Search

To investigate genetic mutations in patients with dilated nonischemic cardiomyopathy (NICM) and history of left ventricular assist device (LVAD) implant. 76 patients were included in this retrospective study. Clinical data was obtained to determine environmental exposures (i.e. infectious, chemotherapy, pregnancy, significant alcohol and drug use), family history, native heart function, and the attributed etiology of their cardiomyopathy as determined by each patient's cardiologist. Genetic testing was obtained using a third-party 168-gene cardiomyopathy panel. Variants were classified into five categories: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign. The VUS were investigated with population allele frequencies from Gnomad or Varsome databases and two modeling scores, Rare Exome Variant Ensemble Learner (Revel) and Combined Annotation Dependent Depletion (CADD), to investigate the likely pathogenicity. The patients were majority male (68%) and racially diverse, with 59% Black, 21% Hispanic, 17% White, and 3% Asian. Family history of heart failure was positive in 29% of the sample. The possible etiology of heart failure was 16% environmental, 8% familial, and 75% idiopathic or unknown. Regarding genetic testing results, 67 (88%) patients had variants identified; the remaining 9 patients (12%) had negative results. There were a total 170 variants for those 67 patients, with an average of 2.6 variants per patient. Of the 170 variants, 14 (8%) were pathogenic/likely pathogenic and 7 of these were titin (TTN) mutations. 151 (89%) of variants were VUS. Allele frequencies were not found for 17% of the VUS, and the remaining variants had total population and race-specific allele frequencies less than 1%. 83 (55%) of VUS were among the top 1% deleterious substitutions by CADD scores, and 27 (18%) of VUS had a Revel score > 0.5, on a scale of 0 to 1, suggesting more likely pathogenic variants. Patients with end-stage dilated NICM undergoing LVAD are likely to have genetic variants. The most frequent pathogenic variants were in TTN but most results are VUS. Due to the high frequency of VUS, augmenting genetic testing in this population may contribute to identification of additional pathogenic variants. [ABSTRACT FROM AUTHOR]

Inflammation drives the initiation and progression of pulmonary hypertension (PH). Platelets, increasingly recognized as immune cells, are activated and increased in the lungs of patients with PH. Platelet activation leads to the release of α-granule chemokines, many of which are implicated in PH. We hypothesized that hypoxia-induced secretion of platelet α-granule stored proteins and PH would be prevented in Nbeal2 -/-, α-granule deficient mice. WT and Nbeal2 -/- mice were maintained in normoxia or exposed to 10% hypobaric hypoxia for 3, 14, 21, or 35 days. We observed macrothrombocytopenia, increased circulating neutrophils and monocytes, and increased lung interstitial macrophages in Nbeal2 -/- mice at baseline. Hypoxia-induced platelet activation was attenuated, and hypoxia-induced increase in lung PF4 and platelets was delayed in Nbeal2 -/- mice compared to WT mice. Finally, although pulmonary vascular remodeling (PVR) and PH were attenuated at day 21, Nbeal2 -/- mice were not protected against hypoxia-induced PVR and PH at day 35. While this mutation also impacted circulating monocytes, neutrophils, and lung IMs, all of which are critical in the development of experimental PH, we gained further support for the role of platelets and α-granule proteins, such as PF4, in PH progression and pathogenesis and made several observations that expand our understanding of α-granule deficient mice in chronic hypoxia., (Copyright © 2024 American Society of Hematology.)

Acute respiratory distress syndrome (ARDS) is a serious illness accounting for 10% of ICU admissions and high mortality of 31-45% with a paucity of pharmacologic treatment options. Dysregulated inflammation and oxidative stress are hallmark features of ARDS. We previously showed that transgenic mice expressing a naturally occurring polymorphism of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), are protected against Staphylococcus aureus (S. aureus) pneumonia, acute lung injury, and pulmonary neutrophilia. In this mouse strain, an R213G amino acid substitution leads to lower tissue binding affinity and elevated alveolar and plasma EC-SOD levels, though the redox-regulated mechanisms responsible for protection against S. aureus are not yet elucidated. Neutrophils are recruited to the areas of injury and inflammation, in part by activated platelets, which contain multiple redox-sensitive targets. Thus, we hypothesize that increased circulating EC-SOD due to the EC-SOD R213G variant protects against S. aureus pneumonia by reducing platelet activation and subsequent neutrophil recruitment to the lung. We demonstrate that, compared to WT mice with S. aureus pneumonia, platelet activation, formation of platelet-neutrophil aggregates (PNAs), and influx of neutrophils and PNAs into the lung are decreased in the infected R213G mice. Furthermore, pre-treatment with a MnTE-2-PyP SOD mimetic protects against S. aureus-induced platelet activation, pulmonary neutrophilia, and acute lung injury. Our data highlight the redox regulation of platelet activation as a driver of S. aureus -induced acute lung injury.

