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BACKGROUND: Postpartum readmission is an important indicator of postpartum morbidity. The likelihood of postpartum readmission is highest for Black individuals. However, it is unclear whether the likelihood of postpartum readmission has changed over time according to race/ethnicity. Little is also known about the factors that contribute to these trends. OBJECTIVE: This study aimed to: (1) examine trends in postpartum readmission by race/ethnicity, (2) examine if prenatal or clinical factors explain the trends, and (3) investigate if racial/ethnic disparities changed over time. STUDY DESIGN: We examined trends in postpartum readmission, defined as hospitalization within 42 days after birth hospitalization discharge, using live birth and fetal death certificates linked to delivery discharge records from 10,711,289 births in California from 1997 to 2018. We used multivariable logistic regression models that included year and year-squared (to allow for nonlinear trends), overall and stratified by race/ethnicity, to estimate the annual change in postpartum readmission during the study period, represented by odds ratios and 95% confidence intervals. We then adjusted models for prenatal (eg, patient demographics) and clinical (eg, gestational age, mode of birth) factors. To determine whether racial/ethnic disparities changed over time, we calculated risk ratios for 1997 and 2018 by comparing the predicted probabilities from the race-specific, unadjusted logistic regression models. RESULTS: The overall incidence of postpartum readmission was 10 per 1000 births (17.4/1000 births for non-Hispanic Black, 10/1000 for non-Hispanic White, 7.9/1000 for non-Hispanic Asian/Pacific Islander, and 9.6/1000 for Hispanic individuals). Odds of readmission increased for all groups during the study period; the increase was greatest for Black individuals (42% vs 21%–29% for the other groups). After adjustment for prenatal and clinical factors, the increase in odds was similar for Black and White individuals (12%). The disparity in postpartum readmission rates relative to White individuals increased for Black individuals (risk ratio, 1.68 in 1997 and 1.90 in 2018) and more modestly for Hispanic individuals (risk ratio, 1.02 in 1997 and 1.05 in 2018) during the study period. Asian/Pacific Islander individuals continued to have lower risk than White individuals during the study period (risk ratio, 0.87 in 1997 and 0.82 in 2018). CONCLUSION: The rate of postpartum readmissions increased from 1997 to 2018 in California across all racial/ethnic groups, with the greatest increase observed for Black individuals. Racial/ethnic differences in the trend were more modest after adjustment for prenatal and clinical factors. It is important to find ways to prevent further increases in postpartum readmission, especially among groups at highest risk.

Purpose: Brain metastases are common among adult patients with solid malignancies and are increasingly being treated with stereotactic radiosurgery (SRS). As more patients with brain metastases are becoming eligible for SRS, there is a need for practical review of patient selection and treatment considerations. Methods and Materials: Two patient cases were identified to use as the foundation for a discussion of a wide and representative range of management principles: (A) SRS alone for 5 to 15 lesions and (B) a large single metastasis to be treated with pre- or postoperative SRS. Patient selection, fractionation, prescription dose, treatment technique, and dose constraints are discussed. Literature relevant to these cases is summarized to provide a framework for treatment of similar patients. Results: Treatment of brain metastases with SRS requires many considerations including optimal patient selection, fractionation selection, and plan optimization. Conclusions: Case-based practice guidelines developed by the Radiosurgery Society provide a practical guide to the common scenarios noted above affecting patients with metastatic brain tumors.

Purpose: Electric bicycles (e-bikes) achieve higher speeds than pedal bicycles, but few studies have investigated the impact on injury rates specific to the pediatric population. Utilizing the National Electronic Injury Surveillance System (NEISS), we compared rates of pediatric injury for e-bikes, bicycles, and gas-engine bicycles (mopeds) from 2011 to 2020. Methods: Descriptive and bivariate inferential analyses were performed upon NEISS estimates of e-bike, bicycle, and moped injuries in children aged 2–18 years. Analyses were stratified by patient age and helmet usage. The Mann-Kendall test of trends was used. Results: We identified 3945 e-bike, 23,389 moped, and 2.05 million bicycle injuries. Over time, the incidence of injury increased for e-bikes (Kendall's τ=0.73, p = 0.004), decreased for pedal bicycles (Kendall's τ= − 0.91, p = 0.0003), and did not change for mopeds (Kendall's τ = 0.06, p = 0.85). Males accounted for 82.5 % of e-bike injuries. The age group most commonly affected by e-bike injury (44.3 %) was 10–13 years old. The proportion of injuries requiring hospitalization was significantly higher for e-bikes (11.5 %), compared to moped and bicycle (7.0 and 4.8 %, respectively, p

