Health Advantages and Disparities in Preterm Birth Among Immigrants Despite Disparate Sociodemographic, Behavioral, and Maternal Risk Factors in San Diego, California.

INTRODUCTION: Reproductive health advantages have been reported among selected immigrants, but few studies have included new immigrants and refugees, nor simultaneously adjusted for socioeconomic, behavioral, and medical disparities. METHODS: We examined the risk of preterm birth (PTB, < 37 weeks' gestation) among singleton live births in San Diego County from 2007 to 2012. Multivariable regression was used to compare PTB (1) by nativity within racial/ethnic groups and (2) among immigrants compared to United States (US) born Whites, while adjusting for sociodemographic, behavioral, reproductive and medical variables. RESULTS: Among 230,878 singleton live births, overall PTB prevalence was highest among parturient women who were US-born Blacks (10.9%), Philippine (10.8%) and US-born Filipinas (10.7%), and US-born Asians (8.6%) despite differences in socioeconomic and maternal risk factors, and lowest among Somali (5.5%) migrants. Blacks born in Somalia or outside of the US, had significantly lower overall PTB prevalence compared to US-born Blacks (5.5% vs 7.6% vs 10.9%). Compared to US-born Whites, spontaneous PTB risk was significantly lower among Somali migrants (4.8% vs 3.7%, adjusted relative risk, aRR 0.7 [95% Confidence Intervals 0.5-0.9]), but higher among Philippine migrants (4.8% vs 7.7%, aRR 1.4 [1.3-1.6]). The strongest risk factor for overall PTB among nulliparous US-born Blacks was preexisting diabetes (aRR 3.81 [2.05-7.08]), and preexisting hypertension among Filipinas (aRR: 3.27 [2.36-4.54] and US-born Asians (aRR: 3.64 [1.61-8.24]). CONCLUSION: Black migrants had lower PTB prevalence compared to US-born Blacks, but this immigrant advantage was not observed in other racial/ethnic groups. Compared to US-born Whites, Somali migrants had significantly lower risk of spontaneous PTB while Filipinas had elevated risk.

Clinical characterization of children with resistant airflow obstruction, a multicenter study.

OBJECTIVE: To characterize a cohort of children with airflow limitation resistant to bronchodilator (BD) therapy. METHODS: Pulmonary function tests performed in children 6-17 years of age at 15 centers in a clinical research consortium were screened for resistant airflow limitation, defined as a post-BD FEV1 and/or an FEV1/FVC less than the lower limits of normal. Demographic and clinical data were analyzed for associations with pulmonary function. RESULTS: 582 children were identified. Median age was 13 years (IQR: 11, 16), 60% were males; 62% were Caucasian, 28% were African-American; 19% were obese; 32% were born prematurely and 21% exposed to second hand smoke. Pulmonary diagnoses included asthma (93%), prior significant pneumonia (28%), and bronchiectasis (5%). 65% reported allergic rhinitis, and 11% chronic sinusitis. Subjects without a history of asthma had significantly lower post-BD FEV1% predicted (p = 0.008). Subjects without allergic rhinitis had lower post-BD FEV1% predicted (p = 0.003). Children with allergic rhinitis, male sex, obesity and Black race had better pulmonary function post-BD. There was lower pulmonary function in children after age 11 years without a history of allergic rhinitis, as compared to those with a history of allergic rhinitis. CONCLUSIONS: The most prevalent diagnosis in children with BD-resistant airflow limitation is asthma. Allergic rhinitis and premature birth are common co-morbidities. Children without a history of asthma, as well as those with asthma but no allergic rhinitis, had lower pulmonary function. Children with BD-resistant airflow limitation may represent a sub-group of children with persistent obstruction and high risk for life-long airway disease.

Structured Data Entry in the Electronic Medical Record: Perspectives of Pediatric Specialty Physicians and Surgeons.

The Epic electronic health record (EHR) platform supports structured data entry systems (SDES), which allow developers, with input from users, to create highly customized patient-record templates in order to maximize data completeness and to standardize structure. There are many potential advantages of using discrete data fields in the EHR to capture data for secondary analysis and epidemiological research, but direct data acquisition from clinicians remains one of the largest obstacles to leveraging the EHR for secondary use. Physician resistance to SDES is multifactorial. A 35-item questionnaire based on Unified Theory of Acceptance and Use of Technology, was used to measure attitudes, facilitation, and potential incentives for adopting SDES for clinical documentation among 25 pediatric specialty physicians and surgeons. Statistical analysis included chi-square for categorical data as well as independent sample t-tests and analysis of variance for continuous variables. Mean scores of the nine constructs demonstrated primarily positive physician attitudes toward SDES, while the surgeons were neutral. Those under 40 were more likely to respond that facilitating conditions for structured entry existed as compared to the two older age groups (p = .02). Pediatric surgeons were significantly less positive than specialty physicians about SDES effects on Performance (p = .01) and the effect of Social Influence (p = .02); but in more agreement that use of forms was voluntary (p = .02). Attitudinal differences likely reflect medical training, clinical practice workflows, and division specific practices. Identified resistance indicate efforts to increase SDES adoption should be discipline-targeted rather than a uniform approach.

