Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long-Read Sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Utility of healthcare-worker-targeted antimicrobial stewardship interventions in hospitals of low- and lower-middle-income countries: a scoping review of systematic reviews.

BACKGROUND: Antimicrobial stewardship (AMS) initiatives in hospitals often include the implementation of clustered intervention components to improve the surveillance and targeting of antibiotics. However, impacts of the individual components of AMS interventions are not well known, especially in low- and lower-middle-income countries (LLMICs). OBJECTIVE: A scoping review was conducted to summarize evidence from systematic reviews (SRs) on the impact of common hospital-implemented healthcare-worker-targeted components of AMS interventions that may be appropriate for LLMICs. METHODS: Major databases were searched systematically for SRs of AMS interventions that were evaluated in hospitals. For SRs to be eligible, they had to report on at least one intervention that could be categorized according to the Effective Practice and Organisation of Care taxonomy. Clinical and process outcomes were considered. Primary studies from LLMICs were consulted for additional information. RESULTS: Eighteen SRs of the evaluation of intervention components met the inclusion criteria. The evidence shows that audit and feedback, and clinical practice guidelines improved several clinical and process outcomes in hospitals. An unintended consequence of interventions was an increase in the use of antibiotics. There was a cumulative total of 547 unique studies, but only 2% (N=12) were conducted in hospitals in LLMICs. Two studies in LLMICs reported that guidelines and educational meetings were effective in hospitals. CONCLUSION: Evidence from high- and upper-middle-income countries suggests that audit and feedback, and clinical practice guidelines have the potential to improve various clinical and process outcomes in hospitals. The lack of evidence in LLMIC settings prevents firm conclusions from being drawn, and highlights the need for further research.

Spatiotemporal and genetic regulation of A-to-I editing throughout human brain development.

Posttranscriptional RNA modifications by adenosine-to-inosine (A-to-I) editing are abundant in the brain, yet elucidating functional sites remains challenging. To bridge this gap, we investigate spatiotemporal and genetically regulated A-to-I editing sites across prenatal and postnatal stages of human brain development. More than 10,000 spatiotemporally regulated A-to-I sites were identified that occur predominately in 3' UTRs and introns, as well as 37 sites that recode amino acids in protein coding regions with precise changes in editing levels across development. Hyper-edited transcripts are also enriched in the aging brain and stabilize RNA secondary structures. These features are conserved in murine and non-human primate models of neurodevelopment. Finally, thousands of cis-editing quantitative trait loci (edQTLs) were identified with unique regulatory effects during prenatal and postnatal development. Collectively, this work offers a resolved atlas linking spatiotemporal variation in editing levels to genetic regulatory effects throughout distinct stages of brain maturation.

Cellular and genetic drivers of RNA editing variation in the human brain.

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantify base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence-derived GABAergic neurons, and oligodendrocytes. We identify more selective editing and hyper-editing in neurons relative to oligodendrocytes. RNA editing patterns are highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites is confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites are enriched in GTEx RNA-sequencing data, edited ~twentyfold higher than all other sites, and variation in RNA editing is largely explained by neuronal proportions in bulk brain tissue. Finally, we uncover 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.

Retraction Note to: High Pulsatility Flow Promotes Vascular Fibrosis by Triggering Endothelial EndMT and Fibroblast Activation.

[This retracts the article DOI: 10.1007/s12195-015-0386-7.].

Abnormalities in autonomic function in obese boys at-risk for insulin resistance and obstructive sleep apnea.

