Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

Computationally Efficient Demographic History Inference from Allele Frequencies with Supervised Machine Learning.

Inferring past demographic history of natural populations from genomic data is of central concern in many studies across research fields. Previously, our group had developed dadi, a widely used demographic history inference method based on the allele frequency spectrum (AFS) and maximum composite-likelihood optimization. However, dadi's optimization procedure can be computationally expensive. Here, we present donni (demography optimization via neural network inference), a new inference method based on dadi that is more efficient while maintaining comparable inference accuracy. For each dadi-supported demographic model, donni simulates the expected AFS for a range of model parameters then trains a set of Mean Variance Estimation neural networks using the simulated AFS. Trained networks can then be used to instantaneously infer the model parameters from future genomic data summarized by an AFS. We demonstrate that for many demographic models, donni can infer some parameters, such as population size changes, very well and other parameters, such as migration rates and times of demographic events, fairly well. Importantly, donni provides both parameter and confidence interval estimates from input AFS with accuracy comparable to parameters inferred by dadi's likelihood optimization while bypassing its long and computationally intensive evaluation process. donni's performance demonstrates that supervised machine learning algorithms may be a promising avenue for developing more sustainable and computationally efficient demographic history inference methods.

Treatment of Multidrug-resistant or Rifampicin-resistant Tuberculosis With an All-oral 9-month Regimen Containing Linezolid or Ethionamide in South Africa: A Retrospective Cohort Study.

BACKGROUND: In 2019, the South African tuberculosis program replaced ethionamide with linezolid as part of an all-oral 9-month regimen. We evaluated treatment outcomes for patients assigned to regimens including linezolid in 2019 and ethionamide in 2017. METHODS: This retrospective cohort study included patients treated for multidrug-resistant/rifampicin-resistant tuberculosis throughout South Africa between 1 January and 31 December 2017 and 1 January to 31 December 2019. The cohort treated with a 9-month regimen containing ethionamide for four months, was compared with a cohort treated with a 9-month regimen containing linezolid for 2 months. The regimens were otherwise identical. Inverse probability weighting of propensity scores was used to adjust for potential confounding. A log-binomial regression model was used to estimate adjusted relative risk (aRR) comparing 24-month outcomes between cohorts including treatment success, death, loss to follow up, and treatment failure. Adverse event data were available for the linezolid cohort. FINDINGS: In total, 817 patients were included in the cohort receiving ethionamide and 4244 in the cohort receiving linezolid. No evidence for a difference was observed between linezolid and ethionamide regimens for treatment success (aRR = 0.96, 95% confidence interval [CI] .91-1.01), death (aRR = 1.01, 95% CI .87-1.17) or treatment failure (aRR = 0.87, 95% CI .44-1.75). Loss to follow-up was more common in the linezolid group, although estimates were imprecise (aRR = 1.22, 95% CI .99-1.50). CONCLUSIONS: No significant differences in treatment success and survival were observed with substitution of linezolid for ethionamide as a part of an all-oral 9-month regimen. Linezolid is an acceptable alternative to ethionamide in this shorter regimen for treatment of multidrug-resistant/rifampicin-resistant tuberculosis.

Highly Activated Terminal Carbon Monoxide Ligand in an Iron-Sulfur Cluster Model of FeMco with Intermediate Local Spin State at Fe.

Nitrogenases, the enzymes that convert N2 to NH3, also catalyze the reductive coupling of CO to yield hydrocarbons. CO-coordinated species of nitrogenase clusters have been isolated and used to infer mechanistic information. However, synthetic FeS clusters displaying CO ligands remain rare, which limits benchmarking. Starting from a synthetic cluster that models a cubane portion of the FeMo cofactor (FeMoco), including a bridging carbyne ligand, we report a heterometallic tungsten-iron-sulfur cluster with a single terminal CO coordination in two oxidation states with a high level of CO activation (νCO = 1851 and 1751 cm-1). The local Fe coordination environment (2S, 1C, 1CO) is identical to that in the protein making this system a suitable benchmark. Computational studies find an unusual intermediate spin electronic configuration at the Fe sites promoted by the presence the carbyne ligand. This electronic feature is partly responsible for the high degree of CO activation in the reduced cluster.

Hospital Capabilities Associated With Behavioral Health Integration Within Emergency Departments.

