Measurement of proximal contact of single crowns to assess interproximal relief: A pilot study.

Background: It is common for dental technicians to adjust the proximal surface of adjacent teeth on casts when fabricating single crowns. However, whether the accuracy of the proximal contact is affected if this step is eliminated is unclear. Objective: To evaluate the accuracy of the proximal contact of single crowns for mandibular first molars fabricated from four different restorative materials, without adjustment of the proximal surface of the adjacent teeth by the laboratory/dental technician. Methods: This study was in vitro; all the clinical procedures were conducted on a dentoform. The mandibular first molar tooth on the dentoform was prepared using diamond burs and a high speed handpiece. Twenty single crowns were fabricated, five for each group (monolithic zirconia, lithium disilicate, metal ceramic, and cast gold). No proximal surface adjacent to the definitive crowns was adjusted for tight contact in the dental laboratory. Both the qualitative analyses, using dental floss and shimstock, and the quantitative analyses, using a stereo microscope, were performed to evaluate the accuracy of the proximal contact of the restoration with the adjacent teeth. In the quantitative analysis, one-way analysis of variance was used to compare mean values at a significance level of 0.05. Results: In quantitative analysis, the differences between the proximal contact tightness of the four groups was not statistically significant (P = 0.802 for mesial contacts, P = 0.354 for distal contacts). In qualitative analysis, in most crowns, dental floss passed through the contact with tight resistance and only one film of shimstock could be inserted between the adjacent teeth and the restoration. However, one specimen from the cast gold crown had open contact. Conclusions: Even without proximal surface adjustment of the adjacent teeth during the crown fabrication process, adequate proximal contact tightness between the restoration and adjacent teeth could be achieved.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

Single-Cell and Spatial Transcriptomic Analysis of Human Skin Delineates Intercellular Communication and Pathogenic Cells.

Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

ATAC-seq Data Processing.

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has gained wide popularity as a fast, straightforward, and efficient way of generating genome-wide maps of open chromatin and guiding identification of active regulatory elements and inference of DNA protein binding locations. Given the ubiquity of this method, uniform and standardized methods for processing and assessing the quality of ATAC-seq datasets are needed. Here, we describe the data processing pipeline used by the ENCODE (Encyclopedia of DNA Elements) consortium to process ATAC-seq data into peak call sets and signal tracks and to assess the quality of these datasets.

Deep Learning on Chromatin Accessibility.

DNA accessibility has been a powerful tool in locating active regulatory elements in a cell type, but dissecting the combinatorial logic within these regulatory elements has been a continued challenge in the field. Deep learning models have been shown to be highly predictive models of regulatory DNA and have led to new biological insights on regulatory syntax and logic. Here, we provide a framework for deep learning in genomics that implements best practices and focuses on ease of use, versatility, and compatibility with existing tools for inference on DNA sequence.

Sensitized 1-Acyl-7-nitroindolines with Enhanced Two-Photon Cross Sections for Release of Neurotransmitters.

Precise photochemical control, using two-photon excitation (2PE), of the timing and location of activation of glutamate is useful for studying the molecular and cellular physiology of the brain. Antenna-based light harvesting strategies represent a general method to increase the sensitivity to 2PE of otherwise insensitive photoremovable protecting groups (PPGs). This was applied to the most commonly used form of "caged" glutamate, MNI-Glu. Computational investigation showed that a four- or six-carbon linker attached between the 4-position of thioxanthone (THX) and the 4-position of the 5-methyl derivative of MNI-Glu (MMNI-Glu) would position the antenna and PPG close to one another to enable Dexter energy transfer. Nine THX-MMNI-Glu conjugates were prepared and their photochemical properties determined. Installation of the THX antenna resulted in a red shift of the absorption (λmax = 385-405 nm) along with increased quantum yield compared to the parent compound MNI-Glu (λmax = 347 nm). The THX-MMNI-Glu conjugate with a four-carbon linker and attachment to the 4-position of THX underwent photolysis via 1PE at 405 and 430 nm and via 2PE at 770 and 860 nm, yielding glutamate. The two-photon uncaging action cross section (δu) was 0.11 and 0.29 GM at 770 and 860, respectively, which was greater than for MNI-Glu (0.06 and 0.072 GM at 720 and 770 nm, respectively). The THX sensitizer harvested the light via 2PE and transferred its resulting triplet energy to MMNI-Glu. Release of glutamate through 2PE at 860 nm from the compound (100 µM) activated iGluSnFR, a genetically encoded, fluorescent glutamate sensor, on the surface of cells in culture, portending its usefulness in studies of neurophysiology in acute brain slice.