Despite extensive research into the genetic underpinnings of neurodevelopmental disorders (NDD), many clinical cases remain unresolved. We studied a female proband with a NDD, mildly dysmorphic facial features, and brain stem hypoplasia on neuroimaging. Comprehensive genomic analyses revealed a terminal 5p loss and terminal 18q gain in the proband while a diploid copy number for chromosomes 5 and 18 in both parents. Genomic investigations in the proband identified an unbalanced translocation t(5;18) with additional genetic material from chromosome 2 (2q31.3) inserted at the breakpoint, pointing to a complex chromosomal rearrangement (CCR) involving 5p15.2, 2q31.3, and 18q21.32. Breakpoint junction analyses enabled by long read genome sequencing unveiled the presence of four distinct junctions in the father, who is carrier of a balanced CCR. The proband inherited from the father both the abnormal chromosome 5 resulting in segmental aneusomies of chr5 (loss) and chr18 (gain) and a der(2) homologue. Evidences suggest a chromoplexy mechanism for this CCR derivation, involving double-strand breaks (DSBs) repaired by non-homologous end joining (NHEJ) or alternative end joining (alt-EJ). The complexity of the CCR and the segregation of homologues elucidate the genetic model for this family. This study demonstrates the importance of combining multiple genomic technologies to uncover genetic causes of complex neurodevelopmental syndrome and to better understand genetic disease mechanisms., Competing Interests: Additional Declarations: J.R.L. has stock ownership in 23andMe, is a paid consultant for Genome International, and is a co-inventor on multiple U.S. and European patents related to molecular diagnostics for inherited neuropathies, genomic disorders, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis and clinical genomic sequencing (both ES and GS) offered in the Baylor Genetics Laboratory (http://bmgl.com). J.R.L. serves on the Scientific Advisory Board of BG.

Background: Guidelines addressing magnetic resonance imaging (MRI) in patients with cardiac implantable electronic devices (CIEDs) provide algorithms for imaging pediatric and congenital heart disease (CHD) patients. Guideline acceptance varies by institution. Guidelines also do not support routine MRI scans in patients with epicardial or abandoned leads, common in pediatric and CHD patients., Objective: The purpose of this study was to determine the incidence of MRI-related complications in pediatric and CHD patients with CIEDs, including epicardial and/or abandoned leads., Methods: A multicenter retrospective review included patients with CIEDs who underwent any MRI between 2007 and 2022 at congenital cardiac centers. The primary outcome was any patient adverse event or clinically significant CIED change after MRI, defined as pacing lead capture threshold increase >0.5 V with output change, P- or R- wave amplitude decrease >50% with sensitivity change, or impedance change >50%., Results: Across 14 institutions, 314 patients (median age 18.8 [1.3; 31.4] years) underwent 389 MRIs. There were 288 pacemakers (74%) and 87 implantable cardioverter-defibrillators (22%); 52% contained epicardial leads, and 14 (4%) were abandoned leads only. Symptoms or CIED changes occurred in 4.9% of MRI scans (6.1% of patients). On 9 occasions (2%), warmth or pain occurred. Pacing capture threshold or lead impedance changes occurred in 1.4% and 2.0% of CIEDs post-MRI and at follow-up., Conclusion: Our data provide evidence that MRIs can be performed in pediatric and CHD patients with CIEDs, including non-MRI-conditional CIEDs and epicardial and/or abandoned leads, with rare minor symptoms or CIED changes but no other complications., (Copyright © 2023 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Outpatient chemotherapy often leaves patients to grapple with a range of complex side effects at home. Leveraging tailored evidence-based content to monitor and manage these symptoms remains an untapped potential among patients with gastrointestinal (GI) cancer., Objective: This study aims to bridge the gap in outpatient chemotherapy care by integrating a cutting-edge text messaging system with a chatbot interface. This approach seeks to enable real-time monitoring and proactive management of side effects in patients with GI cancer undergoing intravenous chemotherapy., Methods: Real-Time Chemotherapy-Associated Side Effects Monitoring Supportive System (RT-CAMSS) was developed iteratively, incorporating patient-centered inputs and evidence-based information. It synthesizes chemotherapy knowledge, self-care symptom management skills, emotional support, and healthy lifestyle recommendations. In a single-arm 2-month pilot study, patients with GI cancer undergoing chemotherapy received tailored intervention messages thrice a week and a weekly Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events-based symptom assessment via a chatbot interface. Baseline and postintervention patient surveys and interviews were conducted., Results: Out of 45 eligible patients, 34 were enrolled (76% consent rate). The mean age was 61 (SD 12) years, with 19 (56%) being females and 21 (62%) non-Hispanic White. The most common cancer type was pancreatic (n=18, 53%), followed by colon (n=12, 35%) and stomach (n=4, 12%). In total, 27 (79% retention rate) participants completed the postintervention follow-up. In total, 20 patients texted back at least once to seek additional information, with the keyword "chemo" or "support" texted the most. Among those who used the chatbot system checker, fatigue emerged as the most frequently reported symptom (n=15), followed by neuropathy (n=7). Adjusted for multiple comparisons, patients engaging with the platform exhibited significantly improved Patient Activation Measure (3.70, 95% CI -6.919 to -0.499; P=.02). Postintervention interviews and satisfaction surveys revealed that participants found the intervention was user-friendly and were provided with valuable information., Conclusions: Capitalizing on mobile technology communication holds tremendous scalability for enhancing health care services. This study presents initial evidence of the engagement and acceptability of RT-CAMSS, warranting further evaluation in a controlled clinical trial setting., (©Sameh Gomaa, James Posey, Babar Bashir, Atrayee Basu Mallick, Eleanor Vanderklok, Max Schnoll, Tingting Zhan, Kuang-Yi Wen. Originally published in JMIR Formative Research (https://formative.jmir.org), 10.11.2023.)

