CSSQ: a ChIP-seq signal quantifier pipeline.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has revolutionized the studies of epigenomes and the massive increase in ChIP-seq datasets calls for robust and user-friendly computational tools for quantitative ChIP-seq. Quantitative ChIP-seq comparisons have been challenging due to noisiness and variations inherent to ChIP-seq and epigenomes. By employing innovative statistical approaches specially catered to ChIP-seq data distribution and sophisticated simulations along with extensive benchmarking studies, we developed and validated CSSQ as a nimble statistical analysis pipeline capable of differential binding analysis across ChIP-seq datasets with high confidence and sensitivity and low false discovery rate with any defined regions. CSSQ models ChIP-seq data as a finite mixture of Gaussians faithfully that reflects ChIP-seq data distribution. By a combination of Anscombe transformation, k-means clustering, estimated maximum normalization, CSSQ minimizes noise and bias from experimental variations. Further, CSSQ utilizes a non-parametric approach and incorporates comparisons under the null hypothesis by unaudited column permutation to perform robust statistical tests to account for fewer replicates of ChIP-seq datasets. In sum, we present CSSQ as a powerful statistical computational pipeline tailored for ChIP-seq data quantitation and a timely addition to the tool kits of differential binding analysis to decipher epigenomes.

Pediatric multicenter cohort comparison of percutaneous endoscopic and non-endoscopic gastrostomy technique outcomes.

Enteral access is one of the mainstays of nutritional support. Several different modalities for gastrostomy placement are established. In pediatrics, however, there is a limited evidence base supporting the choice of 1 modality over the others. We retrospectively compared elective percutaneous endoscopically placed gastrostomy (PEG) with surgical and interventional radiology-placed gastrostomy outcomes using the Pediatric Hospital Inpatient Sample multicenter administrative database (Pediatric Health Information System). Pediatric patients (<18 years) undergoing planned elective gastrostomy (2010-2015) were included. Coded gastrostomy procedure subtype, patient demographic characteristics, chronic comorbidities and subsequent related outcomes, mortality, readmission, length of stay and total cost of admission were analyzed. Univariate analysis differentiated among gastrostomy techniques. The effect of gastrostomy on mortality and 30-day readmission were determined using a forward, stepwise, binary logistic regression. Generalized linear models were used to estimate the effect of gastrostomy type on length of stay and total cost. During the study period, 11,712 children underwent gastrostomy, including PEG (27%). Patients with chronic comorbidities were more, or as likely to undergo non-PEG procedures. Postoperatively, PEG patients were less likely to require mechanical ventilation and total parenteral nutrition (TPN). Gastrostomy type was not predictive of mortality; predictors included non-White race and need for mechanically assisted ventilation. Readmission following gastrostomy was common (29.5%), and more likely in PEG patients (OR 1.31). Predictors of readmission included earlier TPN (OR 1.39), cardiovascular (OR 1.17) and oncology (OR 4.17) comorbidities. Our study suggests that PEG placement entails similar length of stay and cost as in non-PEG gastrostomy. Patients undergoing PEG were less likely to require mechanical ventilation and TPN postoperatively. Mortality is similar in both groups although more likely with specific comorbidities. Racial background appeared to be associated with choice of gastrostomy, length of stay and mortality.

Substrate stiffness directs diverging vascular fates.

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvß1, and not αvß6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. STATEMENT OF SIGNIFICANCE: Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvß1 activation mediates this stiffness-directed vascular differentiation.

TiO2 nanoparticles generate superoxide and alter gene expression in human lung cells.

TiO2 nanoparticles are widely used in consumer products and industrial applications, yet little is understood regarding how the inhalation of these nanoparticles impacts long-term health. This is especially important for the occupational safety of workers who process these materials. We used RNA sequencing to probe changes in gene expression and fluorescence microscopy to image intracellular reactive oxygen species (ROS) in human lung cells incubated with low, non-cytotoxic, concentrations of TiO2 nanoparticles. Experiments were designed to measure changes in gene expression following an acute exposure to TiO2 nanoparticles and changes inherited by progeny cells. We observe that TiO2 nanoparticles lead to significant (>2000 differentially expressed genes) changes in gene expression following a 24 hour incubation. Following this acute exposure, the response dissipates with only 34 differentially expressed genes in progeny cells. The progeny cells adapt to this initial exposure, observed when re-challenged with a second acute TiO2 nanoparticle exposure. Accompanying these changes in gene expression is the production of intracellular ROS, specifically superoxide, along with changes in oxidative stress-related genes. These experiments suggest that TiO2 nanoparticles adapt to oxidative stress through transcriptional changes over multiple generations of cells.

One-day oral polyethylene glycol based cleanout is effective for pre-colonoscopy preparation in children.