An Endoscopic and Histologic Study on Healing of Radiofrequency Ablation Wounds in Patients With Barrett's Esophagus.

INTRODUCTION: Radiofrequency ablation (RFA) of Barrett's esophagus (BE) inflicts a wound spanning 3 epithelial types (stratified squamous, Barrett's metaplasia, gastric epithelium), yet the esophageal injury heals almost completely with squamous epithelium. Knowledge of how this unique wound heals might elucidate mechanisms underlying esophageal metaplasia. We aimed to prospectively and systematically characterize the early endoscopic and histologic features of RFA wound healing. METHODS: Patients with nondysplastic BE had endoscopy with systematic esophageal photographic mapping, biopsy, and volumetric laser endomicroscopy performed before and at 1, 2, and 4 weeks after RFA. RESULTS: Seven patients (6 men; mean age 56.1 ± 10.9 years) completed this study. Squamous re-epithelialization of RFA wounds did not only progress exclusively through squamous cells extending from the proximal wound edge but also progressed through islands of squamous epithelium sprouting throughout the ablated segment. Volumetric laser endomicroscopy revealed significant post-RFA increases in subepithelial glandular structures associated with the squamous islands. In 2 patients, biopsies of such islands revealed newly forming squamous epithelium contiguous with immature-appearing squamous cells arising from esophageal submucosal gland ducts. Subsquamous intestinal metaplasia (SSIM) was found in biopsies at 2 and/or 4 weeks after RFA in 6 of 7 patients. DISCUSSION: RFA wounds in BE are re-epithelialized, not just by squamous cells from the proximal wound margin but by scattered squamous islands in which esophageal submucosal gland duct cells seem to redifferentiate into the squamous progenitors that fuel squamous re-epithelialization. SSIM can be found in most patients during the healing process. We speculate that this SSIM might underlie Barrett's recurrences after apparently successful eradication.

Intracellular Trafficking and Distribution of Cd and InP Quantum Dots in HeLa and ML-1 Thyroid Cancer Cells.

The study of the interaction of engineered nanoparticles, including quantum dots (QDs), with cellular constituents and the kinetics of their localization and transport, has provided new insights into their biological consequences in cancers and for the development of effective cancer therapies. The present study aims to elucidate the toxicity and intracellular transport kinetics of CdSe/ZnS and InP/ZnS QDs in late-stage ML-1 thyroid cancer using well-tested HeLa as a control. Our XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) viability assay (Cell Proliferation Kit II) showed that ML-1 cells and non-cancerous mouse fibroblast cells exhibit no viability defect in response to these QDs, whereas HeLa cell viability decreases. These results suggest that HeLa cells are more sensitive to the QDs compared to ML-1 cells. To test the possibility that transporting rates of QDs are different between HeLa and ML-1 cells, we performed a QD subcellular localization assay by determining Pearson's Coefficient values and found that HeLa cells showed faster QDs transporting towards the lysosome. Consistently, the ICP-OES test showed the uptake of CdSe/ZnS QDs in HeLa cells was significantly higher than in ML-1 cells. Together, we conclude that high levels of toxicity in HeLa are positively correlated with the traffic rate of QDs in the treated cells.

Timing of Resumption of Anticoagulation After Polypectomy and Frequency of Post-procedural Complications: A Post-hoc Analysis.

BACKGROUND: Optimal timing for anticoagulation resumption after polypectomy is unclear. We explored the association between timing of anticoagulation resumption and occurrence of delayed post-polypectomy bleeding (PPB) and thromboembolic (TE) events. METHODS: We performed a post-hoc analysis of patients in an earlier study whose anticoagulants were interrupted for polypectomy. We compared rates of clinically important delayed PPB and TE events in relationship to timing of anticoagulant resumption. Late resumption was defined as > 2 days after polypectomy. RESULTS: Among 437 patients, 351 had early and 86 late resumption. Compared to early resumers, late resumers had greater polypectomy complexity. PPB rate was higher (but not significantly) in the late versus early resumers (2.3% vs. 0.9%, 1.47% greater, 95% CI [- 2.58 to 5.52], p = 0.26). TE events were more frequent in late versus early resumers [0% vs. 1.2% at 30 days, 0% vs. 2.3%, 95% CI 0.3-8, (p = 0.04) at 90 days]. On multivariate analysis, timing of restarting anticoagulation was not a significant predictor of PPB (OR 0.97, 95% CI 0.61-1.44, p = 0.897). Significant predictors were number of polyps ≥ 1 cm (OR 4.14, 95% CI 1.27-13.66, p = 0.014) and use of fulguration (OR 11.43, 95% CI 1.35-80.80, p = 0.014). CONCLUSIONS: Physicians delayed anticoagulation resumption more commonly after complex polypectomies. The timing of restarting anticoagulation was not a significant risk factor for PPB and late resumers had significantly higher rates of TE events within 90 days. Considering the potentially catastrophic consequences of TE events and the generally benign outcome of PPBs, clinicians should be cautious about delaying resumption of anticoagulation after polypectomy.