Background: Recent reports of the utilisation of pyrvinium pamoate (PP), an FDA-approved anti-helminth, have shown that it inhibits pancreatic ductal adenocarcinoma (PDAC) cell growth and proliferation in-vitro and in-vivo in preclinical models. Here, we report about an ongoing phase I open-label, single-arm, dose escalation clinical trial to determine the safety and tolerability of PP in PDAC surgical candidates., Methods and Analysis: In a 3+3 dose design, PP is initiated 3 days prior to surgery. The first three patients will be treated with the initial dose of PP at 5 mg/kg orally for 3 days prior to surgery. Dose doubling will be continued to a reach a maximum of 20 mg/kg orally for 3 days, if the previous two dosages (5 mg/kg and 10 mg/kg) were tolerated. Dose-limiting toxicity grade≥3 is used as the primary endpoint. The pharmacokinetic and pharmacodynamic (PK/PD) profile of PP and bioavailability in humans will be used as the secondary objective. Each participant will be monitored weekly for a total of 30 days from the final dose of PP for any side effects. The purpose of this clinical trial is to examine whether PP is safe and tolerable in patients with pancreatic cancer, as well as assess the drug's PK/PD profile in plasma and fatty tissue. Potential implications include the utilisation of PP in a synergistic manner with chemotherapeutics for the treatment of pancreatic cancer., Ethics and Dissemination: This study was approved by the Thomas Jefferson Institutional Review Board. The protocol number for this study is 20F.041 (Version 3.1 as of 27 October 2021). The data collected and analysed from this study will be used to present at local and national conferences, as well as, written into peer-reviewed manuscript publications., Trial Registration Number: ClinicalTrials.gov: NCT05055323., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Hepatocellular carcinoma (HCC) is a leading cause of mortality, with Hepatitis B virus (HBV) infection as a dominant etiology worldwide. Effective early detection strategies may facilitate curative therapies and improve survival. We investigated genomic aberrations in circulating tumor DNA (ctDNA) as potential diagnostic markers of HCC in HBV-infected patients., Methods: We identified early stage (BCLC 0-A) HCC cases (n = 21) and patients without HCC (n = 14) from a cohort of Asian patients with HBV, undergoing surveillance between 2013 and 2017. Circulating cell-free DNA was isolated from blood samples, and assayed by next-generation sequencing of 23 genes implicated in HCC pathogenesis. Somatic mutations were identified using a computational pipeline. Using area under the curve (AUC) in receiver operating characteristic (ROC) analysis, we evaluated gene alterations and clinical factors in an exploratory early HCC detection model., Results: Mutant ARID1A, CTNNB1, TP53 genes were increased in HCC cases vs. non-HCC patients (85.7% vs 42.9%, P = 0.011; 42.9% vs 0%, P = 0.005; 100% vs 71.4%, P = 0.019, respectively). Using these three genes, AUC for discriminating HCC from non-HCC patients was 0.844 (95% confidence interval [CI]: 0.7317-0.9553). When combining these genes with clinical factors in an exploratory early HCC detection model, AUC increased from 0.7415 (using clinical factors alone) to 0.9354 (P = 0.041)., Conclusion: Genomic aberrations in ctDNA were more prevalent in HBV-infected HCC patients compared with patients without HCC. Combining these alterations with clinical factors may identify HCC in HBV-infected patients at an early stage. These findings warrant validation in future studies., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to disclose., (Copyright © 2023. Published by Elsevier Masson SAS.)

