Stringent Cutoff Research Articles

Clinical value of anti-DSG2 antibodies in arrhythmogenic right ventricular cardiomyopathy: a real-life experience

Abstract Background/Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease leading to ventricular arrhythmias and heart failure. The diagnosis is challenging and currently based on a set of clinical criteria with limited sensitivity and specificity. Anti-desmoglein-2 (DSG2) antibodies were previously found in patients diagnosed with ARVC, while they were absent in both healthy controls and athletes. Purpose The study aimed to evaluate the diagnostic value of anti-DSG2 antibodies by comparing their concentration in patients with ARVC and other right ventricular diseases. Additionally, the prognostic value of the biomarker was assessed. Methods A cohort of 101 patients with a definite diagnosis of ARVC according to the 2010 Task Force criteria was enrolled (64 males, mean age 47 ± 16 years). The control group included patients with Ebstein anomaly and Eisenmenger syndrome (16 and 21 subjects, respectively). Anti-DSG2 antibody concentration was assessed using enzyme-linked immunosorbent assay (ELISA). After that, an ARVC cohort was followed for 3.9 ± 1.6 years for the occurrence of the primary endpoint of cardiac death or heart transplantation (HTx) and major arrhythmic events (MAEs) defined as ventricular fibrillation, sustained ventricular tachycardia (sVT) or appropriate implantable cardioverter-defibrillator intervention. Results The median anti-DSG2 antibody concentration presented as ELISA optical density (OD) reached 1.36 [0.54-2.15] in the ARVC cohort, while it was 0.4 [0.34-0.54] in Ebstein anomaly group and 0.36 [0.28-0.58] in the Eisenmenger syndrome group. There was a significant difference in antibody levels between ARVC and both control groups (p < 0.001, Figure 1). The cutoff value for the diagnosis of ARVC was 0.774, with 72.3% sensitivity and 100% specificity (AUC = 0.823). After dividing the ARVC cohort according to the designated cutoff point, the anti-DSG2-positive subgroup was significantly younger (44.5 ± 15.4 vs. 52.8 ± 15.5 years, p = 0.017) and more likely to have a history of sVT (68.5% vs. 46.4%, p = 0.041). There was no difference regarding sex, presence of PKP2 or DSG2 mutations, previous sports activities, history of sudden cardiac arrest or syncope, right ventricular dimensions and systolic function, left ventricular ejection fraction and heart failure symptoms. This threshold was not predictive for the primary endpoint and MAEs. However, a more stringent cutoff value for anti-DSG2 level (1.021) was found to predict death or HTx (HR [95% CI] 4.56 [1.05-19.76], p = 0.026, Figure 2). Conclusions The concentration of anti-DSG2 antibodies is significantly higher in ARVC compared to other right ventricular diseases. A high antibody level is prognostic for death or HTx in ARVC.Figure 1Figure 2

Revisiting the functional annotation of TriTryp using sequence similarity tools

Trypanosomatids are the causative agents of deadly diseases in humans and livestock. Given the high phylogenetic distance of trypanosomatids from model organisms, these organisms have ample unannotated genes. Manual functional annotation is time-consuming, highlighting the importance of automated functional annotation tools. The development of automated functional tools is a hot research topic, and multiple tools have been developed for the task. PANNZER2 is an automated functional annotation tool that merely relies on the sequence similarity of the query to the annotated proteins. We tried PANNZER2 on Trypanosoma brucei, the most studied organism among trypanosomatids, to see if it could improve our knowledge of the functions of the genes.Even with the availability of automated annotation tools like InterPro2GO in databases such as TriTrypDB, PANNZER2 has made surprisingly confident predictions for some hypothetical proteins in T. brucei. In this study, we identify gaps in such annotations because of not employing pairwise sequence alignment tools in TriTrypDB's automated annotation process. Our findings demonstrate that even the use of stringent cutoffs can successfully annotate a significant number of proteins. Additionally, we discovered that adjusting the open reading frames in certain genes leads to sequences with increased sequence signature coverage—characterized by the length covered by at least one sequence signature—compared to the original sequences. This enhanced sequence signature coverage suggests these genomic fragments could be pseudogenes. To facilitate further exploration, we developed a script to help identify potential pseudogenes within an organism's genome, offering researchers a new tool for genomic analysis and understanding. We extended all our analysis to Trypanosoma cruzi and Leishmania major to assess the impact of this approach across different species.Our study demonstrates that by utilizing pairwise sequence similarity alignment, even with stringent cutoffs, we can attribute 2986, 3953, and 3798 new GO terms to the genomes of T. brucei, T. cruzi, and L. major. Additionally, we found that 210, 239, and 29 genes exhibit increased sequence signature coverage following frame correction, suggesting the presence of pseudogenes.