Ethnic/racial and sex disparities in obstructive sleep apnea among adolescents in southern California.

STUDY OBJECTIVES: Pediatric obstructive sleep apnea (OSA) is common, however, inclusion of adolescents and especially those of ethnic/racial minorities in research is scarce. We hypothesized that ethnic/racial minority adolescents undergoing polysomnography (PSG) have higher prevalence and more severe OSA compared to those who are non-Hispanic (NH) White. METHODS: Retrospective review of 1,745 adolescents undergoing diagnostic PSG. Demographic characteristics, age, body mass index percentile (BMIp), and PSG parameters were obtained. Descriptive statistics comparing race/ethnicity were analyzed. Linear regression of log-transformed obstructive apnea-hypopnea index (OAHI), and logistic regression of moderate-severe OSA (OAHI ≥ 5 events/h) adjusting for covariates were analyzed. RESULTS: 58.2% adolescents were Hispanic, 24.1% NH-White, 4.3% NH-Asian/Pacific Islander (PI), 4.2% NH-Black/African American (AA), and 6.6% NH-other. Compared to the NH-White group, the Hispanic group had higher OAHI and any level of OSA severity; the Black/AA group had higher moderate-severe and severe OSA, and the NH-Asian group had higher moderate-severe OSA. Multiple linear regression of log-OAHI identified an association with Hispanic ethnicity (ß: 0.25, P-value < .05). Compared to the NH-White group, the Hispanic and the Asian/PI groups were 1.45 (95% CI: 1.10, 1.93) and 1.81 (95% CI: 1.05, 3.10) times more likely to have moderate-severe OSA, respectively, after adjusting for relevant covariates. Stratified analysis by sex identified an association only among males between Hispanic ethnicity (OR: 1.85, 95% CI: 1.27, 2.70) and Asian/PI ethnicity (OR: 2.62, 95% CI: 1.35, 5.11) and moderate-severe OSA, compared to the NH-White group. CONCLUSIONS: Among adolescents undergoing PSG evaluation, we identified OSA racial/ethnic and sex disparities in Hispanic and NH-Asian adolescents. Community level studies with adequate representation of these minority groups are needed to identify factors associated with the reported increased susceptibility.

Using machine learning to improve our understanding of COVID-19 infection in children.

PURPOSE: Children are at elevated risk for COVID-19 (SARS-CoV-2) infection due to their social behaviors. The purpose of this study was to determine if usage of radiological chest X-rays impressions can help predict whether a young adult has COVID-19 infection or not. METHODS: A total of 2572 chest impressions from 721 individuals under the age of 18 years were considered for this study. An ensemble learning method, Random Forest Classifier (RFC), was used for classification of patients suffering from infection. RESULTS: Five RFC models were implemented with incremental features and the best model achieved an F1-score of 0.79 with Area Under the ROC curve as 0.85 using all input features. Hyper parameter tuning and cross validation was performed using grid search cross validation and SHAP model was used to determine feature importance. The radiological features such as pneumonia, small airways disease, and atelectasis (confounded with catheter) were found to be highly associated with predicting the status of COVID-19 infection. CONCLUSIONS: In this sample, radiological X-ray films can predict the status of COVID-19 infection with good accuracy. The multivariate model including symptoms presented around the time of COVID-19 test yielded good prediction score.

Congenital CMV associated with diaphragm dysfunction: a rare cause of tachypnoea.

A late preterm infant with intrauterine growth restriction developed respiratory distress, tachypnoea and hypoxia after birth, requiring supplemental oxygen. Chest radiographs demonstrated persistent elevation of the right hemidiaphragm. Chest ultrasound initially demonstrated symmetrical bilateral diaphragm motion, but subsequent ultrasounds showed asymmetrical excursion with weaker movement of the right hemidiaphragm. Placental pathology demonstrated chronic infectious villitis secondary to cytomegalovirus (CMV), and subsequent CMV testing on the infant was positive. The infant was microcephalic and head imaging revealed intracranial calcifications, consistent with congenital CMV infection.CMV is the most common congenital infection and has a wide array of clinical manifestations. This report highlights the rarely described association between congenital CMV infection and respiratory distress due to underlying diaphragm dysfunction. In neonates with respiratory distress and features of congenital CMV infection, clinicians should have a high index of suspicion for diaphragm dysfunction.