Background: Patients undergoing total joint arthroplasty (TJA) are at increased risk for venous thromboembolism (VTE). Prediction tools such as the Caprini Risk Assessment Model (RAM) have been developed to identify patients at higher risk. However, studies have reported heterogeneous results when assessing its efficacy for TJA. Patients treated in an urban health safety net hospital have increased medical complexity, advanced degenerative joint disease, and severe disability prior to TJA increasing the risk of VTE. We hypothesize that use of a tool designed to account for these conditions—the Boston Medical Center (BMC) VTE score—will more accurately predict VTE in this patient population. Methods: A retrospective case-control study was performed including subjects 18 years of age and older who underwent primary or revision TJA in an urban academic health safety net hospital. Patients with hemiarthroplasties, simultaneous bilateral TJA, and TJA after acute trauma were excluded. A total of 80 subjects were included: 40 who developed VTE after TJA (VTE+) and 40 who did not develop VTE (controls). Subjects were matched by age, gender, and surgical procedure. Results: There was a statistically significant difference between the mean BMC VTE score for VTE+ and controls (4.40 and 3.13, respectively, P = .036). Conversely, there was no statistical difference between the mean Caprini scores for VTE+ and controls (9.50 and 9.35, respectively, P = .797). Conclusions: In a health safety-net patient population, an institutional RAM—the BMC VTE score—was found to be more predictive of VTE than the modified Caprini RAM following TJA. The BMC-VTE score should be externally validated to confirm its reliability in VTE prediction in similar patient populations.

OBJECTIVE:. Hypotension affects approximately 40% of critically ill patients undergoing emergency intubation and is associated with an increased risk of death. The objective of this study was to examine the association between prophylactic vasopressor administration and the incidence of peri-intubation hypotension and other clinical outcomes. DESIGN:. A secondary analysis of two multicenter randomized clinical trials. The clinical effect of prophylactic vasopressor administration was estimated using a one-to-one propensity-matched cohort of patients with and without prophylactic vasopressors. SETTING:. Seven emergency departments and 17 ICUs across the United States. PATIENTS:. One thousand seven hundred ninety-eight critically ill patients who underwent emergency intubation at the study sites between February 1, 2019, and May 24, 2021. INTERVENTIONS:. None. MEASUREMENTS AND MAIN RESULTS:. The primary outcome was peri-intubation hypotension defined as a systolic blood pressure less than 90 mm Hg occurring between induction and 2 minutes after tracheal intubation. A total of 187 patients (10%) received prophylactic vasopressors prior to intubation. Compared with patients who did not receive prophylactic vasopressors, those who did were older, had higher Acute Physiology and Chronic Health Evaluation II scores, were more likely to have a diagnosis of sepsis, had lower pre-induction systolic blood pressures, and were more likely to be on continuous vasopressor infusions prior to intubation. In our propensity-matched cohort, prophylactic vasopressor administration was not associated with reduced risk of peri-intubation hypotension (41% vs 32%; p = 0.08) or change in systolic blood pressure from baseline (–12 vs –11 mm Hg; p = 0.66). CONCLUSIONS:. The administration of prophylactic vasopressors was not associated with a lower incidence of peri-intubation hypotension in our propensity-matched analysis. To address potential residual confounding, randomized clinical trials should examine the effect of prophylactic vasopressor administration on peri-intubation outcomes.