Lattice Dynamics of Quasi-2D Perovskites from First Principles.

We present the vibrational properties and phonon dispersion for quasi-2D hybrid organic-inorganic perovskites (BA)2CsPb2I7, (HA)2CsPb2I7, (BA)2(MA)Pb2I7, and (HA)2(MA)Pb2I7 calculated from first principles. Given the highly complex nature of these compounds, we first perform careful benchmarking and convergence testing to identify suitable parameters to describe their structural features and vibrational properties. We find that the inclusion of van der Waals corrections on top of generalized gradient approximation (GGA) exchange-correlation functionals provides the best agreement for the equilibrium structure relative to experimental data. We also investigate the impact of the molecular orientation on the equilibrium structure of these layered perovskite systems. Our results suggest ground state ferroelectric alignment of molecular dipoles in the out-of-plane direction is unlikely and support the assignment of the centrosymmetric space group for the low-temperature phase of (HA)2(MA)Pb2I7. Finally, we compute vibrational properties under the harmonic approximation. We find that stringent energy cut-offs are required to obtain well-converged phonon properties, and once converged, the harmonic approximation can capture key physics for such a large, hybrid inorganic-organic system with vastly different atom types, masses, and interatomic interactions. We discuss the obtained phonon modes and dispersion behavior in the context of known properties for bulk 3D perovskites and ligand molecular crystals. While many vibrational properties are inherited from the parent systems, we also observe unique coupled vibrations that cannot be associated with vibrations of the pure constituent perovskite and ligand subphases. Energy dispersion of the low energy phonon branches primarily occurs in the in-plane direction and within the perovskite subphase and arises from bending and breathing modes of the equatorial Pb-I network within the perovskite octahedral plane. The analysis herein provides the foundation for future investigations on this class of materials, such as exciton-phonon coupling, phase transitions, and general temperature-dependent properties.

Cross-Sectional and Longitudinal Association of Clinical and Neurocognitive Factors With Apathy in Patients With Parkinson Disease.

A robust understanding of the natural history of apathy in Parkinson disease (PD) is foundational for developing effective clinical management tools. However, large longitudinal studies are lacking while the literature is inconsistent about even cross-sectional associations. We aimed to determine the longitudinal predictors of apathy development in a large cohort of people with PD and its cross-sectional associations and trajectories over time, using sophisticated Bayesian modeling techniques. People with PD followed up in the longitudinal New Zealand Parkinson's progression project were included. Apathy was defined using the neuropsychiatric inventory subscale ≥4, and analyses were also repeated using a less stringent cutoff of ≥1. Both MoCA and comprehensive neuropsychological testing were used as appropriate to the model. Depression was assessed using the hospital anxiety and depression scale. Cross-sectional Bayesian regressions were conducted, and a multistate predictive model was used to identify factors that predict the initial onset of apathy in nonapathetic PD, while also accounting for the competing risk of death. The relationship between apathy presence and mortality was also investigated. Three hundred forty-six people with PD followed up for up to 14 years across a total of 1,392 sessions were included. Apathy occurrence did not vary significantly across the disease course (disease duration odds ratio [OR] = 0.55, [95% CI 0.28-1.12], affecting approximately 11% or 22% of people at any time depending on the NPI cutoff used. Its presence was associated with a significantly higher risk of death after controlling for all other factors (hazard ratio [HR] = 2.92 [1.50-5.66]). Lower cognition, higher depression levels, and greater motor severity predicted apathy development in those without motivational deficits (HR [cognition] = 0.66 [0.48-0.90], HR [depression] = 1.45 [1.04-2.02], HR [motor severity] = 1.37 [1.01-1.86]). Cognition and depression were also associated with apathy cross-sectionally, along with male sex and possibly lower dopaminergic therapy level, but apathy still occurred across the full spectrum of each variable (OR [cognition] = 0.58 [0.44-0.76], OR [depression] = 1.43 [1.04-1.97], OR [female sex] = 0.45 [0.22-0.92], and OR [levodopa equivalent dose] = 0.78 [0.59-1.04]. Apathy occurs across the PD time course and is associated with higher mortality. Depressive symptoms and cognitive impairment in particular predict its future development in those with normal motivation.

