DNA mismatch and damage patterns revealed by single-molecule sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

Ultra-Rapid Droplet Digital PCR Enables Intraoperative Tumor Quantification.

The diagnosis and treatment of tumors often depends on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections. To address this gap, we developed Ultra-Rapid droplet digital PCR (UR-ddPCR), which can be completed in 15 minutes from tissue to result with an accuracy comparable to standard ddPCR. We demonstrate UR-ddPCR assays for the IDH1 R132H and BRAF V600E clonal mutations that are present in many low-grade gliomas and melanomas, respectively. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 13 glioma cases. We further combine UR-ddPCR measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities in addition to tumor cell percentages. We anticipate that UR-ddPCR, along with future refinements in assay instrumentation, will enable novel point-of-care diagnostics and the development of molecularly-guided surgeries that improve clinical outcomes.

Chitosan coated selenium: A versatile nano-delivery system for molecular cargoes.

The use of nanoscale delivery platforms holds tremendous potential to overcome the current limitations associated with the conventional delivery of genetic materials and hydrophobic compounds. Therefore, there is an imperative need to develop a suitable alternative nano-enabled delivery platform to overcome these limitations. This work reports the first one-step hydrothermal synthesis of chitosan functionalized selenium nanoparticles (Selenium-chitosan, SeNP) that are capable of serving as a versatile nanodelivery platform for different types of active ingredients. The chitosan functionalization modified the surface charge to allow the loading of active ingredients and improve biocompatibility. The effective loading of the SeNP was demonstrated using genetic material, a hydrophobic small molecule, and an antibiotic. Furthermore, the loading of active ingredients showed no detrimental effect on the specific properties (fluorescence and bactericidal) of the studied active ingredients. In vitro antimicrobial inhibitory studies exhibited good compatibility between the SeNP delivery platform and Penicillin G (Pen), resulting in a reduction of the minimum inhibitory concentration (MIC) from 32 to 16 ppm. Confocal microscopy images showed the uptake of the SeNP by a macrophage cell line (J774A.1), demonstrating trackability and intracellular delivery of an active ingredient. In summary, the present work demonstrates the potential of SeNP as a suitable delivery platform for biomedical and agricultural applications.

Receptor Tyrosine Kinase EPHA2 Drives Epidermal Differentiation through Regulation of EGFR Signaling.

Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.

Examining cefazolin utilization and perioperative anaphylaxis in patients with and without a penicillin allergy label: A cross-sectional study.

STUDY OBJECTIVE: To compare the occurrence of cefazolin perioperative anaphylaxis (POA) in patients with and without a penicillin allergy label (PAL) to determine whether the prevalence of cefazolin POA differs based on the presence of a PAL. DESIGN: Cross-sectional study. SETTING: A large U.S. healthcare system in the Baltimore-D.C. region, July 2017 to July 2020. PATIENTS: 112,817 surgical encounters across inpatient and outpatient settings in various specialties, involving 90,089 patients. Of these, 4876 (4.3%) encounters had a PAL. INTERVENTIONS: Perioperative cefazolin administration within 4 h before surgery to 4 h after the procedure began. MEASUREMENTS: The primary outcome was cefazolin POA in patients with and without PALs. Potential POA cases were identified based on tryptase orders or diphenhydramine administrations within the initial cefazolin administration to 6 h postoperatively. Verification included two validation steps. The first checked for hypersensitivity reaction (HSR) documentation, and the second, led by Allergy specialists, identified POA and the probable culprit. The secondary outcome looked at cefazolin use trends in patients with a PAL, stratified by setting and specialty. MAIN RESULTS: Of 112,817 encounters, 1421 (1.3%) had possible cefazolin HSRs. Of these, 22 (1.5%) had POA, resulting in a 0.02% prevalence. Of these, 13 (59.1%) were linked to cefazolin and 9 (40.9%) attributed to other drugs. Only one cefazolin POA case had a PAL, indicating no significant difference in cefazolin POA prevalence between patients with and without PALs (p = 0.437). Perioperative cefazolin use in patients with PALs steadily increased from 2.6% to 6.0% between 2017 and 2020, specifically in academic settings. CONCLUSIONS: The prevalence of cefazolin POA does not exhibit significant differences between patients with and without PALs, and notably, the incidence remains remarkably low. Based on these findings, it is advisable to view cefazolin as an acceptable choice for prophylaxis in patients carrying a PAL.

