New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

Variation among strains of Borrelia burgdorferi in host tissue abundance and lifetime transmission determine the population strain structure in nature.

Pathogen life history theory assumes a positive relationship between pathogen load in host tissues and pathogen transmission. Empirical evidence for this relationship is surprisingly rare due to the difficulty of measuring transmission for many pathogens. The comparative method, where a common host is experimentally infected with a set of pathogen strains, is a powerful approach for investigating the relationships between pathogen load and transmission. The validity of such experimental estimates of strain-specific transmission is greatly enhanced if they can predict the pathogen population strain structure in nature. Borrelia burgdorferi is a multi-strain, tick-borne spirochete that causes Lyme disease in North America. This study used 11 field-collected strains of B. burgdorferi, a rodent host (Mus musculus, C3H/HeJ) and its tick vector (Ixodes scapularis) to determine the relationship between pathogen load in host tissues and lifetime host-to-tick transmission (HTT). Mice were experimentally infected via tick bite with 1 of 11 strains. Lifetime HTT was measured by infesting mice with I. scapularis larval ticks on 3 separate occasions. The prevalence and abundance of the strains in the mouse tissues and the ticks were determined by qPCR. We used published databases to obtain estimates of the frequencies of these strains in wild I. scapularis tick populations. Spirochete loads in ticks and lifetime HTT varied significantly among the 11 strains of B. burgdorferi. Strains with higher spirochete loads in the host tissues were more likely to infect feeding larval ticks, which molted into nymphal ticks that had a higher probability of B. burgdorferi infection (i.e., higher HTT). Our laboratory-based estimates of lifetime HTT were predictive of the frequencies of these strains in wild I. scapularis populations. For B. burgdorferi, the strains that establish high abundance in host tissues and that have high lifetime transmission are the strains that are most common in nature.

Transforming the Future of Surgeon-Scientists.

OBJECTIVE: To create a blueprint for surgical department leaders, academic institutions, and funding agencies to optimally support surgeon-scientists. BACKGROUND: Scientific contributions by surgeons have been transformative across many medical disciplines. Surgeon-scientists provide a distinct approach and mindset toward key scientific questions. However, lack of institutional support, pressure for increased clinical productivity, and growing administrative burden are major challenges for the surgeon-scientist, as is the time-consuming nature of surgical training and practice. METHODS: An American Surgical Association Research Sustainability Task Force was created to outline a blueprint for sustainable science in surgery. Leaders from top NIH-sponsored departments of surgery engaged in video and in-person meetings between January and April 2023. A strength, weakness, opportunities, threats analysis was performed, and workgroups focused on the roles of surgeons, the department and institutions, and funding agencies. RESULTS: Taskforce recommendations: (1) SURGEONS: Growth mindset : identifying research focus, long-term planning, patience/tenacity, team science, collaborations with disparate experts; Skill set : align skills and research, fill critical skill gaps, develop team leadership skills; DEPARTMENT OF SURGERY (DOS): (2) MENTORSHIP: Chair : mentor-mentee matching/regular meetings/accountability, review of junior faculty progress, mentorship training requirement, recognition of mentorship (eg, relative value unit equivalent, awards; Mentor: dedicated time, relevant scientific expertise, extramural funding, experience and/or trained as mentor, trusted advisor; Mentee : enthusiastic/eager, proactive, open to feedback, clear about goals; (3) FINANCIAL SUSTAINABILITY: diversification of research portfolio, identification of matching funding sources, departmental resource awards (eg, T-/P-grants), leveraging of institutional resources, negotiation of formalized/formulaic funds flow investment from academic medical center toward science, philanthropy; (4) STRUCTURAL/STRATEGIC SUPPORT: Structural: grants administrative support, biostats/bioinformatics support, clinical trial and research support, regulatory support, shared departmental laboratory space/equipment; Strategic: hiring diverse surgeon-scientist/scientists faculty across DOS, strategic faculty retention/ recruitment, philanthropy, career development support, progress tracking, grant writing support, DOS-wide research meetings, regular DOS strategic research planning; (5) COMMUNITY AND CULTURE: Community: right mix of faculty, connection surgeon with broad scientific community; Culture: building research infrastructure, financial support for research, projecting importance of research (awards, grand rounds, shoutouts); (6) THE ROLE OF INSTITUTIONS: Foundation: research space co-location, flexible start-up packages, courses/mock study section, awards, diverse institutional mentorship teams; Nurture: institutional infrastructure, funding (eg, endowed chairs), promotion friendly toward surgeon-scientists, surgeon-scientists in institutional leadership positions; Expectations: RVU target relief, salary gap funding, competitive starting salaries, longitudinal salary strategy; (7) THE ROLE OF FUNDING AGENCIES: change surgeon research training paradigm, offer alternate awards to K-awards, increasing salary cap to reflect market reality, time extension for surgeon early-stage investigator status, surgeon representation on study section, focused award strategies for professional societies/foundations. CONCLUSIONS: Authentic recommitment from surgeon leaders with intentional and ambitious actions from institutions, corporations, funders, and society is essential in order to reap the essential benefits of surgeon-scientists toward advancements of science.