Beyond RCTs: Short-term dual antiplatelet therapy in secondary prevention of ischemic stroke and transient ischemic attack.

Randomized controlled trials (RCTs) proved the efficacy of short-term dual antiplatelet therapy (DAPT) in secondary prevention of minor ischemic stroke or high-risk transient ischemic attack (TIA). We aimed at evaluating effectiveness and safety of short-term DAPT in real-world, where treatment use is broader than in RCTs. READAPT (REAl-life study on short-term Dual Antiplatelet treatment in Patients with ischemic stroke or Transient ischemic attack) (NCT05476081) was an observational multicenter real-world study with a 90-day follow-up. We included patients aged 18+ receiving short-term DAPT soon after ischemic stroke or TIA. No stringent NIHSS and ABCD2 score cut-offs were applied but adherence to guidelines was recommended. Primary effectiveness outcome was stroke (ischemic or hemorrhagic) or death due to vascular causes, primary safety outcome was moderate-to-severe bleeding. Secondary outcomes were the type of ischemic and hemorrhagic events, disability, cause of death, and compliance to treatment. We included 1920 patients; 69.9% started DAPT after an ischemic stroke; only 8.9% strictly followed entry criteria or procedures of RCTs. Primary effectiveness outcome occurred in 3.9% and primary safety outcome in 0.6% of cases. In total, 3.3% cerebrovascular ischemic recurrences occurred, 0.2% intracerebral hemorrhages, and 2.7% bleedings; 0.2% of patients died due to vascular causes. Patients with NIHSS score ⩽5 and those without acute lesions at neuroimaging had significantly higher primary effectiveness outcomes than their counterparts. Additionally, DAPT start >24 h after symptom onset was associated with a lower likelihood of bleeding. In real-world, most of the patients who receive DAPT after an ischemic stroke or a TIA do not follow RCTs entry criteria and procedures. Nevertheless, short-term DAPT remains effective and safe in this population. No safety concerns are raised in patients with low-risk TIA, more severe stroke, and delayed treatment start.

HER2/ERBB2 copy number analysis by targeted next-generation sequencing in breast cancer.

A combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) is the current standard of care for HER2 evaluation in breast cancer. Here, we investigate the potential clinical utility of next-generation sequencing (NGS)-derived HER2/ERBB2 copy number (CN) data for predicting HER2 status as defined by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. In total, 294 locally recurrent and metastatic breast cancers previously tested by targeted hybrid capture-based NGS and by HER2 IHC/FISH were included. Analyses focused on the ERBB2 median log2 ratios and start-end genomic coordinates from NGS, average HER2 CN and HER2/CEP17 ratios from FISH, and the HER2 IHC scores. We also determined a more stringent log2 ratio cutoff to predict HER2-positive status with 100% specificity. Sixty-four (22%) cases were HER2 positive and 230 (78%) were HER2 negative by ASCO/CAP guidelines. The ERBB2 median log2 ratios from NGS strongly correlated with HER2 status by IHC/FISH (area under receiver operator characteristic curve = 0.951). ERBB2 log2 ratio more than 1.7 was 100% specific for HER2-positive results by IHC/FISH. Start and end genomic coordinates for regions of gain near ERBB2 by NGS also predicted HER2 status. Copy number data from our NGS panel strongly correlate with HER2 status. Using a stringent cutoff, ERBB2 log2 ratio accurately predicts HER2 positivity with high specificity. The NGS CN assessment may have utility in determining HER2 status in certain clinical settings.

Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.

