Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events

Assessment of healthcare worker protocol deviations and self-contamination during personal protective equipment donning and doffing

OBJECTIVETo evaluate healthcare worker (HCW) risk of self-contamination when donning and doffing personal protective equipment (PPE) using fluorescence and MS2 bacteriophage.DESIGNProspective pilot study.SETTINGTertiary-care hospital.PARTICIPANTSA total of 36 HCWs were included in this study: 18 donned/doffed contact precaution (CP) PPE and 18 donned/doffed Ebola virus disease (EVD) PPE.INTERVENTIONSHCWs donned PPE according to standard protocols. Fluorescent liquid and MS2 bacteriophage were applied to HCWs. HCWs then doffed their PPE. After doffing, HCWs were scanned for fluorescence and swabbed for MS2. MS2 detection was performed using reverse transcriptase PCR. The donning and doffing processes were videotaped, and protocol deviations were recorded.RESULTSOverall, 27% of EVD PPE HCWs and 50% of CP PPE HCWs made ≥1 protocol deviation while donning, and 100% of EVD PPE HCWs and 67% of CP PPE HCWs made ≥1 protocol deviation while doffing (P=.02). The median number of doffing protocol deviations among EVD PPE HCWs was 4, versus 1 among CP PPE HCWs. Also, 15 EVD PPE protocol deviations were committed by doffing assistants and/or trained observers. Fluorescence was detected on 8 EVD PPE HCWs (44%) and 5 CP PPE HCWs (28%), most commonly on hands. MS2 was recovered from 2 EVD PPE HCWs (11%) and 3 CP PPE HCWs (17%).CONCLUSIONSProtocol deviations were common during both EVD and CP PPE doffing, and some deviations during EVD PPE doffing were committed by the HCW doffing assistant and/or the trained observer. Self-contamination was common. PPE donning/doffing are complex and deserve additional study.Infect Control Hosp Epidemiol 2017;38:1077–1083</jats:sec

Alternative Splicing Events Identified in Human Embryonic Stem Cells and Neural Progenitors

Human embryonic stem cells (hESCs) and neural progenitor (NP) cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation. Alternative RNA splicing (AS), a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function. Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays. We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol), to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC. Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%. REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events. Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes. In addition, we found that REAP predictions are enriched in genes encoding serine/threonine kinase and helicase activities. An example is a REAP-predicted alternative exon in the SLK (serine/threonine kinase 2) gene that is differentially included in hESC, but skipped in NP as well as in other differentiated tissues. Lastly, comparative sequence analysis revealed conserved intronic cis-regulatory elements such as the FOX1/2 binding site GCAUG as being proximal to candidate AS exons, suggesting that FOX1/2 may participate in the regulation of AS in NP and hESC. In summary, a new methodology for exon array analysis was introduced, leading to new insights into the complexity of AS in human embryonic stem cells and their transition to neural stem cells

Assessment of antibiotic-resistant organism transmission among rooms of hospitalized patients, healthcare personnel, and the hospital environment utilizing surrogate markers and selective bacterial cultures

OBJECTIVE: To assess potential transmission of antibiotic-resistant organisms (AROs) using surrogate markers and bacterial cultures. DESIGN: Pilot study. SETTING: A 1,260-bed tertiary-care academic medical center. PARTICIPANTS: The study included 25 patients (17 of whom were on contact precautions for AROs) and 77 healthcare personnel (HCP). METHODS: Fluorescent powder (FP) and MS2 bacteriophage were applied in patient rooms. HCP visits to each room were observed for 2-4 hours; hand hygiene (HH) compliance was recorded. Surfaces inside and outside the room and HCP skin and clothing were assessed for fluorescence, and swabs were collected for MS2 detection by polymerase chain reaction (PCR) and selective bacterial cultures. RESULTS: Transfer of FP was observed for 20 rooms (80%) and 26 HCP (34%). Transfer of MS2 was detected for 10 rooms (40%) and 15 HCP (19%). Bacterial cultures were positive for 1 room and 8 HCP (10%). Interactions with patients on contact precautions resulted in fewer FP detections than interactions with patients not on precautions (P \u3c .001); MS2 detections did not differ by patient isolation status. Fluorescent powder detections did not differ by HCP type, but MS2 was recovered more frequently from physicians than from nurses (P = .03). Overall, HH compliance was better among HCP caring for patients on contact precautions than among HCP caring for patients not on precautions (P = .003), among nurses than among other nonphysician HCP at room entry (P = .002), and among nurses than among physicians at room exit (P = .03). Moreover, HCP who performed HH prior to assessment had fewer fluorescence detections (P = .008). CONCLUSIONS: Contact precautions were associated with greater HCP HH compliance and reduced detection of FP and MS2

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3′ untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.National Institutes of Health (U.S.) (Grant HG007005

Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity

<p>Abstract</p> <p>Background</p> <p>The human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.</p> <p>Methods</p> <p>To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.</p> <p>Results</p> <p>We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.</p> <p>Conclusions</p> <p>This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.</p