Background: A subset of patients who develop post-surgical heart block have recovery of atrioventricular node function. Factors predicting recovery are not understood. We investigated our centre's incidence of post-surgical heart block and examine factors associated with recovery of atrioventricular node function., Methods: We conducted a single-centre retrospective study of patients 0 - 21 years who underwent cardiac surgery between January 2010 and December 2019 and experienced post-operative heart block. Data including patient and clinical characteristics and operative variables were collected and analysed., Results: Of 6333 surgical hospitalisations, 128 (2%) patients developed post-operative heart block. Of the 128 patients, 90 (70%) had return of atrioventricular node function, and 38 (30%) had pacemaker placement. Of the 38 patients who underwent pacemaker placement, 6 (15.8%) had recovery of atrioventricular node function noted on long-term follow-up. Median time from onset of heart block to late atrioventricular node recovery was 13 days (Interquartile range: 5 - 117). Patients with single-ventricle physiology (p = 0.04), greater weight (p = 0.03), and shorter cardiopulmonary bypass time (p = 0.015) were more likely to have recovery. The use of post-operative steroids was similar between all groups (p = 0.445). Infectious or wound complications were similar between pacemaker groups (p = 1)., Conclusions: Two per cent of patients who underwent congenital cardiac surgery developed post-operative heart block, and 0.6% underwent pacemaker placement. Early recovery of atrioventricular node was associated with greater weight at the time of surgery, single-ventricle physiology, and shorter cardiopulmonary bypass time. Late recovery of atrioventricular node conduction following pacemaker placement occurred in 15.8% of patients.

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex., Competing Interests: Declaration of interests S.E.A. is a co-founder and CEO of Medigenome, Swiss Institute of Genomic Medicine, and serves in the Scientific Advisory Board of the “Imagine” Institute in Paris. The Department of Medical and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories. J.R.L. has stock ownership in 23andMe; is a paid consultant for Regeneron Genetics Center; and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting, and serves on the Scientific Advisory Board of Baylor Genomics., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications., (© 2022. The Author(s).)

Diabetes mellitus, caused by loss or dysfunction of the insulin-producing beta cells of the pancreas, is a promising target for recombinant adeno-associated virus (rAAV)-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell type-specific expression control element is needed. In this study, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INS × 2) and microRNA (miRNA) recognition elements (MREs). The INS × 2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2 × -MRE expression vector was highly specific to human beta cells and stem cell-derived beta cells.

Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction., Methods: A candidate gene approach was used to identify mutations as associated with resistance, or consistent with susceptibility, for 13 WHO endorsed anti-tuberculosis drugs. 38,215 MTBC isolates with paired whole-genome sequencing and phenotypic drug susceptibility testing data were amassed from 45 countries. For each mutation, a contingency table of binary phenotypes and presence or absence of the mutation computed positive predictive value, and Fisher's exact tests generated odds ratios and Benjamini-Hochberg corrected p-values. Mutations were graded as Associated with Resistance if present in at least 5 isolates, if the odds ratio was >1 with a statistically significant corrected p-value, and if the lower bound of the 95% confidence interval on the positive predictive value for phenotypic resistance was >25%. A series of expert rules were applied for final confidence grading of each mutation., Findings: 15,667 associations were computed for 13,211 unique mutations linked to one or more drugs. 1,149/15,667 (7·3%) mutations were classified as associated with phenotypic resistance and 107/15,667 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was >80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were classified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs., Interpretation: This first WHO endorsed catalogue of molecular targets for MTBC drug susceptibility testing provides a global standard for resistance interpretation. Its existence should encourage the implementation of molecular diagnostics by National Tuberculosis Programmes., Funding: UNITAID, Wellcome, MRC, BMGF., Competing Interests: Conflicts of interest C.U.K. is a consultant Becton Dickinson, the Foundation for Innovative New Diagnostics and the TB Alliance. C.U.K. is collaborating with Janssen, PZA Innovation and Thermo Fisher Scientific. C.U.K. worked as a consultant for QuantuMDx, the Stop TB Partnership, the World Health Organization (WHO) Global TB Programme and the WHO Regional Office for Europe. C.U.K. gave a paid educational talk for Oxford Immunotec. Hain Lifescience covered C.U.K.’s and accommodation to present at a meeting. C.U.K. is an unpaid advisor to BioVersys and GenoScreen. E.R. is employed by Public Health England and holds an honorary contract with Imperial College London. I.F.L. is Director of the Scottish Mycobacteria Reference Laboratory. S.N. receives funding from German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation, Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG)tion EXC 2167. P.S. is a consultant at Genoscreen. T.R. is funded by NIH and DoD and receives salary support from the non-profit organization FIND. T.R. is a co-founder, board member and shareholder of Verus Diagnostics Inc, a company that was founded with the intent of developing diagnostic assays. Verus Diagnostics was not involved in any way with data collection, analysis or publication of the results. T.R. has not received any financial support from Verus Diagnostics. UCSD Conflict of Interest office has reviewed and approved T.R.’s role in Verus Diagnostics Inc. T.R. is a co-inventor of a provisional patent for a TB diagnostic assay (provisional patent #: 63/048.989). T.R. is a co-inventor on a patent associated with the processing of TB sequencing data (European Patent Application No. 14840432.0 & USSN 14/912,918). T.R. has agreed to “donate all present and future interest in and rights to royalties from this patent” to UCSD to ensure that he does not receive any financial benefits from this patent. S.S. is working and holding ESOPs at HaystackAnalytics Pvt. Ltd. (Product: Using whole genome sequencing for drug susceptibility testing for Mycobacterium tuberculosis). G.F.G. is listed as an inventor on patent applications for RBD-dimer-based CoV vaccines. The patents for RBD-dimers as protein subunit vaccines for SARS-CoV-2 have been licensed to Anhui Zhifei Longcom Biopharmaceutical Co. Ltd, China. No other authors declare a conflict of interest. I.C. is a consultant for the Foundation for Innovative New Diagnostics. DAC reports funding from GlaxoSmithKline and consultancy fees from Biobeats, Oxford University Innovation, Sensyne Health.C.C. reports funding from FIND to his institution (Pathology Queensland, Queensland Department of Health) for his laboratory to perform molecular analytic studies (limits of detection) for new molecular platforms manufactured by Cepheid and Bioneer.

Tuberculosis (TB) control programs use whole-genome sequencing (WGS) of Mycobacterium tuberculosis ( Mtb ) for detecting and investigating TB case clusters. Existence of few genomic differences between Mtb isolates might indicate TB cases are the result of recent transmission. However, the variable and sometimes long duration of latent infection, combined with uncertainty in the Mtb mutation rate during latency, can complicate interpretation of WGS results. To estimate the association between infection duration and single nucleotide polymorphism (SNP) accumulation in the Mtb genome, we first analyzed pairwise SNP differences among TB cases from Los Angeles County, California, with strong epidemiologic links. We found that SNP distance alone was insufficient for concluding that cases are linked through recent transmission. Second, we describe a well-characterized cluster of TB cases in California to illustrate the role of genomic data in conclusions regarding recent transmission. Longer presumed latent periods were inconsistently associated with larger SNP differences. Our analyses suggest that WGS alone cannot be used to definitively determine that a case is attributable to recent transmission. Methods for integrating clinical, epidemiologic, and genomic data can guide conclusions regarding the likelihood of recent transmission, providing local public health practitioners with better tools for monitoring and investigating TB transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict ofinterest., (Copyright © 2022 Nelson, Talarico, Poonja, McDaniel, Cilnis, Chang, Raz, Noboa, Cowan, Shaw, Posey and Silk.)

Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site., Competing Interests: Declaration of interests M.G. has a financial interest in Yecuris Corp. (Tigard, Oregon), a company that has commercialized the humanized mouse liver model used in this work. MG also is a consultant for LogicBio Therapeutics. Other authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Pyrazinamide is a potent sterilising agent that shortens the treatment duration needed to cure tuberculosis. It is synergistic with novel and existing drugs for tuberculosis. The dose of pyrazinamide that optimises efficacy while remaining safe is uncertain, as is its potential role in shortening treatment duration further.Pharmacokinetic data, sputum culture, and safety laboratory results were compiled from Tuberculosis Trials Consortium (TBTC) studies 27 and 28 and Pan-African Consortium for the Evaluation of Antituberculosis Antibiotics (PanACEA) multi-arm multi-stage tuberculosis (MAMS-TB), multi-centre phase 2 trials in which participants received rifampicin (range 10-35 mg·kg -1 ), pyrazinamide (range 20-30 mg·kg -1 ), plus two companion drugs. Pyrazinamide pharmacokinetic-pharmacodynamic (PK-PD) and pharmacokinetic-toxicity analyses were performed.In TBTC studies (n=77), higher pyrazinamide maximum concentration (C max ) was associated with shorter time to culture conversion (TTCC) and higher probability of 2-month culture conversion (p-value<0.001). Parametric survival analyses showed that relationships varied geographically, with steeper PK-PD relationships seen among non-African than African participants. In PanACEA MAMS-TB (n=363), TTCC decreased as pyrazinamide C max increased and varied by rifampicin area under the curve (p-value<0.01). Modelling and simulation suggested that very high doses of pyrazinamide (>4500 mg) or increasing both pyrazinamide and rifampicin would be required to reach targets associated with treatment shortening. Combining all trials, liver toxicity was rare (3.9% with grade 3 or higher liver function tests (LFT)), and no relationship was seen between pyrazinamide C max and LFT levels.Pyrazinamide's microbiological efficacy increases with increasing drug concentrations. Optimising pyrazinamide alone, though, is unlikely to be sufficient to allow tuberculosis treatment shortening; rather, rifampicin dose would need to be increased in parallel., Competing Interests: Conflict of interest: N. Zhang has nothing to disclose. Conflict of interest: R.M. Savic has nothing to disclose. Conflict of interest: M.J. Boeree has nothing to disclose. Conflict of interest: C.A. Peloquin has nothing to disclose. Conflict of interest: M. Weiner has nothing to disclose. Conflict of interest: N. Heinrich reports grants from EDCTP (EU), during the conduct of the study; personal fees for lectures from AstraZeneca, outside the submitted work. Conflict of interest: E. Bliven-Sizemore has nothing to disclose. Conflict of interest: P.P.J. Phillips reports grants from EDCTP, during the conduct of the study. Conflict of interest: M. Hoelscher has nothing to disclose. Conflict of interest: W. Whitworth has nothing to disclose. Conflict of interest: G. Morlock has nothing to disclose. Conflict of interest: J. Posey has nothing to disclose. Conflict of interest: J.E. Stout has nothing to disclose. Conflict of interest: W. Mac Kenzie has nothing to disclose. Conflict of interest: R. Aarnoutse has nothing to disclose. Conflict of interest: K.E. Dooley has nothing to disclose., (The content of this work is not subject to copyright. Design and branding are copyright ©ERS 2021. For commercial reproduction rights and permissions contact permissions@ersnet.org.)

Understanding tuberculosis (TB) transmission chains can help public health staff target their resources to prevent further transmission, but currently there are few tools to automate this process. We have developed the Logically Inferred Tuberculosis Transmission (LITT) algorithm to systematize the integration and analysis of whole-genome sequencing, clinical, and epidemiological data. Based on the work typically performed by hand during a cluster investigation, LITT identifies and ranks potential source cases for each case in a TB cluster. We evaluated LITT using a diverse dataset of 534 cases in 56 clusters (size range: 2-69 cases), which were investigated locally in three different U.S. jurisdictions. Investigators and LITT agreed on the most likely source case for 145 (80%) of 181 cases. By reviewing discrepancies, we found that many of the remaining differences resulted from errors in the dataset used for the LITT algorithm. In addition, we developed a graphical user interface, user's manual, and training resources to improve LITT accessibility for frontline staff. While LITT cannot replace thorough field investigation, the algorithm can help investigators systematically analyze and interpret complex data over the course of a TB cluster investigation. Code available at: https://github.com/CDCgov/TB&#95;molecular&#95;epidemiology/tree/1.0; https://zenodo.org/badge/latestdoi/166261171., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Winglee, McDaniel, Linde, Kammerer, Cilnis, Raz, Noboa, Knorr, Cowan, Reynolds, Posey, Sullivan Meissner, Poonja, Shaw, Talarico and Silk.)