OBJECTIVES: To identify hospital capabilities associated with behavioral health (BH) processes in emergency departments (EDs). RESEARCH DESIGN: Six hundred two hospital responses to the 2017/2018 National Survey of Healthcare Organizations and Systems were linked to 2017 American Hospital Association Annual Survey data. Separate multivariable regressions estimated how hospital capabilities (the use of quality improvement methods, approaches to disseminate best patient-care practices, barriers to using care delivery innovations, and inpatient beds for psychiatric or substance use) were associated with each of 4 ED-based BH processes: mental health and substance use disorder screening, team-based approaches to BH, telepsychiatry, and direct referrals to community-based BH clinicians. Models controlled for hospital structural characteristics and area-level socioeconomic factors. RESULTS: Most hospitals screened for BH conditions and provided direct referrals to community-based BH clinicians. Approximately half of the hospitals used a team approach to BH. A minority had implemented telepsychiatry. Each additional process used to disseminate best patient-care practices was associated with more screening for BH conditions (an increase of 4.07 points on the screening index, P <0.01) and greater likelihood of using a team approach to BH [4.41 percentage point ( P <0.01) increase]. Hospitals reporting more barriers to the use of care delivery innovations reported less screening and use of a team approach [a decrease of 0.15 points on the screening index ( P <0.01) and 0.28 percentage points reduction in likelihood of team approach use ( P <0.001) for 1-point increase in the barrier index]. CONCLUSIONS: Research and interventions focused on removing innovation barriers or adding processes to disseminate best practices offer a path to accelerate BH integration in hospital EDs.

Prenatal opioid use as a predictor of postpartum suicide attempts among reproductive-age women enrolled in Oregon Medicaid.

BACKGROUND: The rates of suicide and opioid use disorder (OUD) among pregnant and postpartum women continue to increase. This research characterized OUD and suicide attempts among Medicaid-enrolled perinatal women and examined prenatal OUD diagnosis as a marker for postpartum suicide attempts. METHODS: Data from Oregon birth certificates, Medicaid eligibility and claims files, and hospital discharge records were linked and analyzed. The sample included Oregon Medicaid women aged 15-44 who became pregnant and gave live births between January 2008 and January 2016 (N = 61,481). Key measures included indicators of suicide attempts (separately for any means and opioid poisoning) and OUD diagnosis, separately assessed during pregnancy and the one-year postpartum period. Probit regression was used to examine the overall relationship between prenatal OUD diagnosis and postpartum suicide attempts. A simultaneous equations model was employed to explore the link between prenatal OUD diagnosis and postpartum suicide attempts, mediated by postpartum OUD diagnosis. RESULTS: Thirty-three prenatal suicide attempts by any means were identified. Postpartum suicide attempts were more frequent with 58 attempts, corresponding to a rate of 94.3 attempts per 100,000. Of these attempts, 79% (46 attempts) involved opioid poisoning. A total of 1,799 unique women (4.6% of the sample) were diagnosed with OUD either during pregnancy or one-year postpartum with 53% receiving the diagnosis postpartum. Postpartum suicide attempts by opioid poisoning increased from 55.5 per 100,000 in 2009 to 105.1 per 100,000 in 2016. The rate of prenatal OUD also almost doubled over the same period. Prenatal OUD diagnosis was associated with a 0.15%-point increase in the probability of suicide attempts by opioid poisoning within the first year postpartum. This increase reflects a three-fold increase compared to the rate for women without a prenatal OUD diagnosis. A prenatal OUD diagnosis was significantly associated with an elevated risk of postpartum suicide attempts by opioid poisoning via a postpartum OUD diagnosis. CONCLUSIONS: The risk of suicide attempt by opioid poisoning is elevated for Medicaid-enrolled reproductive-age women during pregnancy and postpartum. Women diagnosed with prenatal OUD may face an increased risk of postpartum suicides attempts involving opioid poisoning.

Partial synthetic models of FeMoco with sulfide and carbyne ligands: Effect of interstitial atom in nitrogenase active site.

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe7S8C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe3S3C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M'Fe3S3X]n-, where M' = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M' results in minor changes, the chelating, µ3-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Comparing narrative storytelling ability in individuals with autism and fetal alcohol spectrum disorders.