BACKGROUND: The adequacy of pre-procedure preparation is the principal determinant of the quality of colonoscopy in pediatric as in adult patients. There is a lack of consensus, among providers on a standard pre-procedure regimen. Professional society guidelines include the use of Polyethylene glycol (PEG). Herein we report on the provider-assessed adequacy of a one day, age-categorized dosing, PEG based cleanout regimen in children undergoing colonoscopy in a tertiary institution. METHODS: The standard bowel preparation regime at our institution includes an age dependent minimum PEG dosing regimen in addition to clear liquids the day prior to the procedure. We retrospectively abstracted relevant indices including patient demographics, prep quality, procedure impairment, duration and completion from an institutional quality monitoring survey tool between 2015 and 2016 and similarly abstracted prospectively recorded indices that included the dataset above as well as additional fields for procedure deviations and additional laxative use. RESULTS: A total of 642 procedures (mean age 12.2 years; F: 380) were accrued, nonadherence to the cleanout regimen (7.3%) and additional laxative use (3.1%) were observed in a small proportion of the prospective dataset subjects, adequate cleanout defined as thin or thick liquid but no solids present was reported in 79.5% and 15.8% of cases and impaired study from inadequate cleanout was reported in 11.8% of studies albeit the cecum was reached and the terminal ileum was intubated in 97.8 and 93.6% of studies. The duration of the study was significantly longer with the presence of a fellow trainee assisting in the procedure. Patient age and gender did not correlate with prep adequacy or cecal and ileal intubation rates, inadequate cleanout was significantly associated with impairment and incomplete studies. CONCLUSION: A one day, single agent, osmotic laxative (Polyethylene glycol) based cleanout regimen is effective in routine pre-procedure cleanout for standard colonoscopy in pediatric age range patients.

Necessity of Repeat Computed Tomography Imaging in Isolated Mild Traumatic Subarachnoid Hemorrhage.

BACKGROUND: The standard of care for mild isolated traumatic subarachnoid hemorrhage (itSAH) involves serial head computed tomography (CT) examinations at dedicated time intervals to monitor neurologic status and hemorrhagic progression. Considering recent evidence questioning such an aggressive monitoring protocol, this study aimed to assess the necessity of repeat head CT imaging in managing itSAH. METHODS: All patients who presented to our academic institution with mild traumatic brain injury were evaluated. Patients ≥18 years of age presenting with mild, blunt itSAH (Glasgow Coma Scale [GCS] score 13-15) were included. Patients with significant polytrauma or coagulopathy were excluded. Patient demographics (age and sex), imaging results (CT progression), and outcomes (final GCS score, discharge status, interventions, and 30-day readmission) were all recorded. RESULTS: Of 190 cases identified for inclusion, 58 (30.5%) met all study criteria (age, 59.5 ± 18.0 y; 30 men). Five (8.6%) patients presented with repeat CT progression, with none reporting 30-day readmission or adverse event on follow-up. Comparison of cases with and without CT progression found no meaningful differences in age or sex. Of the 3 patients readmitted, none demonstrated CT progression, and cause of readmission was unrelated to head trauma in all cases. Two patients demonstrated clinical deterioration (final GCS score <14), and neither case showed progression on CT, was readmitted, or received neurosurgical intervention of any kind. CONCLUSIONS: Our findings suggest that serial CT imaging has little efficacy in changing mild itSAH management and is poorly correlated with clinical progression. A less aggressive management protocol may be more appropriate for managing this patient population.

Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells.

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1- cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

Gastrointestinal Bowel Obstruction in Acute Ischemic Stroke: Incidence, Risk Factors, and Outcomes in a U.S. Nationwide Analysis of 3,998,667 Hospitalizations.

OBJECTIVE: The prognosis from acute ischemic stroke (AIS) is worsened by poststroke medical complications. The incidence, risk factors, and outcomes of gastrointestinal bowel obstruction (GIBO) in AIS are not known. METHODS: We queried the Nationwide Inpatient Sample (2002-2011) to identify all patients with a primary diagnosis of AIS and subsets with and without a secondary diagnosis of GIBO without hernia. Multivariable analysis was utilized to identify risk factors for GIBO in AIS patients and the association between GIBO, in-hospital complications, and outcomes. RESULTS: We identified 16,987 patients with GIBO (.43%) among 3,988,667 AIS hospitalizations and 4.2% of these patients underwent surgery. In multivariable analysis, patients with 75+ years of age were two times as likely to suffer GIBO compared to younger patients (P < .0001). African Americans were 42% more likely to have GIBO compared to Whites (P < .0001). Stroke patients with pre-existing comorbidities (coagulopathy, cancer, blood loss anemia, and fluid/electrolyte disorder) were more likely to experience GIBO (all P < .0001). AIS patients with GIBO were 184% and 39% times more likely to face moderate-to-severe disability and in-hospital death, respectively (P < .0001). GIBO occurrence increased length of stay and total costs by an average of 9.7 days and $22,342 (P < .0001). CONCLUSION: GIBO is a rare but burdensome complication of AIS, associated with complications, disability, and mortality. The risk factors identified in this study aim to encourage the monitoring of patients at highest risk for GIBO. The predominant form of stroke-related GIBO is nonmechanical obstruction, although the causative relationship remains unknown.

