Correcting signal biases and detecting regulatory elements in STARR-seq data

High-throughput reporter assays such as self-transcribing active regulatory region sequencing (STARR-seq) have made it possible to measure regulatory element activity across the entire human genome at once. The resulting data, however, present substantial analytical challenges. Here, we identify technical biases that explain most of the variance in STARR-seq data. We then develop a statistical model to correct those biases and to improve detection of regulatory elements. This approach substantially improves precision and recall over current methods, improves detection of both activating and repressive regulatory elements, and controls for false discoveries despite strong local correlations in signal.

Gene regulation is of foundational importance to nearly all biological processes, and variation in gene regulatory activity plays a major role in human disease risk (Lee and Young 2013; Parker et al. 2013; Finucane et al. 2015). A major step toward measuring regulatory activity across the human genome has been the development of high-throughput reporter assays such as STARR-seq (Arnold et al. 2013) that allow regulatory element activity to be quantified with high-throughput sequencing rather than with optical detection of a fluorescent or luminescent signal.High-throughput reporter assays create substantial analytical challenges that are distinct from other sequencing-based genomic assays. There is significant local variation in high-throughput reporter assay signal. We show here that, across data from several laboratories, most of that variation can be explained by features of the underlying genomic sequence and experimental procedures rather than by regulatory element activity. For example, nucleotide composition can alter PCR efficiency leading to under- and overrepresentation of some sequences. Meanwhile, highly repetitive sequences often do not align uniquely to the human reference genome, also biasing signal estimates. Additional analytical challenges include that STARR-seq signals can be both positive and negative, reflecting activation and repression, and the boundaries of regulatory elements are typically unknown and must therefore be estimated from the data. Those challenges together impact signal representations, hinder estimation of regulatory element activity, and cause false positives and false negatives when left unaddressed.Taken together, key requirements of statistical methods to analyze STARR-seq data are the ability to identify and estimate the effect of both activating and repressing regulatory elements while also correcting for underlying sequence biases in high-throughput reporter assays. A statistical model was recently introduced that corrects technical biases and detects regulatory elements in STARR-seq, but the model is limited to detecting only activating regulatory elements (Lee et al. 2020). Considering repression is a crucial gene regulation mechanism (Courey and Jia 2001), overlooking repressive elements may limit understanding of gene regulation with STARR-seq. To overcome that challenge, our correcting reads and analysis of differentially active elements (CRADLE) model takes a two-step approach. First, CRADLE uses a generalized linear regression model to estimate and correct major biases that we have identified in STARR-seq data. Next, CRADLE detects regions with statistically significant regulatory activity from the bias-corrected signals while rigorously controlling FDR. In doing so, CRADLE substantially improves the use of STARR-seq by providing a robust estimation of regulatory activity and improved visualization of raw signals.     

Fas-mediated apoptosis of neutrophils in sera of patients with infection

In the presence of infection, neutropenia is considered to be a marker of poor prognosis; conversely, neutrophilia may not be a determinant of a better prognosis. Since apoptotic neutrophils are compromised functionally, we evaluated the effect of infection on neutrophil apoptosis. The rate of apoptosis was greater for neutrophils isolated from patients with infection than for healthy controls. Escherichia coli did not directly modulate the rate of neutrophil apoptosis. However, sera from infected patients promoted (P < 0.001) neutrophil apoptosis. Interestingly, the sera of patients with different types of infection (gram negative, gram positive, or culture negative) exerted a more or less identical response on neutrophil apoptosis. Sera of infected patients showed a fivefold greater content of FasL compared to controls. Moreover, anti-FasL antibody partly attenuated the infected-serum-induced neutrophil apoptosis. In in vitro studies, E. coli enhanced monocyte FasL expression. Moreover, conditioned media prepared from activated macrophages from control mice showed enhanced apoptosis of human as well as mouse neutrophils. On the contrary, conditioned media prepared from activated macrophages isolated from FasL-deficient mice induced only a mild degree of neutrophil apoptosis. These results suggest that neutrophils in patients with infection undergo apoptosis at an accelerated rate. Infection not only promoted monocyte expression of FasL but also increased FasL content of the serum. Because the functional status of apoptotic cells is compromised, a significant number of neutrophils may not be participating in the body's defense. Since neutrophils play the most important role in innate immunity, their compromised status in the presence of infection may transfer the host defense burden from an innate response to acquired immunity. The present study provides some insight into the lack of correlation between neutrophilia and the outcome of infection.     

Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats

Kidney aging has been recognized as a chronic process of compromised renal function and structural changes in the tubulointerstitium and glomerulus. Cell senescence is associated with alterations in cell structure and function, including expression of cytokines and structural and regulatory components of extracellular matrix proteins. In this investigation, we tested the hypothesis that senescent renal cells may accumulate in vivo with advancing age. We also evaluated the expression of transforming growth factor (TGF)-beta1 and p21WAF1/CIP1 in aging kidneys. Sprague-Dawley rats at the ages of 3, 12, and 24 months were used for this study. Renal tissues were processed for morphometric and senescence analysis. Expression of TGF-beta1 and p21WAF1/CIP1 was evaluated by Northern or Western blot analysis and immunohistochemistry. Substantial tubulointerstitial injury occurred at the age of 12 months, but significant glomerular structure alteration was observed at the age of 24 months. Tubular cells developed senescence, which was detected by beta-galactosidase staining. This staining increased in frequency and intensity with age. Renal cortices showed a significant increase in the mRNA expression for TGF-beta1 and protein level for p21WAF1/CIP1. The enhanced expression of TGF-beta1 and p21WAF1/CIP1 was localized in the tubulointersititial cells. These data suggest that tubular cells undergo senescence and express increased TGF-beta1 and p21WAF1/CIP1 with advancing age. These age-related cellular and molecular alterations may play an important role in the initiation and/or progression of tubulointerstitial fibrosis and glomerulosclerosis in aging.     

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis

The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system (CNS) of tadpoles and adult frogs. This cDNA (xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal (Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine (His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cell-specific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15-16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.     

Intracellular potentials of microperfused human sweat duct cells

Intracellular potentials of cells from isolated segments of microperfused human sweat ducts were measured in order to determine the electrical profiles of these cells under resting, transporting, and inhibited conditions. Even though the cells are relatively small (ca. 6–8 m), continuous recordings of intracellular potentials from the same impalement were stable for up to 2 h. In the resting condition in normal Ringer's solution when the lumen of the duct was collapsed and not perfused, the intracellular potential measured across the basal membrane was 34.6±1.5 mV (n=31; mean±SE). In the same bathing medium, when the duct lumen was also perfused with normal Ringer's solution, the basolateral membrane potential (V _b), the apical membrane potential (V _a) and transepithelial potential (V _t) was –33.8±0.47 mV, –23.7±0.48 mV and –9.6±0.9 mV (n=73), respectively. The average input impedence (R _i) of these cells was 19.6±0.4 M (n=36). The frequency distribution ofV _b was unimodal suggesting that only one functional cell type exists in this tissue. Amiloride (0.1 mM) in the lumen hyperpolarized bothV _a andV _b by –40.5±3.6 mV and –33.2±3.7 mV (n=15), respectively, with a slight but significant increase inR _i (15%), while abolishingV _t. Removing luminal Cl^– depolarizedV _a by +37.0±4.2 mV and hyperpolarizedV _b by –19.0±4.2 mV (n=11). Removing Cl^– from the bath hyperpolarizedV _a by –3.3±2.3 mV and depolarizedV _b by +24.3±2.7 mV (n=15). Ouabain caused an initial fast depolarization (+8 mV) followed by a prolonged slow depolarization ofV _b, and an increase inR _i of about 84%. These results not only provide the first electrical profile of the human sweat duct tissue, but they also show that its cell membrane potentials are unusually low. This unusual property of this epithelium appears to be due to the combination of a significant Na⁺ conductance at the apical membrane and a remarkably high tissue Cl^– conductance.     

Prostate cancer: results of external irradiation.