OBJECTIVES:. To analyze the temporal trend in enrollment rates in a COVID-19 platform trial during the first three waves of the pandemic in the United States. DESIGN:. Secondary analysis of data from the I-SPY COVID randomized controlled trial (RCT). SETTING:. Thirty-one hospitals throughout the United States. PATIENTS:. Patients who were approached, either directly or via a legally authorized representative, for consent and enrollment into the I-SPY COVID RCT. INTERVENTIONS:. None. MEASUREMENTS AND MAIN RESULTS:. Among 1,338 patients approached for the I-SPY COVID trial from July 30, 2020, to February 17, 2022, the number of patients who enrolled (n = 1,063) versus declined participation (n = 275) was used to calculate monthly enrollment rates. Overall, demographic and baseline clinical characteristics were similar between those who enrolled versus declined. Enrollment rates fluctuated over the course of the COVID-19 pandemic, but there were no significant trends over time (Mann-Kendall test, p = 0.21). Enrollment rates were also comparable between vaccinated and unvaccinated patients. In multivariable logistic regression analysis, age, sex, region of residence, COVID-19 severity of illness, and vaccination status were not significantly associated with the decision to decline consent. CONCLUSIONS:. In this secondary analysis of the I-SPY COVID clinical trial, there was no significant association between the enrollment rate and time period or vaccination status among all eligible patients approached for clinical trial participation. Additional studies are needed to better understand whether the COVID-19 pandemic has altered clinical trial participation and to develop strategies for encouraging participation in future COVID-19 and critical care clinical trials.

BACKGROUND: To reduce postpartum morbidity and mortality, optimizing routine outpatient postpartum care has become a focus of national attention and a healthcare priority. OBJECTIVE: This study aimed to examine the timing, content, and predictors of routine outpatient postpartum visit attendance within a large, commercially insured patient population. STUDY DESIGN: We performed a retrospective cohort study using a national US database of commercial insurance beneficiaries with a delivery hospitalization between 2011 and 2015. We calculated the proportion of patients who had an outpatient postpartum visit within 8 weeks of hospital discharge. Using a multivariable logistic regression model, we identified independent predictors of an outpatient postpartum visit. To gain insight into the nature and extent of any postpartum medical or surgical morbidity, we also identified the most frequent International Classification of Diseases, Ninth Revision, Clinical Modification codes associated with postpartum visits. RESULTS: The study cohort comprised 431,969 patients who underwent delivery hospitalization, of whom 257,727 (59.7%; 95% confidence interval, 59.5–59.8) had at least 1 outpatient postpartum visit within 8 weeks of hospital discharge. The distribution of postpartum visits was bimodal, occurring most frequently in the first week (23.2%) and sixth week (21.7%) after hospital discharge. The median period between hospital discharge and the postpartum visit was 28 days (interquartile range, 8–41 days). In our multivariable model, patient-level factors that were most strongly associated with a postpartum visit were preexisting medical morbidities, which included: thyroid disease (adjusted odds ratio, 1.62; 95% confidence interval, 1.40–1.52), seizure disorder (adjusted odds ratio, 1.50; 95% confidence interval, 1.33–1.70), chronic hypertension (adjusted odds ratio, 1.46; 95% confidence interval, 1.58–1.67), and psychiatric disease (adjusted odds ratio, 1.41; 95% confidence interval, 1.36–1.47). Between 29% and 42% of patients with preexisting medical morbidity and between 35% and 41% of patients who experienced peri- or postpartum complications did not attend a postpartum visit. CONCLUSION: Our findings indicate that among a large, commercially-insured patient population, postpartum visit attendance was suboptimal. A high proportion with preexisting medical and peripartum morbidities was not evaluated within 8 weeks of hospital discharge. Multifaceted interventions and healthcare reform are suggested to address patients’ concerns and healthcare needs after delivery.