Prediction of lactate concentrations after cardiac surgery using machine learning and deep learning approaches.

Background: Although conventional prediction models for surgical patients often ignore intraoperative time-series data, deep learning approaches are well-suited to incorporate time-varying and non-linear data with complex interactions. Blood lactate concentration is one important clinical marker that can reflect the adequacy of systemic perfusion during cardiac surgery. During cardiac surgery and cardiopulmonary bypass, minute-level data is available on key parameters that affect perfusion. The goal of this study was to use machine learning and deep learning approaches to predict maximum blood lactate concentrations after cardiac surgery. We hypothesized that models using minute-level intraoperative data as inputs would have the best predictive performance. Methods: Adults who underwent cardiac surgery with cardiopulmonary bypass were eligible. The primary outcome was maximum lactate concentration within 24 h postoperatively. We considered three classes of predictive models, using the performance metric of mean absolute error across testing folds: (1) static models using baseline preoperative variables, (2) augmentation of the static models with intraoperative statistics, and (3) a dynamic approach that integrates preoperative variables with intraoperative time series data. Results: 2,187 patients were included. For three models that only used baseline characteristics (linear regression, random forest, artificial neural network) to predict maximum postoperative lactate concentration, the prediction error ranged from a median of 2.52 mmol/L (IQR 2.46, 2.56) to 2.58 mmol/L (IQR 2.54, 2.60). The inclusion of intraoperative summary statistics (including intraoperative lactate concentration) improved model performance, with the prediction error ranging from a median of 2.09 mmol/L (IQR 2.04, 2.14) to 2.12 mmol/L (IQR 2.06, 2.16). For two modelling approaches (recurrent neural network, transformer) that can utilize intraoperative time-series data, the lowest prediction error was obtained with a range of median 1.96 mmol/L (IQR 1.87, 2.05) to 1.97 mmol/L (IQR 1.92, 2.05). Intraoperative lactate concentration was the most important predictive feature based on Shapley additive values. Anemia and weight were also important predictors, but there was heterogeneity in the importance of other features. Conclusion: Postoperative lactate concentrations can be predicted using baseline and intraoperative data with moderate accuracy. These results reflect the value of intraoperative data in the prediction of clinically relevant outcomes to guide perioperative management.

HEXIM1 is an essential transcription regulator during human erythropoiesis.

ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.

Multi-Functional Chitosan Nanovesicles Loaded with Bioactive Manganese for Potential Wound Healing Applications.

Chronic skin wound is a chronic illness that possesses a risk of infection and sepsis. In particular, infections associated with antibiotic-resistant bacterial strains are challenging to treat. To combat this challenge, a suitable alternative that is complementary to antibiotics is desired for wound healing. In this work, we report multi-functional nanoscale chitosan vesicles loaded with manganese (Chi-Mn) that has potential to serve as a new tool to augment traditional antibiotic treatment for skin wound healing. Chi-Mn showed antioxidant activity increase over time as well as antimicrobial activity against E. coli and P. aeruginosa PA01. The modified motility assay that mimicked a skin wound before bacterial colonization showed inhibition of bacterial growth with Chi-Mn treatment at a low area density of 0.04 µg of Mn per cm2. Furthermore, this study demonstrated the compatibility of Chi-Mn with a commercial antibiotic showing no loss of antimicrobial potency. In vitro cytotoxicity of Chi-Mn was assessed with macrophages and dermal cell lines (J774A.1 and HDF) elucidating biocompatibility at a wide range (2 ppm-256 ppm). A scratch wound assay involving human dermal fibroblast (HDF) cells was performed to assess any negative effect of Chi-Mn on cell migration. Confocal microscopy study confirmed that Chi-Mn tested at the MIC (16 ppm Mn) has no effect on cell migration with respect to control. Overall, this study demonstrated the potential of Chi-Mn nanovesicles for wound healing applications.

Single-strand mismatch and damage patterns revealed by single-molecule DNA sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.