STUDY OBJECTIVES: Current evidence in adults suggests that, independent of obesity, obstructive sleep apnea (OSA) can lead to autonomic dysfunction and impaired glucose metabolism, but these relationships are less clear in children. The purpose of this study was to investigate the associations among OSA, glucose metabolism, and daytime autonomic function in obese pediatric subjects. METHODS: Twenty-three obese boys participated in: overnight polysomnography; a frequently sampled intravenous glucose tolerance test; and recordings of spontaneous cardiorespiratory data in both the supine (baseline) and standing (sympathetic stimulus) postures. RESULTS: Baseline systolic blood pressure and reactivity of low-frequency heart rate variability to postural stress correlated with insulin resistance, increased fasting glucose, and reduced beta-cell function, but not OSA severity. Baroreflex sensitivity reactivity was reduced with sleep fragmentation, but only for subjects with low insulin sensitivity and/or low first-phase insulin response to glucose. CONCLUSIONS: These findings suggest that vascular sympathetic activity impairment is more strongly affected by metabolic dysfunction than by OSA severity, while blunted vagal autonomic function associated with sleep fragmentation in OSA is enhanced when metabolic dysfunction is also present.

Upper Airway Narrowing during Central Apnea in Obese Adolescents.

RATIONALE: The use of real-time magnetic resonance imaging (MRI) for the evaluation during sleep-related respiratory events can lead to better understanding of airway dynamics. OBJECTIVES: To investigate the dynamic anatomy of the upper airway during central apnea. METHODS: The study included obese adolescents who snore and were otherwise healthy. Subjects underwent an overnight baseline polysomnogram. Subjects slept during a 24-minute real-time upper airway MRI scan wearing a full face mask attached to a pneumotach. Sleep versus wakefulness during the MRI was inferred from the heart rate and respiratory patterns. Central apneas were scored using tracings of facemask airflow and abdominal bellows. The cross-sectional area of the upper airway before, during, and after each central apnea event was recorded. RESULTS: Eight subjects were studied and 57 central apnea events were observed during real-time MRI scanning during natural sleep. The median age of subjects was 14.0 years (interquartile range [IQR], 13.5 to 15.5). The median average reduction in cross-sectional area during central apnea events was -38% (IQR, -27 to -51) for primary snorers and -45% (IQR, -40 to -54) for subjects with obstructive sleep apnea. The percentage decrease in cross-sectional area of upper airway during a central apnea event was positively correlated to the length of the central apnea (ρ = 0.389; r2 = 0.152; P = 0.003). CONCLUSIONS: We observed that there is upper airway narrowing during central apneas during natural sleep in obese adolescent subjects, using real-time MRI.

Body Fat Composition: A Predictive Factor for Sleep Related Breathing Disorder in Obese Children.

INTRODUCTION: The association between body fat composition as measured by dual energy x-ray absorptiometry (DEXA) scanning and pediatric sleep related breathing disorder (SRBD) is not well established. We investigated the relationship between body mass index (BMI) and DEXA parameters and their association with SRBD in obese children. PATIENTS AND METHODS: Overnight polysomnography was performed on obese/overweight children (10-17 years) with habitual snoring. Total body fat mass (g), trunk fat mass (g), total body % fat, and trunk % fat were determined by DEXA. RESULTS: Forty-one subjects were studied. Logarithm (Log) total arousal index correlated with BMI (p < 0.01, r = 0.473), total body fat mass (p < 0.05, r = 0.331), and trunk fat mass (p < 0.05, r = 0.319). Log desaturation index correlated with BMI (p < 0.05, r = 0.313), total body fat mass (p < 0.05, r = 0.375), and trunk fat mass (p < 0.05, r = 0.391), whereas obstructive apnea hypopnea index (OAHI) did not. In males 10-12 years, there was a significant correlation between Log total arousal index and obesity parameters, but not for males aged 13-17 years. BMI correlated with DEXA parameters in all subjects: total body fat mass (p < 0.001, r = 0.850); total body % fat (p < 0.01, r = 0.425); trunk fat mass (p < 0.001, r = 0.792) and trunk % fat (p < 0.05, r = 0.318) and in 10-12 year old males. This relationship was not significant in males aged 13-17 years. CONCLUSIONS: Total body fat mass and trunk fat mass as well as BMI correlated with total arousal index and desaturation index. BMI correlated with DEXA parameters in 10-12 year old males but not in 13-17 year old males. The value of using DEXA scanning to study the relationship between obesity and SRBD may depend on age and pubertal stage.