Mortality associated with gastrointestinal bleeding in children: A retrospective cohort study.

AIM: To determine the clinical characteristics of children with gastrointestinal bleeding (GIB) who died during the course of their admission. METHODS: We interrogated the Pediatric Hospital Information System database, including International Classification of Diseases, Current Procedural Terminology and Clinical Transaction Classification coding from 47 pediatric tertiary centers extracting the population of patients (1-21 years of age) admitted (inpatient or observation) with acute, upper or indeterminate GIB (1/2007-9/2015). Descriptive statistics, unadjusted univariate and adjusted multivariate analysis of the associations between patient characteristics and treatment course with mortality was performed with mortality as primary and endoscopy a secondary outcome of interest. All analyses were performed using the R statistical package, v.3.2.3. RESULTS: The population with GIB was 19528; 54.6% were male, overall mortality was 2.07%; (0.37% in patients with the principal diagnosis of GIB). When considering only the mortalities in which GIB was the principal diagnosis, 48% (12 of 25 principal diagnosis GIB mortalities) died within the first 3 d of admission, whereas 19.8% of secondary diagnosis GIB patients died with 3 d of admission. Patients who died were more likely to have received octreotide (19.8% c.f. 4.04%) but tended to have not received proton pump inhibitor therapy in the first 48 h, and far less likely to have undergone endoscopy during their admission (OR = 0.489, P < 0.0001). Chronic liver disease associated with a greater likelihood of endoscopy. Mortalities were significantly more likely to have multiple complex chronic conditions. CONCLUSION: GIB associated mortality in children is highest within 7 d of admission. Multiple comorbidities are a risk factor whereas early endoscopy during the admission is protective.

The prevalence and management of angina among patients with chronic coronary artery disease across US outpatient cardiology practices: insights from the Angina Prevalence and Provider Evaluation of Angina Relief (APPEAR) study.

Although eliminating angina is a primary goal in treating patients with chronic coronary artery disease (CAD), few contemporary data quantify prevalence and severity of angina across US cardiology practices. The authors hypothesized that angina among outpatients with CAD managed by US cardiologists is low and its prevalence varies by site. Among 25 US outpatient cardiology clinics enrolled in the American College of Cardiology Practice Innovation and Clinical Excellence (PINNACLE) registry, we prospectively recruited a consecutive sample of patients with chronic CAD over a 1- to 2-week period at each site between April 2013 and July 2015, irrespective of the reason for their appointment. Eligible patients had documented history of CAD (prior acute coronary syndrome, prior coronary revascularization procedure, or diagnosis of stable angina) and ≥1 prior office visit at the practice site. Angina was assessed directly from patients using the Seattle Angina Questionnaire Angina Frequency score. Among 1257 patients from 25 sites, 7.6% (n = 96) reported daily/weekly, 25.1% (n = 315) monthly, and 67.3% (n = 846) no angina. The proportion of patients with daily/weekly angina at each site ranged from 2.0% to 24.0%, but just over half (56.3%) were on ≥2 antianginal medications, with wide variability across sites (0%-100%). One-third of outpatients with chronic CAD managed by cardiologists report having angina in the prior month, and 7.6% have frequent symptoms. Among those with frequent angina, just over half were on ≥2 antianginal medications, with wide variability across sites. These findings suggest an opportunity to improve symptom control.

Bacterial quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone causes direct cytotoxicity and reduced cell motility in human pancreatic carcinoma cells.

In spite of chemotherapeutic and surgical advances, pancreatic cancer continues to have a dismal prognosis. Metastasis due to tumor cell migration remains the most critical challenge in treating pancreatic cancer, and conventional chemotherapy is rarely curative. In the quest for more novel molecules to fight this disease, we tested the hypothesis that the Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone (O-DDHSL) would be cytotoxic to and reduce mobility of pancreatic carcinoma cells (Panc-1 and Aspc-1). Results showed a decrease in cell viability from apoptosis, diminished colony formation, and inhibition of migration of the evaluated pancreatic carcinoma cell lines. Also, cell viability decreased in the presence of O-DDHSL when cells were grown in matrigel basement membrane matrix. While messenger RNA for IQGAP-1 decreased in Panc-1 and HPDE cells upon exposure to O-DDHSL, no change was observed in Aspc-1 cells. Cofilin mRNA expression was found to be increased in both HPDE and Panc-1 cells with marginal decrease in Aspc-1 cells. RhoC, a Rho-family GTPase involved in cell motility, increased in the presence of O-DDHSL, suggesting a possible compensatory response to alteration in other migration associated genes. Our results indicate that O-DDHSL could be an effective biomolecule in eukaryotic systems with multimodal function for essential molecular targeting in pancreatic cancer.

High-resolution mapping of h1 linker histone variants in embryonic stem cells.

H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1(0) displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.