Human-Centered Design of a Clinical Decision Support for Anemia Screening in Children with Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) commonly leads to iron deficiency anemia (IDA). Rates of screening and treatment of IDA are often low. A clinical decision support system (CDSS) embedded in an electronic health record could improve adherence to evidence-based care. Rates of CDSS adoption are often low due to poor usability and fit with work processes. One solution is to use human-centered design (HCD), which designs CDSS based on identified user needs and context of use and evaluates prototypes for usefulness and usability. OBJECTIVES: this study aimed to use HCD to design a CDSS tool called the IBD Anemia Diagnosis Tool, IADx. METHODS: Interviews with IBD practitioners informed creation of a process map of anemia care that was used by an interdisciplinary team that used HCD principles to create a prototype CDSS. The prototype was iteratively tested with "Think Aloud" usability evaluation with clinicians as well as semi-structured interviews, a survey, and observations. Feedback was coded and informed redesign. RESULTS: Process mapping showed that IADx should function at in-person encounters and asynchronous laboratory review. Clinicians desired full automation of clinical information acquisition such as laboratory trends and analysis such as calculation of iron deficit, less automation of clinical decision selection such as laboratory ordering, and no automation of action implementation such as signing medication orders. Providers preferred an interruptive alert over a noninterruptive reminder. CONCLUSION: Providers preferred an interruptive alert, perhaps due to the low likelihood of noticing a noninterruptive advisory. High levels of desire for automation of information acquisition and analysis with less automation of decision selection and action may be generalizable to other CDSSs designed for chronic disease management. This underlines the ways in which CDSSs have the potential to augment rather than replace provider cognitive work.

Serial enrichment of heteroduplex DNA using a MutS-magnetic bead system.

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.

Domains of Frailty Predict Loss of Independence in Older Adults After Noncardiac Surgery.

IMPORTANCE: Preoperative frailty has been consistently associated with death, severe complications, and loss of independence (LOI) after surgery. LOI is an important patient-centered outcome, but it is unclear which domains of frailty are most strongly associated with LOI. Such information would be important to target individual geriatric domains for optimization. OBJECTIVE: To assess whether impairment in individual domains of the Edmonton Frail Scale (EFS) can predict LOI in older adults after noncardiac surgery. DESIGN: Retrospective Cohort Study. SETTING: One Academic Hospital. PARTICIPANTS: Patients aged 65 or older who were living independently and evaluated with the EFS during a preoperative visit to the Center for Preoperative Optimization at the Johns Hopkins Hospital between June 2018 and January 2020. MAIN OUTCOME: LOI defined as discharge to increased level of care outside of the home with new mobility deficit or functional dependence. New mobility deficit and functional dependence were extracted from chart review of the standardized occupational therapy and physical therapy assessment performed before discharge. RESULTS: A total of 3497 patients were analyzed. Age (mean±SD) was 73.4±6.2 years, and 1579 (45.2%) were female. The median total EFS score was 3 (range 0-16), and 725/3497 (27%) were considered frail (EFS≥6). The frequencies of impairment in each EFS domain were functional performance (33.5% moderately impaired, 11% severely impaired), history of hospital readmission (42%), poor self-described health status (37%), and abnormal cognition (17.1% moderately impaired, 13.8% severely impaired). Overall, 235/3497 (6.7%) patients experienced LOI. Total EFS score was associated with LOI (odds ratio: 1.37, 95% CI, 1.30-1.45, P <0.001) in a model adjusted for age, sex, body mass index, American Society of Anesthesiologists rating, congestive heart failure, valvular heart disease, hypertension diagnosis, chronic lung disease, diabetes, renal failure, liver disease, weight loss, anemia, and depression. Using a nested log likelihood approach, the domains of functional performance, functional dependence, social support, health status, and urinary incontinence improved the base multivariable model. In cross-validation, total EFS improved the prediction of LOI with the final model achieving an area under the curve of 0.840. Functional performance was the single domain that most improved outcome prediction, but together with functional dependence, social support, and urinary incontinence, the model resulted in an area under the curve of 0.838. CONCLUSION AND RELEVANCE: Among domains measured by the EFS before a wide range of noncardiac surgeries in older adults, functional performance, functional dependence, social support, and urinary incontinence were independently associated with and improved the prediction of LOI. Clinical initiatives to mitigate LOI may consider screening with the EFS and targeting abnormalities within these domains.