Despite continuous technological improvements in sample preparation, mass-spectrometry-based proteomics for trace samples faces the challenges of sensitivity, quantification accuracy, and reproducibility. Herein, we explored the applicability of turboDDA (a method that uses data-dependent acquisition without dynamic exclusion) for quantitative proteomics of trace samples. After systematic optimization of acquisition parameters, we compared the performance of turboDDA with that of data-dependent acquisition with dynamic exclusion (DEDDA). By benchmarking the analysis of trace unlabeled human cell digests, turboDDA showed substantially better sensitivity in comparison with DEDDA, whether for unfractionated or high pH fractionated samples. Furthermore, through designing an iTRAQ-labeled three-proteome model (i.e., tryptic digest of protein lysates from yeast, human, and E. coli) to document the interference effect, we evaluated the quantification interference, accuracy, reproducibility of iTRAQ labeled trace samples, and the impact of PIF (precursor intensity fraction) cutoff for different approaches (turboDDA and DEDDA). The results showed that improved quantification accuracy and reproducibility could be achieved by turboDDA, while a more stringent PIF cutoff resulted in more accurate quantification but less peptide identification for both approaches. Finally, the turboDDA strategy was applied to the differential analysis of limited amounts of human lung cancer cell samples, showing great promise in trace proteomics sample analysis.

Predicting an Underlying Clonal Plasma Cell Population in Light-Chain Monoclonal Gammopathy of Undetermined Significance Using Free Light-Chain Ratio

Introduction: The management of light-chain (LC) monoclonal gammopathy of undetermined significance (MGUS) presents inherent challenges as the key diagnostic factor - abnormal free-light chain (FLC) levels - only indirectly implies the presence of an underlying clonal cell population. Moreover, the lack of M protein, used in current risk-stratification models, necessitates a distinct approach for risk assessment in LC-MGUS. Using next-generation flow cytometry (NGF), we assessed clonal plasma cell presence in bone marrow (BM) samples from individuals with LC monoclonal gammopathies. This approach enabled us to evaluate the predictive value of FLC ratio for an underlying clonal plasma cell population in LC-MGUS and its prognostic implications. Aim: To evaluate the association between FLC ratio and the presence of clonal plasma cells in the BM of individuals with LC monoclonal gammopathies as well as the risk of progression. Methods: The study is a part of the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study, a population-based screening study for multiple myeloma (MM) precursors and randomized trial of follow-up strategies. We included screened individuals with an abnormal FLC ratio (<0.26 or >1.65) and elevated involved LC (kappa ≥19.4 mg/L or lambda ≥26.3 mg/L) (Freelite, The Binding Site), with no detected M protein. Those with previously known MM or related disorders were excluded. NGF was used to determine clonal plasma cell presence in BM samples. Logistic regression was used to assess the relationship between FLC ratio (used as involved/uninvolved ratio throughout the study) and clonal plasma cell presence. Receiver operating characteristic (ROC) curve analysis was used to assess the predictive ability of FLC ratio for clonal plasma cell presence in LC-MGUS and to determine an optimal cutoff value. Kaplan-Meier analysis was used to assess time to progression to more advanced plasma cell disorder for LC-MGUS, stratified by the FLC ratio cutoff. A linear mixed-effects model was used to evaluate the change in FLC ratio (expressed as fold change relative to the screening sample) over time for those with FLC ratio below the cutoff at screening. Results: After screening 75,422 individuals, 1,965 with LC disease were included, with median age of 71 years (range of 42-100 years), and a median FLC ratio of 1.85. Most (95.4%) had involved FLC of type kappa. Clonal plasma cells were identified by NGF in 8/21 (38.1%) LC-MGUS, and all LC-smoldering MM (SMM) (n=21) and LC-MM (n=7) cases. A significant prediction of clonal plasma cell presence was found for FLC ratio (OR=6.47; p=0.012; n=49), supported by ROC curve analysis for LC-MGUS (AUC=0.94; n=21), identifying FLC ratio of 3.15 as optimal cutoff (92% sensitivity, 100% specificity). Of the 254 individuals randomized to active follow-up within the iStopMM study, baseline diagnosis distribution was 223 (87.8%) LC-MGUS, 22 (8.7%) LC-SMM, 7 (2.8%) LC-MM, 1 (0.4%) solitary plasmacytoma, and 1 (0.4%) chronic lymphocytic leukemia. Among these, 123 (representing 88% of the screened study cohort) had FLC ratio <3.15 at screening, thereof 122 (99.2%) with LC-MGUS and 1 (0.8%) with solitary plasmacytoma. No disease progression was observed for individuals with FLC ratio <3.15 (n=100) over a median follow-up of 46 months. Conversely, disease progression occurred in 13/97 (13.4%) LC-MGUS with ≥3.15 FLC ratio (9 to SMM, 3 to MM, and 1 to smoldering Waldenströms macroglobulinemia) during median follow-up of 42 months (p=0.00018 vs FLC ratio <3.15) (Figure). The linear mixed-effects model of 454 repeated FLC from 128 individuals with <3.15 FLC ratio at screening did not indicate significant change in FLC ratio over a median follow-up of 48 months. Conclusions: In this study, we utilized NGF to confirm an underlying clonal plasma cell population in BM of individuals with LC-MGUS and evaluate its association with involved/uninvolved FLC ratio. Clonal plasma cells were detected in a minority of LC-MGUS (38.1%), for which FLC ratio was found to be highly predictive. An FLC ratio cutoff of 3.15 optimally discriminated based on clonal plasma cell presence, and we found no evidence of progressive disease in LC-MGUS below this cutoff during follow-up. Altogether, the more stringent FLC ratio cutoff can assist in determining the likelihood of an underlying clonal plasma cell population in LC-MGUS and may, as such, help to guide the management of LC-MGUS.