BACKGROUND: Narrative discourse, or storytelling, is used in daily conversation and requires higher-level language and social communication skills that are not always captured by standardised assessments of language. Many autistic individuals and individuals with fetal alcohol spectrum disorders (FASD) have difficulties with both social communication and language skills, and narrative discourse analysis offers an ecologically relevant approach to assessing those challenges. AIMS: This study investigated narrative discourse in individuals with autism and FASD, as well as an age- and sex-matched comparison group. METHODS AND PROCEDURES: Narratives from 45 adolescents and adults, 11 with autism, 11 with FASD and 23 age- and sex-matched comparison participants were elicited using a wordless storybook. They were then transcribed orthographically, formatted to the Systematic Analyses of Language Transcript (SALT) convention and scored based on the SALT Narrative Scoring Scheme (NSS), a standardised language analysis protocol. In addition to the NSS total score, which assesses the overall structure and cohesion of the narratives produced, local and global measures of language ability were also employed. The local language measures included the number of mental state and temporal relation terms produced, while the global language measures included mean length of utterance, total different words, total words, total utterances, rate of speech, the number of mazes (e.g., repetitions, 'um', 'uh' or self-corrections) per total word and the NSS total score. OUTCOMES AND RESULTS: Using the SALT Language Sample Analysis tool, our results revealed that on global language measures, group differences were found on rate of speech, number of mazes per total words and the description of conflict/resolution in the narratives produced. The autism group produced significantly more mazes per total word and scored higher on the NSS conflict/resolution category score compared to the FASD and comparison groups. Both the autism and FASD groups spoke at a lower rate than the comparison group. On local language measures of narrative production, all groups were comparable, on average. CONCLUSIONS AND IMPLICATIONS: While many aspects of narrative discourse in the autism and FASD groups were similar to each other and to the comparison group, we observed group differences on global measures of narrative production and significant individual variability within groups, suggesting that narrative abilities considered at an individual level may provide important clinical information for intervention planning. Future research should also consider additional variables that influence narrative discourse, such as motivation, distractibility or decision-making of individual participants. WHAT THIS PAPER ADDS: What is already known on the subject Narrative discourse, or storytelling, is used in daily conversational interactions and reveals higher-level language skills that may not be well captured by standardised assessments of language. Many autistic individuals and individuals with fetal alcohol spectrum disorders (FASD) show difficulty with pragmatic and expressive language skills. What this paper adds to existing knowledge We found that many aspects of the narratives produced by the adolescents/young adults in the autism and FASD groups were comparable to each other and to the neurotypical group. However, the groups differed on three global measures of narrative production: rate of speech, number of mazes per total words and the description of conflict/resolution in the narratives produced. Also, significant variability was observed within groups, suggesting that narrative abilities should be considered at an individual level as opposed to their clinical groups. What are the potential or actual clinical implications of this work? This study showed that narrative discourse is an appropriate task that can be added to routine clinical assessments of language abilities in autistic adolescents/young adults as well as those with FASD or typical development and has the potential to reveal higher-level, real-world language skills. An important clinical implication of this study is that narrative language abilities should be considered at an individual level and individual-tailored interventions based on ability level due to the variability observed across individuals.

Child Melioidosis Deaths Caused by Burkholderia pseudomallei-Contaminated Borehole Water, Vietnam, 2019.

Within 8 months, 3 children from 1 family in northern Vietnam died from melioidosis. Burkholderia pseudomallei of the same sequence type, 541, was isolated from clinical samples, borehole water, and garden and rice field soil. Boreholes should be properly constructed and maintained to avoid B. pseudomallei contamination.

Community-wide Screening for Tuberculosis in a High-Prevalence Setting.

BACKGROUND: The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress. METHODS: We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012. RESULTS: In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test for Mycobacterium tuberculosis (prevalence ratio, 0.56; 95% confidence interval [CI], 0.40 to 0.78; P<0.001). The prevalence of tuberculosis infection in children born in 2012 was 3.3% in the intervention group and 2.6% in the control group (prevalence ratio, 1.29; 95% CI, 0.70 to 2.36; P = 0.42). CONCLUSIONS: Three years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).

Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis.

Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts' behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts' physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.

The Impact of Continuity of Care on Cervical Cancer Screening: How Visit Pattern Affects Guideline Concordance.

Cervical cancer can be prevented and highly curable if detected early. Current guidelines recommend women to receive cervical cancer screening starting at age 21. Our study aims to investigate how improving continuity of care (COC) may influence guideline concordance of cervical cancer screening. Using the eligibility and claims data, we created a person-month panel data set for women who were enrolled in Oregon Medicaid for at least 80% of the period from 2008 to 2015. We then selected our study cohort following the cervical cancer screening guidelines. Our dependent variable is whether a woman received cervical cancer screening concordant with guidelines in a given month, when she did not receive Pap test in the past 36 months and did not receive co-testing of HPV test plus Pap test in the past 60 months. We used both population-averaged logit model and conditional fixed-effect logit model to estimate the association between the guideline concordance and the COC index, after controlling for high risk, pregnancy, age, race, and ethnicity. A total of 466,526 person-month observations were included in our main models. A 0.1 unit increase of the COC score was significantly associated with a decrease in the odds of receiving guideline-concordant cervical cancer screening (population-averaged logit model: OR = 0.988, p < .001; conditional fixed-effect logit model: OR = 0.966, p < .001). Our findings remain robust to a series of sensitivity analyses. A better COC may not be necessarily beneficial to improving cervical cancer prevention. Educations for both physicians and patients should be supplemented to assure quality of preventive care.