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a Cypor shRNA were administered to neonatal mice. Hepatocytes lacking the essential cofactor of Cyp enzymes, NADPH-cytochrome p450 reductase (Cypor), were selected in vivo by acetaminophen administration, replacing up to 50% of the hepatic mass. Acetaminophen treatment of the mice resulted in over 30-fold expansion of transgene-bearing hepatocytes and achieved therapeutic thresholds in hemophilia B and phenylketonuria. We conclude that therapeutically modified hepatocytes can be selected safely and efficiently in preclinical models with a transient regimen of moderately hepatotoxic acetaminophen., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: To compare overall survival (OS) in patients who underwent surgery for early-stage pancreatic adenocarcinoma (rPca) based on sequence (NAT, neoadjuvant therapy and/or AT, adjuvant therapy) and type (SA, single-agent or MA, multi-agent) of chemotherapy received., Methods: Using the National Cancer Database, patients with clinical stage I/II rPca diagnosed between 2010 and 2014 were identified and five comparison matches (1: NAT vs. upfront resection (UR); 2: multi-agent neoadjuvant (MA NAT) vs. single-agent adjuvant therapy (SA AT), single-agent neoadjuvant therapy (SA NAT), multi-agent adjuvant therapy (MA AT); 3: MA NAT vs. MA AT; 4: NAT + AT vs NAT; 5: NAT + AT vs AT) were constructed using minimum distance matching strategy. Median OS (mOS) was analysed using Kaplan-Meier method, log-rank test and Cox proportional hazard model., Results: A total of 18,470 patients with stage I/II rPca were eligible for analysis. NAT showed a 5 month (mo.) improved OS compared with UR (3271 patients/group, 28.1 vs 23.2 mo. P < 0.0001 hazard ratio [HR]: 0.79). MA-NAT was shown to be superior to other chemotherapy approaches SA AT, SA NAT, and MA AT (1349 patients/group: 30 vs. 25.9 mo., P = 0.0001 [HR: 0.82]). MA NAT showed a survival advantage over MA-AT (1349 patients/group, 30 vs 26.1 mo., P = 0.0008 [HR: 0.86]). The combination of NAT and AT showed a better outcome when compared with NAT alone (1128 patients/group, 31.6 vs 27.4 mo., P = 0.0011 [HR: 0.81]) or AT alone (1128 patients/group, 31.6 vs. 25.2 mo., P < 0.0001 [HR: 0.76])., Conclusions: In patients with stage I/II rPca, MA NAT showed improved mOS compared to UR and all other chemotherapy sequences except both NAT plus AT. These findings support the use of MA NAT in stage I/II rPca patients and warrant prospective trials evaluating MA NAT and post-resection maintenance therapies., Competing Interests: Conflict of interest statement None., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Treatment guidelines for stage I-III esophageal cancer indicate that management should include surgery in appropriate patients. Variations in utilization of surgery may contribute to racial differences observed in survival. We sought to identify factors associated with racial disparities in surgical resection of esophageal cancer and evaluate associated survival differences., Methods: Patients diagnosed with stage I-III esophageal cancer from 2004 to 2015 were identified using the National Cancer Database. Matched patient cohorts were created to reduce confounding. Multivariate logistic regression was used to identify factors associated with receipt of surgery. Multi-level modeling was performed to control for random effects of individual hospitals on surgical utilization., Results: A total of 60,041 patients were included (4402 black; 55,639 white). After 1:1 matching, there were 5858 patients evenly distributed across race. For all stages, significantly fewer black than white patients received surgery. Black race independently conferred lower likelihood of receiving surgery in single-level multivariable analysis (OR (95% CI); stage I, 0.67 (0.48-0.94); stage II, 0.76 (0.60-0.96); stage III, 0.62 (0.50-0.76)) and after controlling for hospital random effects. Hospital-level random effects accounted for one third of the unexplained variance in receipt of surgery. Risk-adjusted 1-, 3-, and 5-year mortality was higher for patients who did not undergo surgery., Conclusion: Black patients with esophageal cancer are at higher risk of mortality compared to white patients. This increased risk may be influenced by decreased likelihood of receiving surgical intervention for resectable disease, in part because of between-hospital differences. Improving access to surgical care may improve disparities in esophageal cancer survival.