Precursor Epithelial Subtypes of Adenocarcinoma Arising from Intraductal Papillary Mucinous Neoplasms (A-IPMN): Clinicopathological Features, Recurrence and Response to Adjuvant Chemotherapy.

BACKGROUND: The clinico-oncological outcomes of precursor epithelial subtypes of adenocarcinoma arising from intraductal papillary mucinous neoplasms (A-IPMN) are limited to small cohort studies. Differences in recurrence patterns and response to adjuvant chemotherapy between A-IPMN subtypes are unknown. METHODS: Clincopathological features, recurrence patterns and long-term outcomes of patients undergoing pancreatic resection (2010-2020) for A-IPMN were reported from 18 academic pancreatic centres worldwide. Precursor epithelial subtype groups were compared using uni- and multivariate analysis. RESULTS: In total, 297 patients were included (median age, 70 years; male, 78.9%), including 54 (18.2%) gastric, 111 (37.3%) pancreatobiliary, 80 (26.9%) intestinal and 52 (17.5%) mixed subtypes. Gastric, pancreaticobiliary and mixed subtypes had comparable clinicopathological features, yet the outcomes were significantly less favourable than the intestinal subtype. The median time to recurrence in gastric, pancreatobiliary, intestinal and mixed subtypes were 32, 30, 61 and 33 months. Gastric and pancreatobiliary subtypes had worse overall recurrence (p = 0.048 and p = 0.049, respectively) compared with the intestinal subtype but gastric and pancreatobiliary subtypes had comparable outcomes. Adjuvant chemotherapy was associated with improved survival in the pancreatobiliary subtype (p = 0.049) but not gastric (p = 0.992), intestinal (p = 0.852) or mixed subtypes (p = 0.723). In multivariate survival analysis, adjuvant chemotherapy was associated with a lower likelihood of death in pancreatobiliary subtype, albeit with borderline significance [hazard ratio (HR) 0.56; 95% confidence interval (CI) 0.31-1.01; p = 0.058]. CONCLUSIONS: Gastric, pancreatobiliary and mixed subtypes have comparable recurrence and survival outcomes, which are inferior to the more indolent intestinal subtype. Pancreatobiliary subtype may respond to adjuvant chemotherapy and further research is warranted to determine the most appropriate adjuvant chemotherapy regimens for each subtype.

Adjuvant chemotherapy for adenocarcinoma arising from intraductal papillary mucinous neoplasia: multicentre ADENO-IPMN study.