Visual Transformers and Convolutional Neural Networks for Disease Classification on Radiographs: A Comparison of Performance, Sample Efficiency, and Hidden Stratification.

Purpose: To compare performance, sample efficiency, and hidden stratification of visual transformer (ViT) and convolutional neural network (CNN) architectures for diagnosis of disease on chest radiographs and extremity radiographs using transfer learning. Materials and Methods: In this HIPAA-compliant retrospective study, the authors fine-tuned data-efficient image transformers (DeiT) ViT and CNN classification models pretrained on ImageNet using the National Institutes of Health Chest X-ray 14 dataset (112 120 images) and MURA dataset (14 656 images) for thoracic disease and extremity abnormalities, respectively. Performance was assessed on internal test sets and 75 000 external chest radiographs (three datasets). The primary comparison was DeiT-B ViT vs DenseNet121 CNN; secondary comparisons included DeiT-Ti (Tiny), ResNet152, and EfficientNetB7. Sample efficiency was evaluated by training models on varying dataset sizes. Hidden stratification was evaluated by comparing prevalence of chest tubes in pneumothorax false-positive and false-negative predictions and specific abnormalities for MURA false-negative predictions. Results: DeiT-B weighted area under the receiver operating characteristic curve (wAUC) was slightly lower than that for DenseNet121 on chest radiograph (0.78 vs 0.79; P < .001) and extremity (0.887 vs 0.893; P < .001) internal test sets and chest radiograph external test sets (P < .001 for each). DeiT-B and DeiT-Ti both performed slightly worse than all CNNs for chest radiograph and extremity tasks. DeiT-B and DenseNet121 showed similar sample efficiency. DeiT-B had lower chest tube prevalence in false-positive predictions than DenseNet121 (43.1% [324 of 5088] vs 47.9% [2290 of 4782]). Conclusion: Although DeiT models had lower wAUCs than CNNs for chest radiograph and extremity domains, the differences may be negligible in clinical practice. DeiT-B had sample efficiency similar to that of DenseNet121 and may be less susceptible to certain types of hidden stratification.Keywords: Computer-aided Diagnosis, Informatics, Neural Networks, Thorax, Skeletal-Appendicular, Convolutional Neural Network (CNN), Feature Detection, Supervised Learning, Machine Learning, Deep Learning Supplemental material is available for this article. © RSNA, 2022.

Collaboration of a Medical School With a Special Forces Group on Annual Training: A Blueprint.

Collaboration on annual training between a medical school and a National Guard Special Forces Group can be accomplished with great benefit to both parties. The authors describe the involvement by the Edward Via College of Osteopathic Medicine in providing training for the 20th Special Forces Group Medical Sergeants of the Alabama Army National Guard.

Association Between Left Ventricular Relative Wall Thickness and Acute Kidney Injury After Noncardiac Surgery.