A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation.

Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in cis-regulatory elements (CREs). The combinations of DNA motifs acting within homeostasis and disease, however, are unclear. Gene expression, chromatin accessibility, TF footprinting, and H3K27ac-dependent DNA looping data were generated and a random-forest-based model was applied to identify 7,531 cell-type-specific cis-regulatory modules (CRMs) across 15 diploid human cell types. A co-enrichment framework within CRMs nominated 838 cell-type-specific, recurrent heterotypic DNA motif combinations (DMCs), which were functionally validated using massively parallel reporter assays. Cancer cells engaged DMCs linked to neoplasia-enabling processes operative in normal cells while also activating new DMCs only seen in the neoplastic state. This integrative approach identifies cell-type-specific cis-regulatory combinatorial DNA motifs in diverse normal and diseased human cells and represents a general framework for deciphering cis-regulatory sequence logic in gene regulation.

The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation.

Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.

Targeting Mitochondrial Damage as a Therapeutic for Ileal Crohn's Disease.

Paneth cell defects in Crohn's disease (CD) patients (called the Type I phenotype) are associated with worse clinical outcomes. Recent studies have implicated mitochondrial dysfunction in Paneth cells as a mediator of ileitis in mice. We hypothesized that CD Paneth cells exhibit impaired mitochondrial health and that mitochondrial-targeted therapeutics may provide a novel strategy for ileal CD. Terminal ileal mucosal biopsies from adult CD and non-IBD patients were characterized for Paneth cell phenotyping and mitochondrial damage. To demonstrate the response of mitochondrial-targeted therapeutics in CD, biopsies were treated with vehicle or Mito-Tempo, a mitochondrial-targeted antioxidant, and RNA transcriptome was analyzed. During active CD inflammation, the epithelium exhibited mitochondrial damage evident in Paneth cells, goblet cells, and enterocytes. Independent of inflammation, Paneth cells in Type I CD patients exhibited mitochondrial damage. Mito-Tempo normalized the expression of interleukin (IL)-17/IL-23, lipid metabolism, and apoptotic gene signatures in CD patients to non-IBD levels. When stratified by Paneth cell phenotype, the global tissue response to Mito-Tempo in Type I patients was associated with innate immune, lipid metabolism, and G protein-coupled receptor (GPCR) gene signatures. Targeting impaired mitochondria as an underlying contributor to inflammation provides a novel treatment approach for CD.

Loci identified by a genome-wide association study of carotid artery stenosis in the eMERGE network.

Carotid artery atherosclerotic disease (CAAD) is a risk factor for stroke. We used a genome-wide association (GWAS) approach to discover genetic variants associated with CAAD in participants in the electronic Medical Records and Genomics (eMERGE) Network. We identified adult CAAD cases with unilateral or bilateral carotid artery stenosis and controls without evidence of stenosis from electronic health records at eight eMERGE sites. We performed GWAS with a model adjusting for age, sex, study site, and genetic principal components of ancestry. In eMERGE we found 1793 CAAD cases and 17,958 controls. Two loci reached genome-wide significance, on chr6 in LPA (rs10455872, odds ratio [OR] (95% confidence interval [CI]) = 1.50 (1.30-1.73), p = 2.1 × 10-8 ) and on chr7, an intergenic single nucleotide variant (SNV; rs6952610, OR (95% CI) = 1.25 (1.16-1.36), p = 4.3 × 10-8 ). The chr7 association remained significant in the presence of the LPA SNV as a covariate. The LPA SNV was also associated with coronary heart disease (CHD; 4199 cases and 11,679 controls) in this study (OR (95% CI) = 1.27 (1.13-1.43), p = 5 × 10-5 ) but the chr7 SNV was not (OR (95% CI) = 1.03 (0.97-1.09), p = .37). Both variants replicated in UK Biobank. Elevated lipoprotein(a) concentrations ([Lp(a)]) and LPA variants associated with elevated [Lp(a)] have previously been associated with CAAD and CHD, including rs10455872. With electronic health record phenotypes in eMERGE and UKB, we replicated a previously known association and identified a novel locus associated with CAAD.