Silk Hydrogels of Tunable Structure and Viscoelastic Properties Using Different Chronological Orders of Genipin and Physical Cross-Linking.

Catering the hydrogel manufacturing process toward defined viscoelastic properties for intended biomedical use is important to hydrogel scaffolding function and cell differentiation. Silk fibroin hydrogels may undergo "physical" cross-linking through ß-sheet crystallization during high pressure carbon dioxide treatment, or covalent "chemical" cross-linking by genipin. We demonstrate here that time-dependent mechanical properties are tunable in silk fibroin hydrogels by altering the chronological order of genipin cross-linking with ß-sheet formation. Genipin cross-linking before ß-sheet formation affects gelation mechanics through increased molecular weight, affecting gel morphology, and decreasing stiffness response. Alternately, genipin cross-linking after gelation anchored amorphous regions of the protein chain, and increasing stiffness. These differences are highlighted and validated through large amplitude oscillatory strain near physiologic levels, after incorporation of material characterization at molecular and micron length scales.

RETRACTED ARTICLE: High Pulsatility Flow Promotes Vascular Fibrosis by Triggering Endothelial EndMT and Fibroblast Activation.

Vascular fibrosis, the formation of excess fibrous tissue on the blood vessel wall, is characterized by unmitigated proliferation of fibroblasts or myofibroblast-like cells exhibiting α-smooth-muscle-actin in vessel lumen and other vascular layers. It likely contributes to vascular unresponsiveness to conventional therapies. This paper demonstrates a new flow-induced vascular fibrosis mechanism. Using our developed flow system which simulates the effect of vessel stiffening and generates unidirectional high pulsatility flow (HPF) with the mean shear flow at a physiological level, we have shown that HPF caused vascular endothelial dysfunction. Herein, we further explored the role of HPF in vascular fibrosis through endothelial-to-mesenchymal transdifferentiation (EndMT). Pulmonary arterial endothelial cells (ECs) were exposed to steady flow and HPF, which have the same physiological mean fluid shear but different in flow pulsatility. Cells were analyzed after being conditioned with flows for 24 or 48 h. HPF was found to induce EndMT of cells after 48 h stimulation; cells demonstrated drastically decreased expression in EC marker CD31, as well as increased transforming growth factor ß, α-SMA, and collagen type-I, in both gene and protein expression profiles. Using the flow media from HPF-conditioned endothelial culture to cultivate arterial adventitial fibroblasts (AdvFBs) and ECs respectively, we found that the conditioned media respectively enhanced migration, proliferation and α-SMA expression of AdvFBs, and induced EndMT of ECs. It was further revealed that cells exposed to HPF exhibited much higher percentage of caspase-positive cells compared to those exposed to steady flow. Apoptotic cells together with remaining, caspase-negative cells suggested the presence of apoptosis-resistant dysfunctional ECs which likely underwent EndMT process and perpetuated fibrosis throughout vascular tissues. Therefore, our results indicate that prolonged HPF stimuli induce vascular fibrosis through triggering EndMT and EC-mediated AdvFB activation and migration, which follows initial endothelial inflammation, dysfunction and apoptosis.

Mechanical and biocompatible characterizations of a readily available multilayer vascular graft.