Inflammation is central to the pathogenesis of pulmonary vascular remodeling and pulmonary hypertension (PH). Inflammation precedes remodeling in preclinical models, thus supporting the concept that changes in immunity drive remodeling in PH. Platelets are recognized as mediators of inflammation, but whether platelets contribute to hypoxia-driven inflammation has not been studied. We utilized a murine hypoxia model to test the hypothesis that platelets drive hypoxia-induced inflammation. We evaluated male and female 9-wk-old normoxic and hypoxic mice and in selected experiments included hypoxic thrombocytopenic mice. Thrombocytopenic mice were generated with an anti-GP1bα rat IgG antibody. We also performed immunostaining of lung sections from failed donor controls and patients with idiopathic pulmonary arterial hypertension. We found that platelets are increased in the lungs of hypoxic mice and hypoxia induces platelet activation. Platelet depletion prevents hypoxia-driven increases in the proinflammatory chemokines CXCL4 and CCL5 and attenuates hypoxia-induced increase in plasma CSF-2. Pulmonary interstitial macrophages are increased in the lungs of hypoxic mice; this increase is prevented in thrombocytopenic mice. To determine the potential relevance to human disease, lung sections from donors and patients with advanced idiopathic pulmonary arterial hypertension (iPAH) were immunostained for the platelet-specific protein CD41. We observed iPAH lungs had a two-fold increase in CD41, compared with controls. Our data provide evidence that the platelet count is increased in the lungs and activated in mice with hypoxia-induced inflammation and provides rationale for the further study of the potential contribution of platelets to inflammatory mediated vascular remodeling and PH.

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: There is a lack of precision medicine in pancreatic ductal adenocarcinoma (PDA) and related cancers, and outcomes for patients with this diagnosis remain poor despite decades of research investigating this disease. Therefore, it is necessary to explore novel therapeutic options for these patients who may benefit from personalized therapies., Objective: Molecular profiling of hepatopancreaticobiliary malignancies at our institution, including but not limited to PDA, was initiated to assess the feasibility of incorporating molecular profiling results into patient oncological therapy planning., Methods: All eligible patients from Thomas Jefferson University (TJU) with hepatopancreaticobiliary tumors including PDA, who agreed to molecular testing profiling, were prospectively enrolled in a registry study from December 2014 to September 2017 and their tumor samples were tested to identify molecular markers that can be used to guide therapy options in the future. Next generation sequencing (NGS) and protein expression in tumor samples were tested at CLIA-certified laboratories. Prospective clinicopathologic data were extracted from medical records and compiled in a de-identified fashion., Results: Seventy eight (78) patients were enrolled in the study, which included 65/78 patients with PDA (local and metastatic) and out of that subset, 52/65 patients had surgically resected PDA. Therapy recommendations were generated based on molecular and clinicopathologic data for all enrolled patients. NGS uncovered actionable alterations in 25/52 surgically resected PDAs (48%) which could be used to guide therapy options in the future. High expression of three proteins, TS (p = 0.005), ERCC1 (p = 0.001), and PD-1 (p = 0.04), was associated with reduced recurrence-free survival (RFS), while TP53 mutations were correlated with longer RFS (p = 0.01)., Conclusions: The goal of this study was to implement a stepwise strategy to identify and profile resected PDAs at our institution. Consistent with previous studies, approximately half of patients with resected PDA harbor actionable mutations with possible targeted therapeutic implications. Ongoing studies will determine the clinical value of identifying these mutations in patients with resected PDA., Competing Interests: Declaration of competing interest Joseph Bender, PhD, Talar Tatarian, MD, Subha Madhavan, PhD, Emanuel F Petricoin 3rd, PhD, Michael J Pishvaian, MD, PhD, and Jonathan R Brody, PhD have affiliations with Perthera, Inc., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)