with greater initial headache intensity. EBP reduced headache intensity quickly, but about 20% of patients needed a second EBP. After 7 days, most patients had no or mild headache. Backache, headache, and analgesic use were more common at 3 months in patients receiving an EBP.

Role of Organizational Support on Implementation of an Environmental Change Intervention to Improve Child Fruit and Vegetable Intake: a Randomized Cross-Over Design.

Little is known about the role of organizational or administrative support in implementation of health promotion interventions, particularly outside of school settings. The purpose was to determine the change in fruit and vegetable (FV) intake among children living in residential children's homes (RCHs) and assess the relationships among change in organizational support, intervention implementation, and child nutrition outcomes. Data were collected from 29 RCHs and 614 children living in RCHs, as part of a group randomized design with delayed intervention, at three cross-sectional waves: 2004, 2006, and 2008. RCH staff made environmental changes to increase intake of FV. Implementation and organizational support data were collected from staff at the RCHs. Child FV intake were measured via 24-h dietary recalls. A two-way (condition by time) repeated measures ANOVA was conducted to test whether FV intake increased in response to the intervention. A two-level path analysis with a robust maximum likelihood estimator was used to explore the relationships among organizational support, intervention implementation fidelity, and child FV intake. There was a significant increase in FV intake within all RCHs from 2004 to 2006 (P = 0.022 for the intervention group, P = 0.015 for the control group). This increase was maintained in both groups from 2006 to 2008 (post-intervention mean servings: intervention = 3.2 vs control = 3.4). Increases in organizational support resulted in greater overall implementation fidelity. When RCH staff, supervisors, and the RCH CEO were perceived to be supportive of the intervention, more environmental changes were made to encourage eating FV. Fostering organizational support may improve implementation of interventions.

The role of repressor kinetics in relief of transcriptional interference between convergent promoters.

Transcriptional interference (TI), where transcription from a promoter is inhibited by the activity of other promoters in its vicinity on the same DNA, enables transcription factors to regulate a target promoter indirectly, inducing or relieving TI by controlling the interfering promoter. For convergent promoters, stochastic simulations indicate that relief of TI can be inhibited if the repressor at the interfering promoter has slow binding kinetics, making it either sensitive to frequent dislodgement by elongating RNA polymerases (RNAPs) from the target promoter, or able to be a strong roadblock to these RNAPs. In vivo measurements of relief of TI by CI or Cro repressors in the bacteriophage λ PR-PRE system show strong relief of TI and a lack of dislodgement and roadblocking effects, indicative of rapid CI and Cro binding kinetics. However, repression of the same λ promoter by a catalytically dead CRISPR Cas9 protein gave either compromised or no relief of TI depending on the orientation at which it binds DNA, consistent with dCas9 being a slow kinetics repressor. This analysis shows how the intrinsic properties of a repressor can be evolutionarily tuned to set the magnitude of relief of TI.

Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter.

Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression.

Road rules for traffic on DNA-systematic analysis of transcriptional roadblocking in vivo.

Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining the magnitude of transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how roadblocking by the lac repressor (LacI) in Escherichia coli cells is controlled by promoter firing rate, the concentration and affinity of the roadblocker protein, the transcription-coupled repair protein Mfd, and promoter-roadblock spacing. Increased readthrough of the roadblock at higher RNAP fluxes requires active dislodgement of LacI by multiple RNAPs. However, this RNAP cooperation effect occurs only for strong promoters because roadblock-paused RNAP is quickly terminated by Mfd. The results are most consistent with a single RNAP also sometimes dislodging LacI, though we cannot exclude the possibility that a single RNAP reads through by waiting for spontaneous LacI dissociation. Reducing the occupancy of the roadblock site by increasing the LacI off-rate (weakening the operator) increased dislodgement strongly, giving a stronger effect on readthrough than decreasing the LacI on-rate (decreasing LacI concentration). Thus, protein binding kinetics can be tuned to maintain site occupation while reducing detrimental roadblocking.

Promoter activation by CII, a potent transcriptional activator from bacteriophage 186.

The lysogeny promoting protein CII from bacteriophage 186 is a potent transcriptional activator, capable of mediating at least a 400-fold increase in transcription over basal activity. Despite being functionally similar to its counterpart in phage λ, it shows no homology at the level of protein sequence and does not belong to any known family of transcriptional activators. It also has the unusual property of binding DNA half-sites that are separated by 20 base pairs, center to center. Here we investigate the structural and functional properties of CII using a combination of genetics, in vitro assays, and mutational analysis. We find that 186 CII possesses two functional domains, with an independent activation epitope in each. 186 CII owes its potent activity to activation mechanisms that are dependent on both the σ(70) and α C-terminal domain (αCTD) components of RNA polymerase, contacting different functional domains. We also present evidence that like λ CII, 186 CII is proteolytically degraded in vivo, but unlike λ CII, 186 CII proteolysis results in a specific, transcriptionally inactive, degradation product with altered self-association properties.

Availability of N-Methyl-d-Aspartate Receptor Coagonists Affects Cocaine-Induced Conditioned Place Preference and Locomotor Sensitization: Implications for Comorbid Schizophrenia and Substance Abuse.

Schizophrenia is associated with high prevalence of substance abuse. Recent research suggests that dysregulation of N-methyl-d-aspartate receptor (NMDAR) function may play a role in the pathophysiology of both schizophrenia and drug addiction, and thus, may account for this high comorbidity. Our laboratory has developed two transgenic mouse lines that exhibit contrasting NMDAR activity based on the availability of the glycine modulatory site (GMS) agonists d-serine and glycine. Glycine transporter 1 knockdowns (GlyT1(+/-)) exhibit NMDAR hyperfunction, whereas serine racemase knockouts (SR(-/-)) exhibit NMDAR hypofunction. We characterized the behavior of these lines in a cocaine-induced (20 mg/kg) conditioned place preference (CPP) and locomotor sensitization paradigm. Compared with wild-type mice, GlyT1(+/-) mice displayed hastened extinction of CPP and robust cocaine-induced reinstatement. SR(-/-) mice appeared to immediately "forget" the learned preference, because they did not exhibit cocaine-induced reinstatement and also displayed attenuated locomotor sensitization. Treatment of GlyT1(+/-) mice with gavestinel (10 mg/kg on day 1; 5 mg/kg on days 2-17), a GMS antagonist, attenuated cocaine-induced CPP and caused them to immediately "forget" the learned preference. Treatment of SR(-/-) mice with d-serine (300 mg/kg on day 1; 150 mg/kg on days 2-17) to normalize brain levels caused them to avoid the cocaine-paired side of the chamber during extinction. These results highlight NMDAR dysfunction as a possible neural mechanism underlying comorbid schizophrenia and substance abuse. Also, these findings suggest drugs that directly or indirectly activate the NMDAR GMS could be an effective treatment of cocaine abuse.