There is always a considerable clinical need for vascular grafts. Considering the availability, physical and mechanical properties, and regenerative potential, we have developed and characterized readily available, strong, and compliant multilayer grafts that support cell culture and ingrowth. The grafts were made from heterogeneous materials and structures, including a thin, dense, nanofibrous core composed of poly-ε-caprolactone (PCL), and a thick, porous, hydrogel sleeve composed of genipin-crosslinked collagen-chitosan (GCC). Because the difference in physicochemical properties between PCL and GCC caused layer separation, the layer adhesion was identified as a determinant to graft property and integrity under physiological conditions. Thus, strategies to modify the layer interface, including increasing porosity of the PCL surface, decreasing hydrophobicity, and increasing interlayer crosslinking, were developed. Results from microscopic images showed that increasing PCL porosity was characterized by improved layer adhesion. The resultant graft was characterized by high compliance (4.5%), and desired permeability (528 mL/cm(2)/min), burst strength (695 mmHg), and suture strength (2.38 N) for readily grafting. Results also showed that PCL mainly contributed to the graft mechanical properties, whereas GCC reduced the water permeability. In addition to their complementary contributions to physical and mechanical properties, the distinct graft layers also provided layer-specific structures for seeding and culture of vascular endothelial and smooth muscle cells in vitro. Acellular graft constructs were readily used to replace abdominal aorta of rabbits, resulting in rapid cell ingrowth and flow reperfusion. The multilayer constructs capable of sustaining physiological conditions and promoting cellular activities could serve as a platform for future development of regenerative vascular grafts.

Diversity within the Redeye Bass, Micropterus coosae (Perciformes: Centrarchidae) species group, with descriptions of four new species.

The Redeye Bass, Micropterus coosae, was described from the Mobile River basin, Chattahoochee, and Savannah rivers in Alabama and Georgia, USA, by Hubbs and Bailey (1940). At that time the authors recognized significant variation in the Black Warrior River population, and noted that with further study this form may be recognized as a separate taxon. An examination of variation in morphology and mitochondrial DNA supported this observation, and highlighted additional species-level variation, resulting in descriptions of a total of four new species: Micropterus cahabae, new sp., restricted to the Cahaba River system; Micropterus tallapoosae, new sp., restricted to the Tallapoosa River system; Micropterus warriorensis, new sp., from the Black Warrior River system; and Micropterus chattahoochae, new sp., from the Chattahoochee River system. Micropterus coosae is restricted to the Coosa River system. The new species differ from each other and from M. coosae by a combination of pigmentation and scale count characteristics, development of the tooth patch, and divergence within the ND2 gene. While two of these species are relatively common in upland streams within their ranges, M. warriorensis, M. cahabae and M. chattahoochae are uncommon and may warrant protection.

Sleep fragmentation and intermittent hypoxemia are associated with decreased insulin sensitivity in obese adolescent Latino males.

BACKGROUND: Although sleep-related breathing disorder (SRBD) has been linked to insulin resistance in adults, this has not been as well established in children. We hypothesized that the severity of SRBD in adolescents was associated with metabolic impairment. METHODS: Polysomnography was performed on obese, Latino males referred for snoring. The frequently sampled intravenous glucose tolerance test was used to assess glucose homeostasis. Total-body dual-energy X-ray absorptiometry was used to quantify adiposity. RESULTS: A total of 22 males (mean age ± SD: 13.4 ± 2.1 y, BMI z-score 2.4 ± 0.3, obstructive apnea hypopnea index 4.1 ± 3.2) were studied. After correcting for age and adiposity in multiple-regression models, Log frequency of desaturation (defined as ≥3% drop in oxygen saturation from baseline) negatively correlated with insulin sensitivity. Sleep efficiency was positively correlated with glucose effectiveness (S(G), the capacity of glucose to mediate its own disposal). The Log total arousal index was positively correlated with Log homeostasis model assessment-estimated insulin resistance. CONCLUSION: Sleep fragmentation and intermittent hypoxemia are associated with metabolic impairment in obese adolescent Latino males independent of age and adiposity. We speculate that SRBD potentiates the risk for development of metabolic syndrome and type 2 diabetes in the obese adolescent population.

MEDIC: medical embedded device for individualized care.