BACKGROUND: There is an increased odds of having a recurrence of clinical chorioamnionitis in patients with a diagnosis of clinical chorioamnionitis compared with those without clinical chorioamnionitis in a previous pregnancy. However, it is unclear how gestational age at delivery of the first pregnancy or interpregnancy interval may contribute to this increased risk. OBJECTIVE: This study aimed to evaluate how gestational age of delivery in a first pregnancy and interpregnancy interval affect the odds of recurrent clinical chorioamnionitis. STUDY DESIGN: Using maternally linked birth record files, Nulliparous patients from California with at least 2 consecutive deliveries between the gestational ages of 20 and 44 weeks from 2007 to 2012 were identified. The rates of clinical chorioamnionitis in the second pregnancy for patients with clinical chorioamnionitis vs those without clinical chorioamnionitis in the first pregnancy, stratified by the gestational age at delivery of the first pregnancy were determined. As a secondary analysis, the analysis by interpregnancy interval (

BACKGROUND: The high maternal mortality and severe morbidity rates in the United States compared with other high-income countries have received national attention. Characterization of postpartum hospital readmissions within the first days after delivery hospitalization discharge could help to identify patients who need additional preparedness for discharge. OBJECTIVE: This study aimed to investigate conditions at birth associated with postpartum readmissions occurring within 0 to 6 days and at 7 to 29 days after discharge from the delivery hospitalization. STUDY DESIGN: We analyzed linked vital statistics and hospital discharge records of patients who gave birth in California during 2007 to 2018. We investigated hospital readmissions within 30 days after birth hospitalization discharge. We used multivariable logistic regression to investigate factors associated with early readmission (0–6 days) and later readmission (7–29 days) compared with no readmission within 30 days (reference). The risk factors assessed included maternal medical or obstetrical conditions before and at birth, birth hospitalization length of stay, and mode of delivery. Severe maternal morbidity was defined as the presence of any of the 21 indicators recommended by the Centers for Disease Control and Prevention. RESULTS: Among 5,248,746 pregnant patients, 23,636 (0.45%) had an early postpartum readmission, whereas 24,712 (0.47%) had a later postpartum readmission. After adjustments, early readmission was most strongly associated with sepsis (adjusted odds ratio, 4.63; 95% confidence interval, 3.87–5.53), severe maternal morbidity (adjusted odds ratio, 3.46; 95% confidence interval, 3.28–3.65) at birth hospitalization, or preeclampsia before birth hospitalization (adjusted odds ratio, 3.67; 95% confidence interval, 3.54–3.81). The associations between later readmission and sepsis and severe maternal morbidity were similar, whereas the association between preeclampsia and later readmission was less strong (adjusted odds ratio, 1.65; 95% confidence interval, 1.57–1.73). CONCLUSION: Pregnant patients with sepsis or severe maternal morbidity during delivery hospitalization or preeclampsia before birth hospitalization were at the highest risk for readmission within 6 days of discharge. These findings may be informative for efforts to improve postpartum care.

Introduction This study reports on the impact of a clinical decision support tool embedded in the electronic medical record and characterizes the demographics, prescribing patterns, and risk factors associated with opioid and benzodiazepine misuse in the older adult population. Significance This study reports on prescribing patterns for patients ≥65 years-old who presented to Emergency Departments (ED) or Urgent Care (UC) facilities across a large healthcare system following a fall (n = 34,334 encounters; n = 25,469 patients). This system implemented a clinical decision support intervention which provides an alert when the patient has an evidence-based risk factor for prescription drug misuse; prescribers can continue, amend or cancel the prescription. Results Of older adults presenting with a fall, 31.4% (N = 7986) received an opioid or benzodiazepine prescription. Women and younger patients (65-74) had a higher likelihood of receiving a prescription ( P < .0001). 11% had ≥1 risk factor. Women were more likely to receive an early refill ( P = .0002) and younger (65-74) men were more likely to have a past positive toxicology ( P < .0001). A prescription was initiated in 8,591 encounters, and 946 (9.0%) triggered an alert. In 58 cases, the alert resulted in a prescription modification, and in 80 the prescription was canceled. Conclusions Documented risk for opioid misuse in the elderly was 10% among patients presenting to the ED/UC after a fall. The dangers associated with opioid/benzodiazepine use increase with age as does fall risk. Awareness of risk factors is an important first step; more work is needed to address potentially hazardous prescriptions in this population.

