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.     

Differences and relationships between the physical properties and the microscopic structure of human femoral,tibial and fibular cortical bone

Coefficients of correlation between certain physical properties and the histological components of the break area were calculated on an IBM 7090 computer for 56 femoral, 79 tibial and 37 fibular specimens of embalmed cortical bone of standardized size and shape. Strong positive correlations (0.01–0.02 significance level) were found between tensile strength and the percentage of interstitial lamellae in the break area; between hardness and the number of osteons/mm²; and between hardness and the percentage of osteons in the break area. Equally high negative correlations were found between tensile strength and percentage of osteons in the break area; between shearing strength and average area/osteon remnant; between elastic modulus and percentage of spaces in the break area; and an even higher correlation (0.001) between hardness and percentage of spaces in the break area. Negative correlations (at slightly more than 0.05 significance level) were found between shearing strength and modulus and average area/osteon. Osteons tend to reduce the tensile strength and elastic modulus of bone while interstitial lamellae tend to increase them. The probable reason is the relatively greater amount of cement lines, which are sites of weakness where failure can occur, in Haversian bone as compared with lamellar bone. The predominant orientation of collagen fibers and the amount and distribution of calcium may also be involved. These factors are now being investigated.     

Two novel mutations in the EPM2A gene in a Korean patient with Lafora's progressive myoclonus epilepsy

The progressive myoclonus epilepsy of the Lafora type (LD; MIM 254780) is a rare autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration, and the presence of periodic acid-Schiff-positive polyglucosan inclusions (Lafora bodies). Mutations in the EPM2A gene have recently been found to cause LD and about 30 or more mutations have been reported thus far. LD is relatively common in countries of the Mediterranean Basin, the Middle East, India, and Pakistan. Although a few sporadic cases with the typical LD phenotype have also been reported in the Far East including Korea and Japan, a recent effort to find mutations in Japanese LD families was not successful. In the present study, we report two novel mutations in a Korean girl with LD; a 1-bp insertion mutation (c.223insC; G75fsX107) in exon 1 and a missense mutation (c.559A>G; T187A) in exon 3 of the EPM2A gene. To our knowledge, this is the first report of a genetically confirmed case of LD in Koreans and also in the Far East.     

Three-dimensional forward solver and its performance analysis for magnetic resonance electrical impedance tomography (MREIT) using recessed electrodes

In magnetic resonance electrical impedance tomography (MREIT), we try to reconstruct a cross-sectional resistivity (or conductivity) image of a subject. When we inject a current through surface electrodes, it generates a magnetic field. Using a magnetic resonance imaging (MRI) scanner, we can obtain the induced magnetic flux density from MR phase images of the subject. We use recessed electrodes to avoid undesirable artefacts near electrodes in measuring magnetic flux densities. An MREIT image reconstruction algorithm produces cross-sectional resistivity images utilizing the measured internal magnetic flux density in addition to boundary voltage data. In order to develop such an image reconstruction algorithm, we need a three-dimensional forward solver. Given injection currents as boundary conditions, the forward solver described in this paper computes voltage and current density distributions using the finite element method (FEM). Then, it calculates the magnetic flux density within the subject using the Biot-Savart law and FEM. The performance of the forward solver is analysed and found to be enough for use in MREIT for resistivity image reconstructions and also experimental designs and validations. The forward solver may find other applications where one needs to compute voltage, current density and magnetic flux density distributions all within a volume conductor.     

Image reconstruction of anisotropic conductivity tensor distribution in MREIT: computer simulation study

We describe a novel method of reconstructing images of an anisotropic conductivity tensor distribution inside an electrically conducting subject in magnetic resonance electrical impedance tomography (MREIT). MREIT is a recent medical imaging technique combining electrical impedance tomography (EIT) and magnetic resonance imaging (MRI) to produce conductivity images with improved spatial resolution and accuracy. In MREIT, we inject electrical current into the subject through surface electrodes and measure the z-component Bz of the induced magnetic flux density using an MRI scanner. Here, we assume that z is the direction of the main magnetic field of the MRI scanner. Considering the fact that most biological tissues are known to have anisotropic conductivity values, the primary goal of MREIT should be the imaging of an anisotropic conductivity tensor distribution. However, up to now, all MREIT techniques have assumed an isotropic conductivity distribution in the image reconstruction problem to simplify the underlying mathematical theory. In this paper, we firstly formulate a new image reconstruction method of an anisotropic conductivity tensor distribution. We use the relationship between multiple injection currents and the corresponding induced Bz data. Simulation results show that the algorithm can successfully reconstruct images of anisotropic conductivity tensor distributions. While the results show the feasibility of the method, they also suggest a more careful design of data collection methods and data processing techniques compared with isotropic conductivity imaging.     