From 1975 to 1982, 205 patients with local prostate cancer were treated at the radiation oncology department, the University of Kansas Medical Center, Kansas City, Kansas. Patients'' median age was 73 years. All of the patients were staged according to American Urologic staging criteria. Twenty-eight patients had stage A2 cancer, 91 patients had stage B cancer, and 86 patients had stage C cancer. All patients were treated using megavoltage radiation (dosage range: 6000 cGy to 7100 cGy). The follow-up period ranged from a minimum of 8 years to a maximum of 15 years (median: 9.4 years). The clinical local control was 96% for stage A2, 94% for stage B, and 90% for stage C disease. The overall and disease-free survival rates were 71% and 60%, respectively. Fourteen patients developed moderate complications with one patient (0.5%) requiring surgical intervention. The local control and survival rates reported in this study are comparable with surgical results, suggesting that external beam irradiation in prostate cancer is safe and effective.     

Surgical management of synchronous coronary artery disease and multiple aortic stenosis: a case report with brief review of the literature

One-stage surgery was successfully performed in a 44-year-old hypertensive man with uncontrolled angina, multiple coarctations of the thoracic and abdominal aorta, and a previous subtotal gastrectomy. There was a gradient of 120 mm Hg between the thoracic and abdominal aorta. A graft was placed retroperitoneally from the infrarenal aorta to the ascending aorta and was followed by a coronary artery bypass graft. Twenty-four months postoperatively, the patient was free of angina, and his hypertension was easily controlled.     

Temporal trends in correlates of HIV testing uptake in South Africa: evaluation and population-level impacts of socio-economic factors and information sources

Aim

The primary objective of this study was to investigate temporal changes in HIV testing rates and quantify the degree to which these trends can be attributed to certain socio-economic characteristics, as well as exposure to information sources.

Subjects and methods

Data from a nationally representative sample of 30,020 sexually active black Africans who participated in the first, second, third and fourth South African National HIV, Behaviour and Health Surveys conducted in 2002, 2005, 2008 and 2012, respectively. Multivariable logistic regression models and population-attributable risks were calculated for the socio-economic characteristics and the information sources.

Results

The socio-economic characteristics of the survey participants remained stable over time, while HIV testing rates increased substantially from 20% in 2002 to 70% in 2012. However, there was little improvement in condom use rates. Combined impact of education, employment and geographical locations were associated with increased levels of HIV testing rates. Most of the survey participants (> 80%) were exposed to several mass-media and interpersonal information sources. The combined impact of mass-media tools on HIV testing rates ranged between 48 and 60%, while 40–50% of the HIV tests were collectively attributed to the interpersonal information sources.

Conclusion

We observed significant temporal changes in population-level impacts of several key socio-economic characteristics and information sources on HIV testing rates. Widespread nationwide HIV awareness efforts led to significant increases in access to testing facilities and substantial increases in HIV testing rates over time. However, this increase was not mirrored in condom use behaviour.

     

Success of Maternal and Child Health Pipeline Training Programs: Alumni Survey Results

Maternal and Child Health Journal - The Maternal and Child Health (MCH) Pipeline Training Program, promotes development of a diverse health workforce by training undergraduate students from...     

Estimate of Burden and Direct Healthcare Cost of Infectious Waterborne Disease in the United States

Provision of safe drinking water in the United States is a great public health achievement. However, new waterborne disease challenges have emerged (e.g., aging infrastructure, chlorine-tolerant and biofilm-related pathogens, increased recreational water use). Comprehensive estimates of the health burden for all water exposure routes (ingestion, contact, inhalation) and sources (drinking, recreational, environmental) are needed. We estimated total illnesses, emergency department (ED) visits, hospitalizations, deaths, and direct healthcare costs for 17 waterborne infectious diseases. About 7.15 million waterborne illnesses occur annually (95% credible interval [CrI] 3.88 million–12.0 million), results in 601,000 ED visits (95% CrI 364,000–866,000), 118,000 hospitalizations (95% CrI 86,800–150,000), and 6,630 deaths (95% CrI 4,520–8,870) and incurring US $3.33 billion (95% CrI 1.37 billion–8.77 billion) in direct healthcare costs. Otitis externa and norovirus infection were the most common illnesses. Most hospitalizations and deaths were caused by biofilm-associated pathogens (nontuberculous mycobacteria, Pseudomonas, Legionella), costing US $2.39 billion annually.