Background: The COVID-19 pandemic disproportionately affected populations served by Federally Qualified Health Centers (FQHCs), with high morbidity and mortality rates in ethnic minority older adults. In response to this pandemic, academic geriatric medicine teams through federally funded Geriatric Workforce Enhancement Program (GWEP) with FQHC partnership implemented new initiatives to improve the care for vulnerable older adults. Objectives: To describe how four FQHC/GWEP teams collaborated in caring for diverse communities of older adults during the pandemic. Methods: Four GWEPs have addressed pandemic response efforts with their respective FQHC partners. These collaborations to meet the increasing numbers of older adults seeking services, and the rising disparities exacerbated during the pandemic are delineated. Results: FQHC/GWEP partnerships enabled access to care, whether in-person or virtually to serve unmet needs of underserved older adults during the pandemic. Partnerships promoted COVID-19 education, testing, and vaccinations. Most FQHCs faced severe staffing shortages, and the digital divide challenged patients with barriers. GWEPs provided direct care, created educational materials, and developed telehealth programs. These partnerships addressed social determinants of health gaps caused by the pandemic. Conclusion: The findings demonstrate that strong partnerships between GWEPs and FQHCs mitigate health inequities for vulnerable ethnic minority and rural older adults during pandemic crises.

Hypothesis/Background: Treatment options for the biceps brachii tendon include tenotomy, arthroscopic tenodesis, and open tenodesis. Few studies to date have compared all treatment options in the context of a rotator cuff repair. Methods: A retrospective review of 100 patients who underwent arthroscopic supraspinatus repair between 2013 and 2018 with a minimum of one-year follow-up was performed. Patients were separated into the following 4 groups: (1) 57 had isolated supraspinatus repair with no biceps tendon surgery (SSP); (2) 16 had supraspinatus repair and biceps tenotomy; (3) 18 had supraspinatus repair and arthroscopic biceps tenodesis; (4) 9 had supraspinatus repair and an open biceps tenodesis (SSP + OT). The primary outcome was operative time. The secondary outcomes were cost analysis, complications, patient-reported outcome measures, range of motion, and strength testing. Results: The operative time for the SSP + OT group was significantly longer than that of the SSP group (P

OBJECTIVES:. To assess the pragmatism of published critical care randomized controlled trials self-described as pragmatic using a validated tool. DATA SOURCES:. Medical Literature Analysis and Retrieval Online database and PubMed interface from inception to November 1, 2021. STUDY SELECTION:. We performed a systematic search of randomized controlled trials evaluating interventions for critically ill adults that self-identified as pragmatic in title or abstract. DATA EXTRACTION:. Reviewers independently performed study selection and data extraction in duplicate; discrepancies were resolved by consensus. Pragmatism was assessed independently in duplicate by trained reviewers using the Pragmatic-Explanatory Continuum Indicator Summary 2 (PRECIS-2), a validated tool designed to represent how explanatory/pragmatic a trial is on the pragmatic to explanatory continuum. Trials were scored in nine domains on a 5-point continuum (from 1 = very explanatory to 5 = very pragmatic). Discrepancies of greater than 2 points were adjudicated by consensus discussion. DATA SYNTHESIS:. The search resulted in 284 studies; 56 met eligibility criteria. Forty-one of the trials had a discrepancy in at least one domain that required consensus discussion, most commonly in domains of eligibility and follow-up. Twelve studies (21.4%) were scored as “overall pragmatic,” defined as score of greater than 4 in five domains provided the scores in the remaining domains were three. The overall PRECIS-2 score of self-identified pragmatic studies increased from 1995 to 2021 suggesting increasing pragmatism over time. Pragmatic trials were more likely to have a waiver of informed consent (p = 0.05). CONCLUSIONS:. The number and pragmatism of self-identified pragmatic trials have increased, particularly in the past decade. However, less than one-quarter of these trials that use the term pragmatic in title or abstract were retrospectively rated as pragmatic. Our results support the concept that trials are designed on a spectrum of pragmatic to explanatory. Advances in the design and reporting of critical care trials are needed to ensure their real-world applicability.