OBJECTIVE: Presented work highlights the development and initial validation of a medical embedded device for individualized care (MEDIC), which is based on a novel software architecture, enabling sensor management and disease prediction capabilities, and commercially available microelectronic components, sensors and conventional personal digital assistant (PDA) (or a cell phone). METHODS AND MATERIALS: In this paper, we present a general architecture for a wearable sensor system that can be customized to an individual patient's needs. This architecture is based on embedded artificial intelligence that permits autonomous operation, sensor management and inference, and may be applied to a general purpose wearable medical diagnostics. RESULTS: A prototype of the system has been developed based on a standard PDA and wireless sensor nodes equipped with commercially available Bluetooth radio components, permitting real-time streaming of high-bandwidth data from various physiological and contextual sensors. We also present the results of abnormal gait diagnosis using the complete system from our evaluation, and illustrate how the wearable system and its operation can be remotely configured and managed by either enterprise systems or medical personnel at centralized locations. CONCLUSION: By using commercially available hardware components and software architecture presented in this paper, the MEDIC system can be rapidly configured, providing medical researchers with broadband sensor data from remote patients and platform access to best adapt operation for diagnostic operation objectives.

Context-aware sensing of physiological signals.

Recent advancement in microsensor technology permits miniaturization of conventional physiological sensors. Combined with low-power, energy-aware embedded systems and low power wireless interfaces, these sensors now enable patient monitoring in home and workplace environments in addition to the clinic. Low energy operation is critical for meeting typical long operating lifetime requirements. Some of these physiological sensors, such as electrocardiographs (ECG), introduce large energy demand because of the need for high sampling rate and resolution, and also introduce limitations due to reduced user wearability. In this paper, we show how context-aware sensing can provide the required monitoring capability while eliminating the need for energy-intensive continuous ECG signal acquisition. We have implemented a wearable system based on standard widely-used handheld computing hardware components. This system relies on a new software architecture and an embedded inference engine developed for these standard platforms. The performance of the system is evaluated using experimental data sets acquired for subjects wearing this system during an exercise sequence. This same approach can be used in context-aware monitoring of diverse physiological signals in a patient's daily life.

Incremental diagnosis method for intelligent wearable sensor systems.

This paper presents an incremental diagnosis method (IDM) to detect a medical condition with the minimum wearable sensor usage by dynamically adjusting the sensor set based on the patient's state in his/her natural environment. The IDM, comprised of a naive Bayes classifier generated by supervised training with Gaussian clustering, is developed to classify patient motion in-context (due to a medical condition) and in real-time using a wearable sensor system. The IDM also incorporates a utility function, which is a simple form of expert knowledge and user preferences in sensor selection. Upon initial in-context detection, the utility function decides which sensor is to be activated next. High-resolution in-context detection with minimum sensor usage is possible because the necessary sensor can be activated or requested at the appropriate time. As a case study, the IDM is demonstrated in detecting different severity levels of a limp with minimum usage of high diagnostic resolution sensors.

A strategy for retrofitting critical structures in reducing earthquake and hurricane risks

With three of the most severe hurricanes of this century visiting the Caribbean in the last five years (Gilbert, Hugo and Andrew), leaving in their wake billions of dollar in damage, scores of lives lost and thousands homeless, one lesson seems to have been well learnt and documented. Today every Caribbean island, without exception, has put in place at least a rudimentary disaster preparedness programme. Over the last three years, programmes aimed at sensitization and education of the public have met with reasonable success. Community groups, service clubs, voluntary organizations such as the Red Cross, St. John's Ambulance Brigade, the Scouts and Girl Guides have all been very active. In some cases detailed, national disaster plans have been developed. We must now begin to take the next step. The retrofitting of critical public buildings such as schools, churches, meeting halls and hospitals must now be put at the top of a priority listing. These structures often serve as emergency shelters and we must do all in our power to ensure not only that they remain safe and functional during the disaster but that they stand a very good chance of surviving any disaster. But we must note that by virtue of the large numbers of persons who congregate in these buildings at any one time the risk of catastrophic loss of life must be reduced to a minimum.(AU)