BACKGROUND: The clinical impact of adjuvant chemotherapy after resection for adenocarcinoma arising from intraductal papillary mucinous neoplasia is unclear. The aim of this study was to identify factors related to receipt of adjuvant chemotherapy and its impact on recurrence and survival. METHODS: This was a multicentre retrospective study of patients undergoing pancreatic resection for adenocarcinoma arising from intraductal papillary mucinous neoplasia between January 2010 and December 2020 at 18 centres. Recurrence and survival outcomes for patients who did and did not receive adjuvant chemotherapy were compared using propensity score matching. RESULTS: Of 459 patients who underwent pancreatic resection, 275 (59.9%) received adjuvant chemotherapy (gemcitabine 51.3%, gemcitabine-capecitabine 21.8%, FOLFIRINOX 8.0%, other 18.9%). Median follow-up was 78 months. The overall recurrence rate was 45.5% and the median time to recurrence was 33 months. In univariable analysis in the matched cohort, adjuvant chemotherapy was not associated with reduced overall (P = 0.713), locoregional (P = 0.283) or systemic (P = 0.592) recurrence, disease-free survival (P = 0.284) or overall survival (P = 0.455). Adjuvant chemotherapy was not associated with reduced site-specific recurrence. In multivariable analysis, there was no association between adjuvant chemotherapy and overall recurrence (HR 0.89, 95% c.i. 0.57 to 1.40), disease-free survival (HR 0.86, 0.59 to 1.30) or overall survival (HR 0.77, 0.50 to 1.20). Adjuvant chemotherapy was not associated with reduced recurrence in any high-risk subgroup (for example, lymph node-positive, higher AJCC stage, poor differentiation). No particular chemotherapy regimen resulted in superior outcomes. CONCLUSION: Chemotherapy following resection of adenocarcinoma arising from intraductal papillary mucinous neoplasia does not appear to influence recurrence rates, recurrence patterns or survival.

Between-hospital care referrals for severe alcohol-related liver disease during the COVID-19 pandemic, 2020 to 2022.

Increased alcohol consumption during the coronavirus disease 2019 pandemic is projected to impact alcohol-related liver disease (ALD) morbidity and mortality. Inter-hospital escalation-of-care referral requests to our tertiary-care hepatology unit were analyzed from January 2020 through December 2022. Most requests to our center were for ALD with an increase in requests from intermediate care units, suggestive of higher acuity illness.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element.

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

Evidence for molecular subtyping in pancreatic ductal adenocarcinoma: a systematic review.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit varied responses to multimodal therapy. RNA gene sequencing has unravelled distinct tumour biology subtypes, forming the focus of this review exploring its impact on survival outcomes. METHODS: A systematic search across PubMed, Medline, Embase, and CINAHL databases targeted studies assessing long-term overall and disease-free survival in PDAC patients with molecular subtyping. RESULTS: Fifteen studies including 2731 patients were identified. Molecular subtyping was performed by RNA sequencing and Immunohistochemistry in 14 studies and by Mass Spectrometry in 1 study. Two main tumour subtypes were identified (classical and basal-like or squamous) with basal like associated with poorer outcomes. Further subtypes were identified in individual studies. Superior survival was seen with classical subtype in all other analyses that compared the classical and basal subtypes. High risk stromal subtypes were identified on further analysis of the stroma and were associated with a worse survival independent of the tumour subtype. CONCLUSION: Molecular subtyping of PDAC specimens can identify patients with high-risk tumour biology and poor survival outcomes. Routine subtyping is limited by the cost of RNA sequencing and the volume of raw data generated which has made its translation into routine clinical practice difficult.

Risk of Recurrence after Surgical Resection for Adenocarcinoma Arising from Intraductal Papillary Mucinous Neoplasia (IPMN) with Patterns of Distribution and Treatment: An International, Multicentre, Observational Study.

OBJECTIVE: This international multicentre cohort study aims to identify recurrence patterns and treatment of first and second recurrence in a large cohort of patients after pancreatic resection for adenocarcinoma arising from IPMN. SUMMARY BACKGROUND DATA: Recurrence patterns and treatment of recurrence post resection of adenocarcinoma arising from IPMN are poorly explored. METHOD: Patients undergoing pancreatic resection for adenocarcinoma from IPMN between January 2010 to December 2020 at 18 pancreatic centres were identified. Survival analysis was performed by the Kaplan-Meier log rank test and multivariable logistic regression by Cox-Proportional Hazards modelling. Endpoints were recurrence (time-to, location, and pattern of recurrence) and survival (overall survival and adjusted for treatment provided). RESULTS: Four hundred and fifty-nine patients were included (median, 70 y; IQR, 64-76; male, 54 percent) with a median follow-up of 26.3 months (IQR, 13.0-48.1 mo). Recurrence occurred in 209 patients (45.5 percent; median time to recurrence, 32.8 months, early recurrence [within 1 y], 23.2 percent). Eighty-three (18.1 percent) patients experienced a local regional recurrence and 164 (35.7 percent) patients experienced distant recurrence. Adjuvant chemotherapy was not associated with reduction in recurrence (HR 1.09;P=0.669) One hundred and twenty patients with recurrence received further treatment. The median survival with and without additional treatment was 27.0 and 14.6 months (P<0.001), with no significant difference between treatment modalities. There was no significant difference in survival between location of recurrence (P=0.401). CONCLUSION: Recurrence after pancreatic resection for adenocarcinoma arising from IPMN is frequent with a quarter of patients recurring within 12 months. Treatment of recurrence is associated with improved overall survival and should be considered.