A data-driven model for early prediction of need for invasive mechanical ventilation in pediatric intensive care unit patients.

RATIONALE: Acute respiratory failure is a life-threatening clinical outcome in critically ill pediatric patients. In severe cases, patients can require mechanical ventilation (MV) for survival. Early recognition of these patients can potentially help clinicians alter the clinical course and lead to improved outcomes. OBJECTIVES: To build a data-driven model for early prediction of the need for mechanical ventilation in pediatric intensive care unit (PICU) patients. METHODS: The study consists of a single-center retrospective observational study on a cohort of 13,651 PICU patients admitted between 1/01/2010 and 5/15/2018 with a prevalence of 8.06% for MV due to respiratory failure. XGBoost (extreme gradient boosting) and a convolutional neural network (CNN) using medication history were used to develop a prediction model that could yield a time-varying "risk-score"-a continuous probability of whether a patient will receive MV-and an ideal global threshold was calculated from the receiver operating characteristics (ROC) curve. The early prediction point (EPP) was the first time the risk-score surpassed the optimal threshold, and the interval between the EPP and the start of the MV was the early warning period (EWT). Spectral clustering identified patient groups based on risk-score trajectories after EPP. RESULTS: A clinical and medication history-based model achieved a 0.89 area under the ROC curve (AUROC), 0.6 sensitivity, 0.95 specificity, 0.55 positive predictive value (PPV), and 0.95 negative predictive value (NPV). Early warning time (EWT) median [inter-quartile range] of this model was 9.9[4.2-69.2] hours. Clustering risk-score trajectories within a six-hour window after the early prediction point (EPP) established three patient groups, with the highest risk group's PPV being 0.92. CONCLUSIONS: This study uses a unique method to extract and apply medication history information, such as time-varying variables, to identify patients who may need mechanical ventilation for respiratory failure and provide an early warning period to avert it.

Leveraging Electronic Health Records for Guideline-Based Asthma Documentation.

BACKGROUND: Asthma is the most common pediatric chronic disease; thus, clinical guidelines have been developed for its assessment and management, which rely on systematic symptom documentation. Electronic health records (EHR) have the potential to record clinical data systematically; however, variability in documentation persists. OBJECTIVE: To identify if the use of a structured asthma template is associated with increased guideline-based asthma documentation and clinical outcomes when compared with the use of nonstructured ones. METHODS: We performed a retrospective case-control study comparing the use of nonstructured templates (NSTs) and asthma-structured templates (ASTs) in new patient and first follow-up encounters, evaluated by pediatric pulmonologists between March 2016 and December 2021. Asthma history items were selected following clinical guidelines, summarized in 29 items for new and 22 items for follow-up encounters. Associations with demographic, spirometry, and health care utilization were explored. RESULTS: A total of 546 initial encounters were included; 450 used structured templates. The use of an AST was associated with higher documentation of asthma items in initial and follow-up encounters. Linear regression analysis showed that the use of ASTs was associated with a 28.2% and 39.65% increase in asthma history completeness (in initial and follow-up encounters, respectively), compared with the use of NSTs. AST use was associated with higher rates of systemic steroid prescriptions within 12 months. No other differences were observed after adjusting for asthma severity. CONCLUSIONS: Using asthma-specific structured templates was associated with increased guideline-based asthma documentation. Leveraging the EHR as a clinical and research tool has the potential to improve clinical practice.

Genome sequence of Shigella flexneri serotype 5a strain M90T Sm.

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).

Lambda Red Recombineering in Shigella flexneri.

Shigellosis remains a major cause of severe diarrheal disease and death throughout the world. Vaccine development against shigellosis has been hampered by an incomplete understanding of the molecular mechanisms by which Shigella spp. causes disease and difficulties in manipulating Shigella spp. genomes. While homologous recombination protocols for the construction of precise gene deletions exist, construction of mutants in S. flexneri has not become commonplace. We describe the steps for construction of gene deletions using λ-red recombination using tools that we have developed in our laboratory.

Clinical implications of pulmonary nodules detected in children.