BACKGROUND: Acute kidney injury (AKI) after major noncardiac surgery is commonly attributed to cardiovascular dysfunction. Identifying novel associations between preoperative cardiovascular markers and kidney injury may guide risk stratification and perioperative intervention. Increased left ventricular relative wall thickness (RWT), routinely measured on echocardiography, is associated with myocardial dysfunction and long-term risk of heart failure in patients with preserved left ventricular ejection fraction (LVEF); however, its relationship to postoperative complications has not been studied. We evaluated the association between preoperative RWT and AKI in high-risk noncardiac surgical patients with preserved LVEF. METHODS: Patients ≥18 years of age having major noncardiac surgery (high-risk elective intra-abdominal or noncardiac intrathoracic surgery) between July 1, 2016, and June 30, 2018, who had transthoracic echocardiography in the previous 12 months were eligible. Patients with preoperative creatinine ≥2 mg/dL or reduced LVEF (<50%) were excluded. The association between RWT and AKI, defined as an increase in serum creatinine by 0.3 mg/dL from baseline within 48 hours or by 50% within 7 days after surgery, was assessed using multivariable logistic regression adjusted for preoperative covariates. An additional model adjusted for intraoperative covariates, which are strongly associated with AKI, especially hypotension. RWT was modeled continuously, associating the change in odds of AKI for each 0.1 increase in RWT. RESULTS: The study included 1041 patients (mean ± standard deviation [SD] age 62 ± 15 years; 59% female). A total of 145 subjects (13.9%) developed AKI within 7 days. For RWT quartiles 1 through 4, respectively, 20 of 262 (7.6%), 40 of 259 (15.4%), 39 of 263 (14.8%), and 46 of 257 (17.9%) developed AKI. Log-odds and proportion with AKI increased across the observed RWT values. After adjusting for confounders (demographics, American Society of Anesthesiologists [ASA] physical status, comorbidities, baseline creatinine, antihypertensive medications, and left ventricular mass index), each RWT increase of 0.1 was associated with an estimated 26% increased odds of developing AKI (odds ratio [OR]; 95% confidence interval [CI]) of 1.26 (1.09-1.46; P = .002). After adjusting for intraoperative covariates (length of surgery, presence of an arterial line, intraoperative hypotension, crystalloid administration, transfusion, and urine output), RWT remained independently associated with the odds of AKI (OR; 95% CI) of 1.28 (1.13-1.47; P = .001). Increased RWT was also independently associated with hospital length of stay and adjusted hazard ratio (HR [95% CI]) of 0.94 (0.89-0.99; P = .018). CONCLUSIONS: Left ventricular RWT is a novel cardiovascular factor associated with AKI within 7 days after high-risk noncardiac surgery among patients with preserved LVEF. Application of this commonly available measurement of risk stratification or perioperative intervention warrants further investigation.

Regulation of RNA polymerase II activity is essential for terminal erythroid maturation.

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

Design and synthesis of C-aryl angular luotonins via a one-pot aza-Nazarov-Friedlander sequence and their Topo-I inhibition studies along with C-aryl vasicinones and luotonins.

A facile one-pot synthesis of C-ring substituted angular luotonins has been realized via a methanesulfonic acid mediated aza-Nazarov-Friedlander condensation sequence on quinazolinonyl enones. Topoisomerase I (topo-I) inhibition studies revealed that the angular luotonin library (7a-7l) and their regioisomeric analogs (linear luotonins, 8a-8l) are weak negative modulators, compared to camptothecin. These results would fare well for the design of topo-I-inert luotonins for non-oncological applications such as anti-fungal and insecticide lead developments. Surprisingly, the tricyclic vasicinones (9h, 9i, and 9j) showed better topo-I inhibition compared to pentacyclic C-aryl luotonins providing a novel pharmacophore for further explorations.

Telemedicine Expansion in Pediatric Gastroenterology in Response to COVID-19: Early Results of an International Physician Survey.

The COVID-19 pandemic triggered an unprecedented expansion of telemedicine, leading to development of new workflows. We conducted a survey of telemedicine practice among pediatric gastroenterology practitioners on March 26, 2020. Responses were coded and analyzed. The survey garnered 33 responses. Most centers were 3 weeks into the implementation. The most commonly used telemedicine software was Zoom followed by FaceTime, telephone, and Epic software. Provider education was through online meetings, webinars, and tip sheets. Patient education was by nonclinical staff at the time of visit scheduling or tip sheets. A major barrier was the need for patients to enroll in an electronic portal. Two thirds of practices offered telemedicine to both new and return patients. Most sites billed based on time. This represents a record of the very early response of the pediatric gastroenterology community to the COVID-19 telemedicine expansion and can inform follow-up studies.

Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.

Synthesis and biofilm inhibition studies of 2-(2-amino-6-arylpyrimidin-4-yl)quinazolin-4(3H)-ones.

Synthesis of novel 4(3H)-quinazolinonyl aminopyrimidine derivatives has been achieved via quinazolinonyl enones which in turn were obtained from 2-acyl-4(3H)-quinazolinone. They have been assayed for biofilm inhibition against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative bacteria (Acinetobacter baumannii). The analogues with 2,4,6-trimethoxy phenyl, 4-methylthio phenyl, and 3-bromo phenyl substituents (5h, 5j & 5k) have been shown to inhibit biofilm formation efficiently in MRSA with IC50 values of 20.7-22.4 µM). The analogues 5h and 5j have demonstrated low toxicity in human cells in vitro and can be investigated further as leads.