Species Delineation and Comparative Genomics within the Campylobacter ureolyticus Complex.

Campylobacter ureolyticus is an emerging pathogen increasingly appreciated as a common cause of gastroenteritis and extra-intestinal infections in humans. Outside the setting of gastroenteritis, little work has been done to describe the genomic content and relatedness of the species, especially regarding clinical isolates. We reviewed the epidemiology of clinical C. ureolyticus cultured by our institution over the past 10 years. Fifty-one unique C. ureolyticus isolates were identified between January 2010 and August 2022, mostly originating from abscesses and blood cultures. To clarify the taxonomic relationships between isolates and to attribute specific genes with different clinical manifestations, we sequenced 19 available isolates from a variety of clinical specimen types and conducted a pangenomic analysis with publicly available C. ureolyticus genomes. Digital DNA:DNA hybridization suggested that these C. ureolyticus comprised a species complex of 10 species clusters (SCs) and several subspecies clusters. Although some orthologous genes or gene functions were enriched in isolates found in different SCs and clinical specimens, no association was significant. Nearly a third of the isolates possessed antimicrobial resistance genes, including the ermA resistance gene, potentially conferring resistance to macrolides, the treatment of choice for severe human campylobacteriosis. This work effectively doubles the number of publicly available C. ureolyticus genomes, provides further clarification of taxonomic relationships within this bacterial complex, and identifies target SCs for future analysis.

Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55).

Macrolides are a mainstay of therapy for infections due to nontuberculous mycobacteria (NTM). Among rapidly growing mycobacteria (RGM), inducible macrolide resistance is associated with four chromosomal 23S rRNA methylase (erm) genes. Beginning in 2018, we detected high-level inducible clarithromycin resistance (MICs of ≥16µg/mL) in clinical isolates of Mycobacterium chelonae, an RGM species not previously known to contain erm genes. Using whole-genome sequencing, we identified a novel plasmid-mediated erm gene. This gene, designated erm(55)P, exhibits <65% amino acid identity to previously described RGM erm genes. Two additional chromosomal erm(55) alleles, with sequence identities of 81% to 86% to erm(55)P, were also identified and designated erm(55)C and erm(55)T. The erm(55)T is part of a transposon. The erm(55)P allele variant is located on a putative 137-kb conjugative plasmid, pMchErm55. Evaluation of 133 consecutive isolates from 2020 to 2022 revealed 5 (3.8%) with erm(55). The erm(55)P gene was also identified in public data sets of two emerging pathogenic pigmented RGM species: Mycobacterium iranicum and Mycobacterium obuense, dating back to 2008. In both species, the gene appeared to be present on plasmids homologous to pMchErm55. Plasmid-mediated macrolide resistance, not described previously for any NTM species, appears to have spread to multiple RGM species. This has important implications for antimicrobial susceptibility guidelines and treatment of RGM infections. Further spread could present serious consequences for treatment of other macrolide-susceptible RGM. Additional studies are needed to determine the transmissibility of pMchErm55 and the distribution of erm(55) among other RGM species.

Patient reflections, challenges, and supports related to early liver transplant for severe alcohol-associated liver disease.