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017 : a systematic analysis for the Global Burden of Disease Study 2017

Background Global development goals increasingly rely on country-specific estimates for benchmarking a nation's progress. To meet this need, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2016 estimated global, regional, national, and, for selected locations, subnational cause-specific mortality beginning in the year 1980. Here we report an update to that study, making use of newly available data and improved methods. GBD 2017 provides a comprehensive assessment of cause-specific mortality for 282 causes in 195 countries and territories from 1980 to 2017. Methods The causes of death database is composed of vital registration (VR), verbal autopsy (VA), registry, survey, police, and surveillance data. GBD 2017 added ten VA studies, 127 country-years of VR data, 502 cancer-registry country-years, and an additional surveillance country-year. Expansions of the GBD cause of death hierarchy resulted in 18 additional causes estimated for GBD 2017. Newly available data led to subnational estimates for five additional countries Ethiopia, Iran, New Zealand, Norway, and Russia. Deaths assigned International Classification of Diseases (ICD) codes for non-specific, implausible, or intermediate causes of death were reassigned to underlying causes by redistribution algorithms that were incorporated into uncertainty estimation. We used statistical modelling tools developed for GBD, including the Cause of Death Ensemble model (CODErn), to generate cause fractions and cause specific death rates for each location, year, age, and sex. Instead of using UN estimates as in previous versions, GBD 2017 independently estimated population size and fertility rate for all locations. Years of life lost (YLLs) were then calculated as the sum of each death multiplied by the standard life expectancy at each age. All rates reported here are age-standardised. Findings At the broadest grouping of causes of death (Level 1), non-communicable diseases (NC Ds) comprised the greatest fraction of deaths, contributing to 73.4% (95% uncertainty interval [UI] 72.5-74.1) of total deaths in 2017, while communicable, maternal, neonatal, and nutritional (CMNN) causes accounted for 186% (17.9-19.6), and injuries 8.0% (7.7-8.2). Total numbers of deaths from NCD causes increased from 2007 to 2017 by 22.7% (21.5-23.9), representing an additional 7.61 million (7. 20-8.01) deaths estimated in 2017 versus 2007. The death rate from NCDs decreased globally by 7.9% (7.08.8). The number of deaths for CMNN causes decreased by 222% (20.0-24.0) and the death rate by 31.8% (30.1-33.3). Total deaths from injuries increased by 2.3% (0-5-4-0) between 2007 and 2017, and the death rate from injuries decreased by 13.7% (12.2-15.1) to 57.9 deaths (55.9-59.2) per 100 000 in 2017. Deaths from substance use disorders also increased, rising from 284 000 deaths (268 000-289 000) globally in 2007 to 352 000 (334 000-363 000) in 2017. Between 2007 and 2017, total deaths from conflict and terrorism increased by 118.0% (88.8-148.6). A greater reduction in total deaths and death rates was observed for some CMNN causes among children younger than 5 years than for older adults, such as a 36.4% (32.2-40.6) reduction in deaths from lower respiratory infections for children younger than 5 years compared with a 33.6% (31.2-36.1) increase in adults older than 70 years. Globally, the number of deaths was greater for men than for women at most ages in 2017, except at ages older than 85 years. Trends in global YLLs reflect an epidemiological transition, with decreases in total YLLs from enteric infections, respirator}, infections and tuberculosis, and maternal and neonatal disorders between 1990 and 2017; these were generally greater in magnitude at the lowest levels of the Socio-demographic Index (SDI). At the same time, there were large increases in YLLs from neoplasms and cardiovascular diseases. YLL rates decreased across the five leading Level 2 causes in all SDI quintiles. The leading causes of YLLs in 1990 neonatal disorders, lower respiratory infections, and diarrhoeal diseases were ranked second, fourth, and fifth, in 2017. Meanwhile, estimated YLLs increased for ischaemic heart disease (ranked first in 2017) and stroke (ranked third), even though YLL rates decreased. Population growth contributed to increased total deaths across the 20 leading Level 2 causes of mortality between 2007 and 2017. Decreases in the cause-specific mortality rate reduced the effect of population growth for all but three causes: substance use disorders, neurological disorders, and skin and subcutaneous diseases. Interpretation Improvements in global health have been unevenly distributed among populations. Deaths due to injuries, substance use disorders, armed conflict and terrorism, neoplasms, and cardiovascular disease are expanding threats to global health. For causes of death such as lower respiratory and enteric infections, more rapid progress occurred for children than for the oldest adults, and there is continuing disparity in mortality rates by sex across age groups. Reductions in the death rate of some common diseases are themselves slowing or have ceased, primarily for NCDs, and the death rate for selected causes has increased in the past decade. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd.Peer reviewe

Population and fertility by age and sex for 195 countries and territories, 1950–2017: a systematic analysis for the Global Burden of Disease Study 2017