No abstract available. Article truncated after 150 words. A 78-year-old- man with cerebral palsy requiring an in-home caregiver presented to the emergency room in hypovolemic shock post-sudden cardiac arrest in the setting of hematemesis. The caregiver noticed the patient become unresponsive after having one episode of bright red emesis. EMS arrived and found the patient to be pulseless and performed three rounds of CPR and gave 1 mg of epinephrine before return of spontaneous circulation was obtained. The caregiver reported the patient had been complaining of diarrhea for the past few days after being started on magnesium citrate for constipation by his PCP. In the ED patient was intubated, sedated, and started on pressors due to undifferentiated shock. CT abdomen pelvis demonstrated diffuse dilation of the colon with massive stool burden and markedly distended rectum with impacted stool and circumferential rectal wall thickening consistent with stercoral colitis (Figures 1 and 2). In addition, there was a large hiatal …

Recent events have reaffirmed that racism is a pervasive disease plaguing the United States and infiltrating the fabric of this nation. As health care professionals dedicated to understanding and alleviating disease, many radiation oncologists have failed to acknowledge how structural racism affects the health and well-being of the patients we aim to serve. The literature is full of descriptive statistics showing the higher incidence and mortality experienced by the Black population for health conditions ranging from infant mortality to infectious disease, including coronavirus disease 2019 (COVID-19). Acknowledgment that the root of health disparities experienced by Black people in this country are based in racism is essential to moving the nation and the field of radiation oncology forward. With this lens, a brief overview of structural and institutional racism shapes a discussion of what radiation oncologists and the organizations that represent them can do to address this scourge. As members of a technological field, we often harness the power of data to advance human health and approach challenging diseases with optimism that multidisciplinary effort can produce cure. A few principles to mitigate the longstanding issues of Black marginalization within the field have been recommended via the ATIP (Acknowledgment, Transparency, Intentionality, and rePresentation) and LEADS (Learn, Engage, Advocate, Defend, Support) approaches. However, additional introspection is encouraged. Just as individuals, practices, and organizations rallied to determine how best to address the issues related to the COVID-19 pandemic, the same investigational fervor must be applied to the issue of racism to combat this sinister and often deadly disease.

Objectives:. Given finite ICU bed capacity, knowledge of ICU bed utilization during the coronavirus disease 2019 pandemic is critical to ensure future strategies for resource allocation and utilization. We sought to examine ICU census trends in relation to ICU bed capacity during the rapid increase in severe coronavirus disease 2019 cases early during the pandemic. Design:. Observational cohort study. Setting:. Thirteen geographically dispersed academic medical centers in the United States. Patients/Subjects:. We obtained daily ICU censuses from March 26 to June 30, 2020, as well as prepandemic ICU bed capacities. The primary outcome was daily census of ICU patients stratified by coronavirus disease 2019 and mechanical ventilation status in relation to ICU capacity. Interventions:. None. Measurements and Main Results:. Prepandemic overall ICU capacity ranged from 62 to 225 beds (median 109). During the study period, the median daily coronavirus disease 2019 ICU census per hospital ranged from 1 to 84 patients, and the daily ICU census exceeded overall ICU capacity for at least 1 day at five institutions. The number of critically ill patients exceeded ICU capacity for a median (interquartile range) of 17 (12–50) of 97 days at these five sites. All 13 institutions experienced decreases in their noncoronavirus disease ICU population, whereas local coronavirus disease 2019 cases increased. Coronavirus disease 2019 patients reached their greatest proportion of ICU capacity on April 12, 2020, when they accounted for 44% of ICU patients across all participating hospitals. Maximum ICU census ranged from 52% to 289% of overall ICU capacity, with three sites less than 80%, four sites 80–100%, five sites 100–128%, and one site 289%. Conclusions:. From March to June 2020, the coronavirus disease 2019 pandemic led to ICU censuses greater than ICU bed capacity at fives of 13 institutions evaluated. These findings demonstrate the short-term adaptability of U.S. healthcare institutions in redirecting limited resources to accommodate a public health emergency.