INTRODUCTION: The management of children found to have pulmonary nodules is not well established. We determined how often diagnostic testing was pursued, the outcome of diagnostic testing, and how often pulmonary nodules were given a definitive diagnosis. METHOD: A retrospective review of patients found to have pulmonary nodules. Patients with oncologic diagnoses were excluded. Data collected included number of nodules, presence of pre-existing systemic disease, laboratory testing, presence of respiratory symptoms, repeat imaging, biopsy result, and final diagnosis. RESULTS: We identified 88 patients, of which 56 (64%) had a single nodule, 21 (24%) had a pre-existing nononcologic systemic disease, and four patients (5%) had a new systemic disease identified at the same time the nodule(s) was found. In otherwise healthy patients presenting with a solitary nodule, 94% did not have a definitive diagnosis and none went on to be diagnosed with systemic disease. Serum infectious work-up result for tuberculosis, coccidioidomycosis, histoplasmosis, or aspergillosis was not significantly different between single and multiple nodule/systemic illness groups. No previously healthy patients presenting with a solitary nodule were later diagnosed with malignancy. CONCLUSION: Diagnostic workup for a solitary pulmonary nodule was often inconclusive, especially if the patient did not have symptoms at presentation. Pulmonary nodules were not the sole presenting sign of systemic disease for any subjects. We suggest that in an otherwise healthy pediatric patient found to have an asymptomatic single pulmonary nodule, observation without laboratory work-up or repeat imaging is a reasonable option.

Electronic Health Records and Pulmonary Function Data: Developing an Interoperability Roadmap. An Official American Thoracic Society Workshop Report.

A workshop "Electronic Health Records and Pulmonary Function Data: Developing an Interoperability Roadmap" was held at the American Thoracic Society 2019 International Conference. "Interoperability" is defined as is the ability of different information-technology systems and software applications to directly communicate, exchange data, and use the information that has been exchanged. At present, pulmonary function test (PFT) equipment is not required to be interoperable with other clinical data systems, including electronic health records (EHRs). For this workshop, we assembled a diverse group of experts and stakeholders, including representatives from patient-advocacy groups, adult and pediatric general and pulmonary medicine, informatics, government and healthcare organizations, pulmonary function laboratories, and EHR and PFT equipment and software companies. The participants were tasked with two overarching Aobjectives: 1) identifying the key obstacles to achieving interoperability of PFT systems and the EHR and 2) recommending solutions to the identified obstacles. Successful interoperability of PFT data with the EHR impacts the full scope of individual patient health and clinical care, population health, and research. The existing EHR-PFT device platforms lack sufficient data standardization to promote interoperability. Cost is a major obstacle to PFT-EHR interoperability, and incentives are insufficient to justify the needed investment. The current vendor-EHR system lacks sufficient flexibility, thereby impeding interoperability. To advance the goal of achieving interoperability, next steps include identifying and standardizing priority PFT data elements. To increase the motivation of stakeholders to invest in this effort, it is necessary to demonstrate the benefits of PFT interoperability across patient care and population health.

Neuronal death during combined intermittent hypoxia/hypercapnia is due to mitochondrial dysfunction.

Breathing-disordered states, such as in obstructive sleep apnea, which are cyclical in nature, have been postulated to induce neurocognitive morbidity in both pediatric and adult populations. The oscillatory nature of intermittent hypoxia, especially when chronic, may mimic the paradigm of ischemia-reperfusion in that tissues and cells are exposed to episodes of low and high O(2) and this may lead to oxidant stress. Therefore, we decided to explore the potential contribution of oxidant stress in our intermittent hypoxia/hypercapnia animal model and the role that mitochondria might play in this stress. Neonatal mice were exposed to intermittent hypoxia/hypercapnia for 10 days and 2 wk. Combined intermittent hypoxia/hypercapnia led to a marked increase in apoptotic cell death in the cerebral cortex. Oxygen consumption studies in isolated mitochondria from intermittent hypoxia/hypercapnia-exposed brains demonstrated significant reductions in both state 4 and state 3 respiratory activities by approximately 60% and 75%, respectively. Electron paramagnetic resonance spectroscopy registered a significant increase in superoxide production during nonphosphorylating state 4 by 37%, although superoxide leakage during state 3 did not increase upon treatment. Neuronal superoxide-specific dihydroethidium oxidation was also greater in exposed animals. These studies indicate that intermittent hypoxia/hypercapnia leads to oxidative stress due to mitochondrial response within the mouse central nervous system.

HIV/HCV therapy with ledipasvir/sofosbuvir after randomized switch to emtricitabine-tenofovir alafenamide-based single-tablet regimens.