Transplant centers conventionally require at least 6 months of alcohol abstinence before offering liver transplants for alcohol-associated liver disease. However, early liver transplant (ELT)-proceeding with a transplant when clinically necessary without first meeting the conventional requirement-is increasingly gaining attention. In our study, we qualitatively assessed ELT recipients' perceived challenges and supports regarding alcohol-associated liver disease, transplant, and posttransplant survivorship. To diversify perspectives based on gender, race/ethnicity, age, time since ELT, and pretransplant abstinence duration, we purposively recruited ELT recipients and conducted semistructured interviews. Recruitment continued until data saturation. We analyzed transcripts using inductive thematic analysis. We interviewed 20 ELT recipients between June and December 2020 and identified themes within 3 participant-characterized time periods. Three themes emerged in life before severe illness: (1) alcohol as a "constant" part of life, (2) alcohol use negatively affecting relationships and work life, and (3) feeling "stuck" in the cycle of drinking. Two themes emerged during the severe illness period: (4) rapidity of health decline and (5) navigating medical care and the 6-month abstinence requirement. Finally, in life after transplant, 4 themes emerged: (6) feelings of shame or stigma and new self-worth, (7) reconnecting with others and redefining boundaries, (8) transplant as a defining point for sobriety, and (9) work-related challenges. Overall, participants expressed gratitude for receiving a gift of life and acknowledged their responsibilities to the new liver. ELT recipient experiences reveal complex psychosocial challenges related to addiction, inadequate support system, and stigma, particularly in the posttransplant period. The care of ELT recipients would be incomplete if focused solely on optimizing patient or graft survival.

Critical interactions between race and the highly granular area deprivation index in liver transplant evaluation.

Neighborhood socioeconomic deprivation may have important implications on disparities in liver transplant (LT) evaluation. In this retrospective cohort study, we constructed a novel dataset by linking individual patient-level data with the highly granular Area Deprivation Index (ADI), which is advantageous over other neighborhood measures due to: specificity of Census Block-Group (versus Census Tract, Zip code), scoring, and robust variables. Our cohort included 1377 adults referred to our center for LT evaluation 8/1/2016-12/31/2019. Using modified Poisson regression, we tested for effect measure modification of the association between neighborhood socioeconomic status (nSES) and LT evaluation outcomes (listing, initiating evaluation, and death) by race and ethnicity. Compared to patients with high nSES, those with low nSES were at higher risk of not being listed (aRR = 1.14; 95%CI 1.05-1.22; p < .001), of not initiating evaluation post-referral (aRR = 1.20; 95%CI 1.01-1.42; p = .03) and of dying without initiating evaluation (aRR = 1.55; 95%CI 1.09-2.2; p = .01). While White patients with low nSES had similar rates of listing compared to White patients with high nSES (aRR = 1.06; 95%CI .96-1.17; p = .25), Underrepresented patients from neighborhoods with low nSES incurred 31% higher risk of not being listed compared to Underrepresented patients from neighborhoods with high nSES (aRR = 1.31; 95%CI 1.12-1.5; p < .001). Interventions addressing neighborhood deprivation may not only benefit patients with low nSES but may address racial and ethnic inequities.

Clinical Outcomes and Treatment Strategies in Patients With Non-Carbapenemase-producing Carbapenem-Resistant Versus Carbapenem-Susceptible Enterobacterales Infections.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are difficult to treat and can cause significant morbidity and mortality, however most data reflect carbapenemase-producing infections. OBJECTIVE: Our objective was to evaluate clinical outcomes of non-carbapenemase-producing CRE (nCP-CRE) compared with carbapenem-susceptible Enterobacterales (CSE) infections. METHODS: This was a retrospective, multicenter, observational study (January 1, 2018 to December 31, 2020). The primary outcome was clinical success at 30 days with secondary outcomes, including clinical success at 90 days, clinical success based on treatment for nCP-CRE, persistent bacteremia, intensive care unit (ICU) admission, length of stay, and rate of Clostridioides difficile or multidrug resistant infections. RESULTS: The final analysis included 211 patients: 142 (67%) with CSE and 69 (33%) with nCP-CRE infections. Prior carbapenem exposure was more common with nCP-CRE (15% vs 4%, P = 0.01). Clinical success at 30 days was similar between groups (77% vs 74%, P = 0.73). There were no differences in secondary outcomes. There was an overall low use of carbapenems (empiric 6%, definitive 7%). Most nCP-CRE infections were treated with a monotherapy carbapenem-sparing regimen (empiric 88%, definitive 90%). Limitations include the retrospective design and the high rate of urinary infections. CONCLUSION AND RELEVANCE: Our study found no difference in clinical outcomes between nCP-CRE and CSE infections. Application of this study with future studies would help in determining optimal regimens for these infections.