Postpartum Care Utilization Among Women with Medicaid-Funded Live Births in Oregon.

INTRODUCTION: Postpartum care is an important strategy for preventing and managing chronic disease in women with pregnancy complications (i.e., gestational diabetes (GDM) and hypertensive disorders of pregnancy (HDP)). METHODS: Using a population-based, cohort study among Oregon women with Medicaid-financed deliveries (2009-2012), we examined Medicaid-financed postpartum care (postpartum visits, contraceptive services, and routine preventive health services) among women who retained Medicaid coverage for at least 90 days after delivery (n = 74,933). We estimated postpartum care overall and among women with and without GDM and/or HDP using two different definitions: 1) excluding care provided on the day of delivery, and 2) including care on the day of delivery. Pearson chi-square tests were used to assess differential distributions in postpartum care by pregnancy complications (p < .05), and generalized estimating equations were used to calculate adjusted odds ratios (aORs) with 95% confidence intervals (CIs). RESULTS: Of Oregon women who retained coverage through 90 days after delivery, 56.6-78.1% (based on the two definitions) received any postpartum care, including postpartum visits (26.5%-71.8%), contraceptive services (30.7-35.6%), or other routine preventive health services (38.5-39.1%). Excluding day of delivery services, the odds of receiving any postpartum care (aOR 1.26, 95% CI 1.08-1.47) or routine preventive services (aOR 1.32, 95% CI 1.14-1.53) were meaningfully higher among women with GDM and HDP (reference = neither). DISCUSSION: Medicaid-financed postpartum care in Oregon was underutilized, it varied by pregnancy complications, and needs improvement. Postpartum care is important for all women and especially those with GDM or HDP, who may require chronic disease risk assessment, management, and referrals.

Using the modified frailty index to predict negative outcomes in free-flap breast reconstruction: A National Surgical Quality Improvement Project-based study.

BACKGROUND: Post-mastectomy free-flap breast reconstruction is becoming increasingly common in the United States. However, predicting which patients may suffer complications remains challenging. We sought to apply the validated modified frailty index (mFI) to free-flap breast reconstruction in breast cancer patients and determine its utility in predicting negative outcomes. METHODS: We conducted a retrospective study using National Surgical Quality Improvement Project (NSQIP). All patients who had a CPT code of 19364, indicative of free tissue transfer for breast cancer reconstruction, were included. Data on preoperative characteristics and postoperative outcomes were collected. Patients were separated based on the number of mFI factors present into three categories: 0, 1, and > 2 factors. Preoperative demographics, clinical status, and other comorbidities were also studied. Negative outcomes were compared using multivariate logistic regression. RESULTS: 11,852 patients (mean age 50.9 ± 9.5) were found; 24.2% had complications, comparable to previous literature. mFI is predictive of all types of negative outcomes. 22.5% of all patients with 0 mFI, 27.7% of patients with 1 mFI and 34.2% of patients with at least two mFI had a negative outcome. The most common factors contributing to the mFI were history of hypertension (24.8%) and diabetes (6.1%). mFI was found to be an isolated risk factor for negative outcomes, along with steroid use, American Society of Anesthesiology (ASA) classification, body mass index, and immediate, and bilateral operations. CONCLUSIONS: This NSQIP-based study for patients undergoing free flap breast reconstruction shows that the mFI holds predictive value regarding negative outcomes. This provides more information to properly counsel patients before free flap breast reconstruction surgery.

Determinants of Boarding of Patients with Severe Mental Illness in Hospital Emergency Departments.