Morbidity and Mortality of Typhoid Intestinal Perforation Among Children in Sub-Saharan Africa 1995-2019: A Scoping Review.

BACKGROUND: Typhoid fever incidence and complications, including intestinal perforation, have declined significantly in high-income countries, with mortality rates <1%. However, an estimated 10.9 million cases still occur annually, most in low- and middle-income countries. With the availability of a new typhoid conjugate vaccine licensed for children and recommended by the World Health Organization, understanding severe complications, including associated mortality rates, is essential to inform country-level decisions on introduction of this vaccine. This scoping review summarizes over 20 years of the literature on typhoid intestinal perforation in sub-Saharan Africa. METHODS: We searched EMBASE, PubMed, Medline, and Cochrane databases for studies reporting mortality rates due to typhoid intestinal perforation in children, under 18 years old, in sub-Saharan Africa published from January 1995 through June 2019. RESULTS: Twenty-four papers from six countries were included. Reported mortality rates ranged from 4.6-75%, with 16 of the 24 studies between 11 and 30%. Thirteen papers included postoperative morbidity rates, ranging from 16-100%. The most documented complications included surgical site infections, intra-abdominal abscesses, and enterocutaneous fistulas. High mortality rates can be attributed to late presentation to tertiary centers, sepsis and electrolyte abnormalities requiring preoperative resuscitation, prolonged perforation-to-surgery interval, and lack of access to critical care or an intensive care unit postoperatively. CONCLUSIONS: Current estimates of mortality related to typhoid intestinal perforation among children in sub-Saharan Africa remain unacceptably high. Prevention of typhoid fever is essential to reduce mortality, with the ultimate goal of a comprehensive approach that utilizes vaccination, improvements in water, sanitation, and hygiene, and greater access to surgical care.

Transcriptome Profile Alteration with Cadmium Selenide/Zinc Sulfide Quantum Dots in Saccharomyces cerevisiae.

Quantum Dots (QDs) are becoming more prevalent in products used in our daily lives, such as TVs and laptops, due to their unique and tunable optical properties. The possibility of using QDs as fluorescent probes in applications, such as medical imaging, has been a topic of interest for some time, but their potential toxicity and long-term effects on the environment are not well understood. In the present study, we investigated the effects of yellow CdSe/ZnS-QDs on Saccharomyces cerevisiae. We utilized growth assays, RNA-seq, reactive oxygen species (ROS) detection assays, and cell wall stability experiments to investigate the potential toxic effects of CdSe/ZnS-QDs. We found CdSe/ZnS-QDs had no negative effects on cell viability; however, cell wall-compromised cells showed more sensitivity in the presence of 10 µg/mL CdSe/ZnS-QDs compared to non-treated cells. In CdSe/ZnS-treated and non-treated cells, no significant change in superoxide was detected, but according to our transcriptomic analysis, thousands of genes in CdSe/ZnS-treated cells became differentially expressed. Four significantly differentiated genes found, including FAF1, SDA1, DAN1, and TIR1, were validated by consistent results with RT-qPCR assays. Our transcriptome analysis led us to conclude that exposure of CdSe/ZnS-QDs on yeast significantly affected genes implicated in multiple cellular processes.

Integrating regulatory DNA sequence and gene expression to predict genome-wide chromatin accessibility across cellular contexts.

MOTIVATION: Genome-wide profiles of chromatin accessibility and gene expression in diverse cellular contexts are critical to decipher the dynamics of transcriptional regulation. Recently, convolutional neural networks have been used to learn predictive cis-regulatory DNA sequence models of context-specific chromatin accessibility landscapes. However, these context-specific regulatory sequence models cannot generalize predictions across cell types. RESULTS: We introduce multi-modal, residual neural network architectures that integrate cis-regulatory sequence and context-specific expression of trans-regulators to predict genome-wide chromatin accessibility profiles across cellular contexts. We show that the average accessibility of a genomic region across training contexts can be a surprisingly powerful predictor. We leverage this feature and employ novel strategies for training models to enhance genome-wide prediction of shared and context-specific chromatin accessible sites across cell types. We interpret the models to reveal insights into cis- and trans-regulation of chromatin dynamics across 123 diverse cellular contexts. AVAILABILITY AND IMPLEMENTATION: The code is available at https://github.com/kundajelab/ChromDragoNN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.