Perturbations in Respiratory Syncytial Virus Activity During the SARS-CoV-2 Pandemic.

Respiratory syncytial virus (RSV) is a common cause of respiratory disease in all age groups, with young children and older adults experiencing the most severe illness. The coronavirus disease 2019 (COVID-19) pandemic resulted in striking changes in the activity of seasonal respiratory viruses, including RSV. After a period of suppression early in the pandemic, an interseasonal surge of RSV occurred in 2021. Viral activity was detected primarily in children and young adults after relaxation of public health measures, but without the usual proportional increases in infections and hospitalizations in older adults who were likely still adhering to stricter public health measures.

IgG Against Human Betacoronavirus Spike Proteins Correlates With SARS-CoV-2 Anti-Spike IgG Responses and COVID-19 Disease Severity.

BACKGROUND: A protective antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to decrease morbidity and mortality from severe coronavirus disease 2019 (COVID-19) disease. The effects of preexisting anti-human coronavirus (HCoV) antibodies on the SARS-CoV-2-specific immunoglobulin G (IgG) responses and severity of disease are currently unclear. METHODS: We profiled anti-spike (S), S1, S2, and receptor-binding domain IgG antibodies against SARS-CoV-2 and 6 HCoVs using a multiplex assay (mPLEX-CoV) with serum samples from SARS-CoV-2 infected (n = 155) and pre-COVID-19 (n = 188) cohorts. RESULTS: COVID-19 subjects showed significantly increased anti-S SARS-CoV-2 IgG levels that were highly correlated with IgG antibodies against OC43 and HKU1 S proteins. However, OC43 and HKU1 anti-S antibodies in pre-COVID-19 era sera did not cross-react with SARS-CoV-2. Unidirectional cross-reactive antibodies elicited by SARS-CoV-2 infection were distinct from the bidirectional cross-reactive antibodies recognizing homologous strains RaTG13 and SARS-CoV-1. High anti-OC43 and anti-S2 antibody levels were associated with both a rapid anti-SARS-CoV-2 antibody response and increased disease severity. Subjects with increased sequential organ failure assessment (SOFA) scores developed a higher ratio of S2- to S1-reactive antibodies. CONCLUSIONS: Early and rapid emergence of OC43 S- and S2-reactive IgG after SARS-CoV-2 infection correlates with COVID-19 disease severity.

HOPE in action: A prospective multicenter pilot study of liver transplantation from donors with HIV to recipients with HIV.

Liver transplantation (LT) from donors-with-HIV to recipients-with-HIV (HIV D+/R+) is permitted under the HOPE Act. There are only three international single-case reports of HIV D+/R+ LT, each with limited follow-up. We performed a prospective multicenter pilot study comparing HIV D+/R+ to donors-without-HIV to recipients-with-HIV (HIV D-/R+) LT. We quantified patient survival, graft survival, rejection, serious adverse events (SAEs), human immunodeficiency virus (HIV) breakthrough, infections, and malignancies, using Cox and negative binomial regression with inverse probability of treatment weighting. Between March 2016-July 2019, there were 45 LTs (8 simultaneous liver-kidney) at 9 centers: 24 HIV D+/R+, 21 HIV D-/R+ (10 D- were false-positive). The median follow-up time was 23 months. Median recipient CD4 was 287 cells/µL with 100% on antiretroviral therapy; 56% were hepatitis C virus (HCV)-seropositive, 13% HCV-viremic. Weighted 1-year survival was 83.3% versus 100.0% in D+ versus D- groups (p = .04). There were no differences in one-year graft survival (96.0% vs. 100.0%), rejection (10.8% vs. 18.2%), HIV breakthrough (8% vs. 10%), or SAEs (all p > .05). HIV D+/R+ had more opportunistic infections, infectious hospitalizations, and cancer. In this multicenter pilot study of HIV D+/R+ LT, patient and graft survival were better than historical cohorts, however, a potential increase in infections and cancer merits further investigation.