BACKGROUND: Boarding of patients in hospital emergency departments (EDs) occurs routinely across the U.S. ED patients with behavioral health conditions are more likely to be boarded than other patients. However, the existing literature on ED boarding of psychiatric patients remains largely descriptive and has not empirically related mental health system capacity to psychiatric boarding. Nor does it show how the mental health system could better address the needs of populations at the highest risk of ED boarding. AIMS OF THE STUDY: We examined extent and determinants of "boarding" of patients with severe mental illness (SMI) in hospital emergency departments (ED) and tested whether greater mental health system capacity may mitigate the degree of ED boarding. METHODS: We linked Oregon's ED Information Exchange, hospital discharge, and Medicaid data to analyze encounters in Oregon hospital EDs from October 2014 through September 2015 by 7,103 persons aged 15 to 64 with SMI (N = 34,207). We additionally utilized Medicaid claims for years 2010-2015 to identify Medicaid beneficiaries with SMI. Boarding was defined as an ED stay over six hours. We estimated a recursive simultaneous-equation model to test the pathway that mental health system capacity affects ED boarding via psychiatric visits. RESULTS: Psychiatric visits were more likely to be boarded than non-psychiatric visits (30.2% vs. 7.4%). Severe psychiatric visits were 1.4 times more likely to be boarded than non-severe psychiatric visits. Thirty-four percent of psychiatric visits by children were boarded compared to 29.6% for adults. Statistical analysis found that psychiatric visit, substance abuse, younger age, black race and urban residence corresponded with an elevated risk of boarding. Discharge destinations such as psychiatric facility and acute care hospitals also corresponded with a higher probability of ED boarding. Greater supply of mental health resources in a county, both inpatient and intensive community-based, corresponded with a reduced risk of ED boarding via fewer psychiatric ED visits. DISCUSSION: Psychiatric visit, severity of psychiatric diagnosis, substance abuse, and discharge destinations are among important predictors of psychiatric ED boarding by persons with SMI. A greater capacity of inpatient and intensive community mental health systems may lead to a reduction in psychiatric ED visits by persons with SMI and thereby decrease the extent of psychiatric ED boarding. IMPLICATIONS FOR HEALTH POLICIES: Continued investment in mental health system resources may reduce psychiatric ED visits and mitigate the psychiatric ED boarding problem.

Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks.

The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.

Exact formula for the horizontal-to-vertical displacement ratio of Rayleigh waves in layered orthotropic half-spaces.

This paper is concerned with the propagation of Rayleigh waves in a compressible orthotropic half-space coated by a compressible orthotropic layer. The main purpose of the paper is to establish an explicit exact formula for the H/V ratio (the horizontal-to-vertical displacement ratio) of the Rayleigh wave. This formula is derived by using the transfer matrix for an orthotropic elastic layer and by the effective boundary condition technique. The formula recovers the H/V formula by Malischewsky and Scherbaum [(2004). Wave Motion 40, 57-67] for the compressible isotropic case and the H/V formula by Love [(1911). Some Problems of Geodynamics (Cambridge University Press, Cambridge)] for the incompressible isotropic case. The effect of anisotropy on the H/V ratio's properties is examined and it is numerically shown that the anisotropy strongly affects the peak and zero frequency of the H/V ratio and its prograde domain as well. As the H/V ratio is a dimensionless quantity and it is easily experimentally measured, the obtained formula will be a useful tool for the nondestructive evaluation of mechanical properties of thin films deposited onto half-spaces. Two numerical examples are performed to show how the obtained formula can be used to extract the material properties of deposited layers from measured values of the H/V ratio.

Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly.

UNLABELLED: Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection. IMPORTANCE: The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

Molecular Level Characterization of the Structure and Interactions in Peptide-Functionalized Metal-Organic Frameworks.

We use density functional theory, newly parameterized molecular dynamics simulations, and last generation 15 N dynamic nuclear polarization surface enhanced solid-state NMR spectroscopy (DNP SENS) to understand graft-host interactions and effects imposed by the metal-organic framework (MOF) host on peptide conformations in a peptide-functionalized MOF. Focusing on two grafts typified by MIL-68-proline (-Pro) and MIL-68-glycine-proline (-Gly-Pro), we identified the most likely peptide conformations adopted in the functionalized hybrid frameworks. We found that hydrogen bond interactions between the graft and the surface hydroxyl groups of the MOF are essential in determining the peptides conformation(s). DNP SENS methodology shows unprecedented signal enhancements when applied to these peptide-functionalized MOFs. The calculated chemical shifts of selected MIL-68-NH-Pro and MIL-68-NH-Gly-Pro conformations are in a good agreement with the experimentally obtained 15 N NMR signals. The study shows that the conformations of peptides when grafted in a MOF host are unlikely to be freely distributed, and conformational selection is directed by strong host-guest interactions.