The alterations of N-Methyl-D-aspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia-reperfusion insult

Delayed neuronal death in the CA1 of the hippocampus following global ischemia has been evoked by both the activation of N-methyl-D-aspartate receptor (NR) and the generate reactive oxygen species in the neurons. In the present study, we investigated whether oxidative DNA damages may be correlated with NR subunits (NR1 and NR2A/B) expression following ischemia insults in vivo. Thirty minutes after ischemia-reperfusion, the intensities of both NR and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivities were markedly increased in neurons of CA1. However, NR2A/B and 8-OHdG immunoreactivities were enhanced in CA1 over 24 h after ischemia although NR1 immunoreactivity was decreased. These results suggest that oxidative stress and excitotoxicity in the CA1 may simultaneously trigger neuronal damages at early time after ischemia, and free radical damage including oxidative DNA damage may eventually promote the delayed neuronal death in this region.     

Effect of Heat-Killed Lactobacillus casei DKGF7 on a Rat Model of Irritable Bowel Syndrome

Non-viable bacteria, referred to as “paraprobiotics,” have attracted attention as potentially safer alternatives to probiotics. The aim of this study was to investigate the efficacy of heat-killed Lactobacillus casei DKGF7 on the symptomatic improvement of irritable bowel syndrome (IBS) in a rat disease model and to elucidate the underlying mechanisms that contribute to the beneficial effects of heat-killed probiotics. Seven male Wistar rats were induced with IBS by restraint stress and administered heat-killed L. casei DKGF7 for four weeks and then compared with seven rats in the control group. Stool consistency measured four weeks after initial treatment was the primary outcome measure. To investigate the mechanism of action of the heat-killed bacteria on IBS, we measured serum corticosterone levels, inflammatory cytokines in colon tissue, and expression of tight junction proteins (TJPs) in the epithelium. The treatment group showed significantly better stool consistency scores than the control group at week 4, as well as at every measured time point (all p values < 0.05). The treatment group showed lower serum corticosterone levels, lower colonic inflammatory cytokine levels, and higher expression of TJPs compared with the control group. Paraprobiotics such as heat-killed L. casei DKGF7 can improve stool consistency in a rat IBS model, which may indicate a potential therapeutic strategy for IBS treatment.     

Surveillance of COVID-19–Associated Multisystem Inflammatory Syndrome in Children,South Korea

A concerning development during the coronavirus disease pandemic has been multisystem inflammatory syndrome in children. Reports of this condition in East Asia have been limited. In South Korea, 3 cases were reported to the national surveillance system for multisystem inflammatory syndrome in children. All case-patients were hospitalized and survived with no major disease sequelae.     

Editorial Commentary: Stem Cell Treatment in Knee Osteoarthritis: What for? Pain Management or Cartilage Regeneration?

The efficacy of mesenchymal stem cells regarding clinical outcomes and cartilage regeneration in knee osteoarthritis remains unclear; however, their theoretical role in multilineage cellular differentiation and immunomodulation of the arthritic cascade has been investigated. Several studies have reported that the use of stem cell therapy for knee osteoarthritis helps in pain improvement, but its effect on cartilage regeneration has not yet been explored. Moreover, numerous studies have reported high heterogeneity in the cell sources, as well as methods of culture expansion or cell concentration, and differences in delivery methods, assessment tools, and concomitant surgical procedures, which could affect the clinical outcomes or evaluation of cartilage regeneration potency. Furthermore, future studies are warranted to examine these factors in detail to interpret the results of mesenchymal stem cell treatment for knee osteoarthritis.