Integrated Multiomics Reveals Glucose Use Reprogramming and Identifies a Novel Hexokinase in Alcoholic Hepatitis.

BACKGROUND & AIMS: We recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism. METHODS: We performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells. RESULTS: Patients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes. CONCLUSIONS: Altered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.

Survival Benefit of Split-Liver Transplantation for Pediatric and Adult Candidates.

Patient and graft survival are similar following whole-liver transplantations (WLTs) versus split-liver transplantations (SLTs) among pediatric and adult recipients, yet SLTs are rarely used. We sought to determine the survival benefit associated with accepting a splittable graft offer for SLT versus declining and waiting for a subsequent offer using 2010 to 2018 Scientific Registry of Transplant Recipients (SRTR) data on 928 pediatric and 1814 adult liver transplantation candidates who were ever offered a splittable graft. We compared eventual mortality, regardless of subsequent transplants, between those patients who accepted versus declined a split liver offer with adjustments for Pediatric End-Stage Liver Disease/Model for End-Stage Liver Disease (MELD) scores, diagnosis, and weight among pediatric candidates and matching for MELD score, height, and offer among adult candidates. Among pediatric candidates ≤7 kg, split liver offer acceptance versus decline was associated with a 63% reduction in mortality (adjusted hazard ratio [aHR], 0.17 0.370.80 [P = 0.01]; 93.1% versus 84.0% 1-year survival after decision). Within 1 year of decline for those ≤7 kg, 6.4% died and 31.1% received a WLT. Among pediatric candidates >7 kg, there was no significant difference associated with acceptance of a split liver offer (aHR, 0.63 1.071.82 [P = 0.81]; 91.7% versus 94.4% 1-year survival after decision). Within 1 year of decline for those >7 kg, 1.8% died and 45.8% received a WLT. Among adult candidates, split liver offer acceptance was associated with a 43% reduction in mortality (aHR, 0.39 0.570.83 [P = 0.005]; 92.2% versus 84.4% 1-year survival after decision). Within 1 year of decline for adult candidates, 7.9% died and 39.3% received a WLT. Accepting split liver offers for SLT could significantly improve survival for small children and adults on the waiting list.

Multicenter study of racial and ethnic inequities in liver transplantation evaluation: Understanding mechanisms and identifying solutions.

Racial and ethnic disparities persist in access to the liver transplantation (LT) waiting list; however, there is limited knowledge about underlying system-level factors that may be responsible for these disparities. Given the complex nature of LT candidate evaluation, a human factors and systems engineering approach may provide insights. We recruited participants from the LT teams (coordinators, advanced practice providers, physicians, social workers, dieticians, pharmacists, leadership) at two major LT centers. From December 2020 to July 2021, we performed ethnographic observations (participant-patient appointments, committee meetings) and semistructured interviews (N = 54 interviews, 49 observation hours). Based on findings from this multicenter, multimethod qualitative study combined with the Systems Engineering Initiative for Patient Safety 2.0 (a human factors and systems engineering model for health care), we created a conceptual framework describing how transplant work system characteristics and other external factors may improve equity in the LT evaluation process. Participant perceptions about listing disparities described external factors (e.g., structural racism, ambiguous national guidelines, national quality metrics) that permeate the LT evaluation process. Mechanisms identified included minimal transplant team diversity, implicit bias, and interpersonal racism. A lack of resources was a common theme, such as social workers, transportation assistance, non-English-language materials, and time (e.g., more time for education for patients with health literacy concerns). Because of the minimal data collection or center feedback about disparities, participants felt uncomfortable with and unadaptable to unwanted outcomes, which perpetuate disparities. We proposed transplant center-level solutions (i.e., including but not limited to training of staff on health equity) to modifiable barriers in the clinical work system that could help patient navigation, reduce disparities, and improve access to care. Our findings call for an urgent need for transplant centers, national societies, and policy makers to focus efforts on improving equity (tailored, patient-centered resources) using the science of human factors and systems engineering.