Summary: Apelin agonism causes systemic vasodilatation and increased cardiac contractility in humans, and improves pulmonary arterial hypertension (PAH) in animal models. Here, the authors examined the short-term pulmonary hemodynamic effects of systemic apelin infusion in patients with PAH. In a double-blind randomized crossover study, 19 patients with PAH received intravenous (Pyr1)apelin-13 and matched saline placebo during invasive right heart catheterization. (Pyr1)apelin-13 infusion caused a reduction in pulmonary vascular resistance and increased cardiac output. This effect was accentuated in the subgroup of patients receiving concomitant phosphodiesterase type 5 inhibition. Apelin agonism is a novel potential therapeutic target for PAH. (Effects of Apelin on the Lung Circulation in Pulmonary Hypertension; NCT01457170) Key Words: apelin, APJ, human, pulmonary arterial hypertension

Lung carcinoid tumors account for approximately 2% of lung cancers, with 10% of the tumors represented by the atypical type. While atypical carcinoids are metastatic to intrathoracic lymph nodes in approximately half of the cases on the initial presentation, distant metastases are seen in only 20% of the patients and are found most frequently in bones, liver, adrenal glands, and brain. We present a case of an unusual metastatic disease to the breast in 51-year-old female who developed a new breast mass 2 years after left lower lobectomy due to atypical carcinoid tumor. Atypical pulmonary carcinoid metastases to the breast are exceptionally uncommon, yet they are important considerations for appropriate management, especially with an anamnesis of this neoplasm.

We assembled the plastome of the temperate, Southern Hemisphere liana Muehlenbeckia australis from high throughput sequencing data (paired-end Illumina reads) generated from total genomic DNA sequencing libraries. M . australis ' chloroplast genome sequence (GenBank: MG604297) is 163,484 bp in length and composed of long single copy (LSC; 88,166 bp) and short single copy (SSC; 13,486 bp) regions flanked by inverted repeats (IR; 30,916 bp each) typical for angiosperms. The plastome includes 131 genes comprising 83 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, two possible pseudogenes, psbL and rpl23 with internal stop codons, and truncated repeats of ndhF and rps19 at IR boundaries., Competing Interests: The authors report no conflicts of interest., (© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Objective: To determine if deliberate practice and simulation would improve pediatric residents' neonatal resuscitation abilities., Study Design: Prospective cohort with pre-post-intervention design. Senior residents from 2008 to 2009 were evaluated and served as controls (C-Senior, N=23). Interns (PL-1, N=28) from 2008 to 2009 received the education and evaluation at the beginning (I-Pre) and end of their NICU rotation (I-Post) and again when they were seniors (I-Senior, N=24). The education, based on deliberate practice, included scored assessments of a resident's ability to assemble delivery room equipment and lead a simulated resuscitation., Result: PL-1 equipment score increased by 30% (I-Pre; 53%, I-Post; 83%) and was not different 1 to 2 years later (I-Senior; 87%). I-Senior equipment score was 22% higher than the C-Senior group (C-Senior; 65%). The PL-1's ability to lead a resuscitation improved after the education (I-Pre; 76%, I-Post; 85%) and was maintained as senior residents (I-Senior; 85%), but was not superior to the C-Senior group (C-Senior; 81%)., Conclusion: The use of deliberate practice and simulation can improve a residents' resuscitation training, particularly for equipment.

Xylanases have several industrial uses, particularly in baking, modification of animal feed and in pulp bleaching in the paper industry. Process conditions in kraft pulp bleaching generally favour an enzyme that is active at high pH values. The activities of several glycosyl hydrolase family 11 xylanases reported to be active under alkaline conditions were determined under optimal conditions and found to have optima in the pH 5-6 range. Only one enzyme tested, BadX, was shown to have an alkaline pH optimum. Significant activity at pH values higher than 8 appears often to be the result of excess enzyme added to the reaction mixtures so that substrate is limiting. The different nature of laboratory and industrial substrates needs to be taken into consideration in designing assay conditions. In some cases, significant differences were observed in pH profiles generated using a small-molecule substrate when compared to those generated using xylan. We conclude that small-molecule substrates are not a suitable proxy for determining the pH profiles of family 11 xylanases., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions. One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases. Gene shuffling between badX and Dictyoglomus thermophilum xynB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6. Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH. With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX. The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 11 xylanases showing enhanced activity at alkaline pH's require multiple sequence adaptations across the protein., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5'-3' exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.

Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P(L). A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.

Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.

A number of thermophilic bacteria have been surveyed for possessing reverse transcriptase genes using a degenerate primer approach derived from an alignment of known group II intron encoded reverse transcriptases (RT) from mesophilic prokaryotes and eukaryotes. Six out of 34 thermophilic isolates gave a PCR product that was indicative of an RT internal fragment on sequencing. A putative RT from Bacillus caldolyticus strain EA1 was isolated by genomic walking and cloned into an Escherichia coli expression vector. The recombinant protein proved to be insoluble and was unable to be recovered from the insoluble fraction of lysates of E. coli. The RT was successfully expressed in a baculovirus vector although yields remained low. We followed RT activity during purification using the poly(rC)*p(dG)(12-18), which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC) )*p(dG)(12-18) when using uninfected Sf21 lysates and conclude that the substrate is not a template for DNA-dependent DNA polymerase. Although a high level of RT activity was detected in the total cell protein, when compared to the activity detected in the soluble fraction, only about 10% of the activity was soluble. Sequence comparisons showed significant differences between the EA1-IEP and a Geobacillus RT expressed by others. We conclude that it may be necessary to isolate the IEP RT as a ribonucleoprotein to obtain sufficient material for further analysis.

Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G and C contents.

Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous ( approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.

Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg2+, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium., (Copyright 2004 Springer-Verlag)

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 ( cbh1 ) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.

Plasmid shuttle vectors that contain both prokaryotic (Escherichia coli) and eukaryotic origins of replication are routinely used in molecular biology since E. coli is generally the organism of choice for manipulation of recombinant DNA. Initial transformation of the shuttle vector into E. coli allows production of microgram quantities of DNA suitable for transformation of low-transformation-efficiency hosts. A shuttle/expression vector for the yeast Kluyveromyces lactis, pCWK1, allows recombinant protein fused to the killer toxin signal sequence to be secreted to the medium. The heterologous genes are transcribed under the control of the K. lactis LAC4 promoter, which is tightly regulated in K. lactis. However, in E. coli the LAC4 promoter functions constitutively, and as a result, uncontrolled transcription and translation of genes that are toxic in E. coli can result in cell death, and subsequent failure to recover intact E. coli transformants. We have constructed and tested a modified shuttle vector that contains a K. lactis ribosomal intron that acts as a translational terminator in E. coli, preventing or reducing the expression of recombinant proteins and avoiding toxicity. When transcribed in K. lactis, the intron is spliced from the mRNA allowing the translation of intact full-length, active recombinant gene product.

A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described. The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design. Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA. This lipase showed less than 20% similarity with other known lipases at the amino acid level. The study also revealed that distantly related members of the alpha/beta hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

Many glycoside hydrolases, which degrade long-chain carbohydrate polymers, possess distinct catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). On the basis of conserved protein secondary structure, we describe here the identification and experimental characterization of novel type of mannanase-associated mannan-binding module and also characterization of two CBM family 4 laminarinase-associated beta-glucan-binding modules. These modules are predicted to belong to a superfamily of CBMs which include families 4, 16, 17, 22 and a proposed new family, family 27.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.

The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 degrees C (XynAd1/2) to 60 degrees C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1, 4-beta-xylanases, but differ in their ability to cleave small xylooligosaccharides.

DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

We show that the N-terminal 'thermostabilizing domain' (TSD) of the xylanase, XynA, from the thermophilic bacterium Caldibacillus cellulovorans also acts as a xylan binding domain. Affinity electrophoresis experiments show that this TSD selectively binds soluble xylan and binds weakly to hydroxyethylcellulose. Based on this, and previously reported evidence, we propose that xylanase-associated TSDs are xylan binding domains.

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.