INTRODUCTION: Guidelines advocate the treatment of HCV in all HIV/HCV co-infected individuals. The aim of this randomized, open-label study (ClinicalTrials.gov identifier: NCT02707601; https://clinicaltrials.gov/ct2/show/NCT02707601) was to evaluate the safety/efficacy of ledipasvir/sofosbuvir (LDV/SOF) co-administered with elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) or rilpivirine/F/TAF (R/F/TAF) in HIV-1/HCV co-infected participants. METHODS: Participants with HIV-1 RNA <50 copies/mL and chronic HCV-genotype (GT) 1 (HCV treatment-naïve ± compensated cirrhosis or HCV treatment-experienced non-cirrhotic) were randomized 1:1 to switch to E/C/F/TAF or R/F/TAF. If HIV suppression was maintained at Week 8, participants received 12 weeks of LDV/SOF. The primary endpoint was sustained HCV virologic response 12 weeks after LDV/SOF completion (SVR12). RESULTS: Of 150 participants, 148 received ≥1 dose of HIV study drug and 144 received LDV/SOF (72 in each F/TAF group; 83% GT1a, 94% HCV treatment-naïve, 12% cirrhotic). Overall, SVR12 was 97% (95% confidence interval: 93-99%). Black race did not affect SVR12. Of four participants not achieving SVR12, one had HCV relapse, one had HCV virologic non-response due to non-adherence, and two missed the post-HCV Week 12 visit. Of 148 participants, 96% receiving E/C/F/TAF and 95% receiving R/F/TAF maintained HIV suppression at Week 24; no HIV resistance was detected. No participant discontinued LDV/SOF or E/C/F/TAF due to adverse events; one participant discontinued R/F/TAF due to worsening of pre-existing hypercholesterolemia. Renal toxicity was not observed in either F/TAF regimen during LDV/SOF co-administration. In conclusion, high rates of HCV SVR12 and maintenance of HIV suppression were achieved with LDV/SOF and F/TAF-based regimens. CONCLUSION: This study supports LDV/SOF co-administered with an F/TAF-based regimen in HIV-1/HCV-GT1 co-infected patients.

Diagnosis of genetic diseases in seriously ill children by rapid whole-genome sequencing and automated phenotyping and interpretation.

By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children's deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient's CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient's genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.

Effect of carbon dioxide on neonatal mouse lung: a genomic approach.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO(2), but only a few at 12% CO(2). Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO(2) seem to depend on the level to which the lung is exposed.

Differential expression of matrix metalloproteinases and their inhibitors in human and mouse lung development.

Lung development is a highly orchestrated process characterized by timed expression and activation of growth factor and protease/antiprotease systems. This interplay is essential in regulating vasculogenesis, alveolarization, and epithelial to mesenchymal transition during lung development. Alterations in the proteolytic/antiproteolytic balance of the lung have been associated with several respiratory diseases characterized by changes in the lung extracellular matrix (ECM). Here, we characterized the expression pattern of matrix metalloproteases (MMP) and their inhibitors, the tissue inhibitors of metalloproteases (TIMP), in human and mouse lung development. Using MMP/TIMP expression arrays, RT-PCR, Western Blotting, and ELISA analyses, we demonstrate that fetal human lung is characterized by a dominant proteolytic profile with high MMP-2 and little TIMP-3 expression. Adult human lung, in contrast, exhibits a more anti-proteolytic profile with decreased MMP-2 and increased TIMP-3 expression. MMP-14, MMP-20, TIMP-1, and TIMP-2 were constitutively expressed, irrespective of the developmental stage. Similar results were obtained using mouse lungs of different developmental stages, with the addition that in mouse lung, TIMP-2 and TIMP-3 were upregulated as lung development progressed. Exposure of neonatal mice to chronic hypoxia (10% O2), a stimulus that leads to an arrest of lung development, resulted in upregulation of MMP-2 with a concomitant downregulation of TIMP-2. These results provide a comprehensive analysis of MMP and TIMP expression during human and mouse lung development. MMP-2, TIMP-2, and TIMP-3 may be key regulatory enzymes during lung development, possibly through their complex action on ECM components, membrane receptor ectodomain shedding, and growth factor bioactivity.

A Shigella flexneri virulence plasmid encoded factor controls production of outer membrane vesicles.

Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA.

Clinical effectiveness and safety of permissive hypercapnia.

Experimental and clinical data indicate that ventilator strategies with permissive hypercapnia may reduce lung injury by a variety of mechanisms. Seven randomized controlled trials in preterm neonates suggest that permissive hypercapnia started early, before the initiation of mechanical ventilation (in conjunction with continuous positive airway pressure), followed by prolonged permissive hypercapnia if mechanical ventilation is needed is an alternative to early ventilation and surfactant. Permissive hypercapnia may improve pulmonary outcomes and survival.