Background: Population estimates underpin demographic and epidemiological research and are used to track progress on numerous international indicators of health and development. To date, internationally available estimates of population and fertility, although useful, have not been produced with transparent and replicable methods and do not use standardised estimates of mortality. We present single-calendar year and single-year of age estimates of fertility and population by sex with standardised and replicable methods. Methods: We estimated population in 195 locations by single year of age and single calendar year from 1950 to 2017 with standardised and replicable methods. We based the estimates on the demographic balancing equation, with inputs of fertility, mortality, population, and migration data. Fertility data came from 7817 location-years of vital registration data, 429 surveys reporting complete birth histories, and 977 surveys and censuses reporting summary birth histories. We estimated age-specific fertility rates (ASFRs; the annual number of livebirths to women of a specified age group per 1000 women in that age group) by use of spatiotemporal Gaussian process regression and used the ASFRs to estimate total fertility rates (TFRs; the average number of children a woman would bear if she survived through the end of the reproductive age span [age 10–54 years] and experienced at each age a particular set of ASFRs observed in the year of interest). Because of sparse data, fertility at ages 10–14 years and 50–54 years was estimated from data on fertility in women aged 15–19 years and 45–49 years, through use of linear regression. Age-specific mortality data came from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 estimates. Data on population came from 1257 censuses and 761 population registry location-years and were adjusted for underenumeration and age misreporting with standard demographic methods. Migration was estimated with the GBD Bayesian demographic balancing model, after incorporating information about refugee migration into the model prior. Final population estimates used the cohort-component method of population projection, with inputs of fertility, mortality, and migration data. Population uncertainty was estimated by use of out-of-sample predictive validity testing. With these data, we estimated the trends in population by age and sex and in fertility by age between 1950 and 2017 in 195 countries and territories. Findings: From 1950 to 2017, TFRs decreased by 49\ub74% (95% uncertainty interval [UI] 46\ub74–52\ub70). The TFR decreased from 4\ub77 livebirths (4\ub75–4\ub79) to 2\ub74 livebirths (2\ub72–2\ub75), and the ASFR of mothers aged 10–19 years decreased from 37 livebirths (34–40) to 22 livebirths (19–24) per 1000 women. Despite reductions in the TFR, the global population has been increasing by an average of 83\ub78 million people per year since 1985. The global population increased by 197\ub72% (193\ub73–200\ub78) since 1950, from 2\ub76 billion (2\ub75–2\ub76) to 7\ub76 billion (7\ub74–7\ub79) people in 2017; much of this increase was in the proportion of the global population in south Asia and sub-Saharan Africa. The global annual rate of population growth increased between 1950 and 1964, when it peaked at 2\ub70%; this rate then remained nearly constant until 1970 and then decreased to 1\ub71% in 2017. Population growth rates in the southeast Asia, east Asia, and Oceania GBD super-region decreased from 2\ub75% in 1963 to 0\ub77% in 2017, whereas in sub-Saharan Africa, population growth rates were almost at the highest reported levels ever in 2017, when they were at 2\ub77%. The global average age increased from 26\ub76 years in 1950 to 32\ub71 years in 2017, and the proportion of the population that is of working age (age 15–64 years) increased from 59\ub79% to 65\ub73%. At the national level, the TFR decreased in all countries and territories between 1950 and 2017; in 2017, TFRs ranged from a low of 1\ub70 livebirths (95% UI 0\ub79–1\ub72) in Cyprus to a high of 7\ub71 livebirths (6\ub78–7\ub74) in Niger. The TFR under age 25 years (TFU25; number of livebirths expected by age 25 years for a hypothetical woman who survived the age group and was exposed to current ASFRs) in 2017 ranged from 0\ub708 livebirths (0\ub707–0\ub709) in South Korea to 2\ub74 livebirths (2\ub72–2\ub76) in Niger, and the TFR over age 30 years (TFO30; number of livebirths expected for a hypothetical woman ageing from 30 to 54 years who survived the age group and was exposed to current ASFRs) ranged from a low of 0\ub73 livebirths (0\ub73–0\ub74) in Puerto Rico to a high of 3\ub71 livebirths (3\ub70–3\ub72) in Niger. TFO30 was higher than TFU25 in 145 countries and territories in 2017. 33 countries had a negative population growth rate from 2010 to 2017, most of which were located in central, eastern, and western Europe, whereas population growth rates of more than 2\ub70% were seen in 33 of 46 countries in sub-Saharan Africa. In 2017, less than 65% of the national population was of working age in 12 of 34 high-income countries, and less than 50% of the national population was of working age in Mali, Chad, and Niger. Interpretation: Population trends create demographic dividends and headwinds (ie, economic benefits and detriments) that affect national economies and determine national planning needs. Although TFRs are decreasing, the global population continues to grow as mortality declines, with diverse patterns at the national level and across age groups. To our knowledge, this is the first study to provide transparent and replicable estimates of population and fertility, which can be used to inform decision making and to monitor progress. Funding: Bill &amp; Melinda Gates Foundation