Are Body Mass Index (BMI) Cutoffs Creating Racial, Ethnic, Gender, or Age Disparities in Patient Eligibility for Total Ankle Arthroplasty?

Category: Ankle; Other Introduction/Purpose: The rising prevalence of obesity may drastically affect arthroplasty eligibility as Body Mass Index (BMI) remains a commonly used screening criterion. As obesity disproportionately affects Black adults and women, using such a criterion without scientific merit may lead to inequitable access to care. Particularly in total ankle arthroplasty – where weight has been previously attributed to aseptic implant failure – close examination of screening criteria is imperative. The purpose of this study was to evaluate for disparities in patient eligibility for total ankle arthroplasty (TAA) based on race, ethnicity, gender, and age by applying commonly used BMI cutoffs to the American College of Surgeons National Surgical Quality Improvement Program (ACS- NSQIP) database. Methods: Patients in the ACS-NSQIP database who underwent TAA from 2011- 2020 were retrospectively reviewed. BMI cutoffs of < 35, < 40, < 45, and < 50 were applied to the cohort to model the rate of surgical eligibility for TAA. Patients were deemed eligible for surgery if their BMI was less than each modeled BMI cutoff and ineligible if their BMI was equal to or greater than the cutoff. The relative eligibility rate, which was defined as the rate of eligibility minus the rate of ineligibility, was examined for each BMI cutoff. The cohorts were then stratified by race, ethnicity, gender, and age. Independent T-tests, Chi-squared tests, and Fisher’s exact tests were performed to compare differences at an α = 0.05. Results: 65.1% of all TAA patients were included after applying the exclusion criteria. Black patients had disproportionately lower rates of eligibility at the most stringent BMI cutoff of < 35kg/m² (p=0.004). Hispanic patients had generally lower rates of eligibility across all BMI cutoffs. In contrast, Asian American and Pacific Islander patients had higher rates of eligibility at the BMI cutoffs of < 35kg/m² (p=0.033) and < 40kg/m² (p=0.039), and White non-Hispanic patients had higher rates of eligibility across all BMI cutoffs. Females had lower eligibility rates across all BMI cutoffs. Ineligible patients were also younger compared to eligible patients across all BMI cutoffs. Conclusion: Stringent BMI cutoffs may disproportionately disqualify Black, female, and younger patients from receiving total ankle arthroplasty. The data suggests that current eligibility criteria may exacerbate disparity in access to TAA for historically vulnerable populations. While eligibility criteria exist to protect patients from interventions that put them at unacceptable risk, these criteria are not uniformly distributed across populations. Given these data and the rising obesity epidemic, additional highly powered studies are needed to determine the safety of TAA in patients with BMI 35-40 to ensure at-risk populations are not disproportionately excluded from mobility-improving surgery.

High unrecognized SARS-CoV-2 exposure of newly admitted and hospitalized psychiatric patients

BackgroundPatients with pre-existing mental disorders are at higher risk for SARS-CoV-2 infection and adverse outcomes, and severe mental illness, including mood and psychosis spectrum disorders, is associated with increased mortality risk. Despite their increased risk profile, patients with severe mental illness have been understudied during the pandemic, with limited estimates of exposure in inpatient settings. ObjectiveThe aim of this study was to describe the SARS-CoV-2 seroprevalence and antibody titers, and pro-inflammatory cytokine concentrations of newly admitted or hospitalized psychiatric inpatients without known history of COVID-19 infection, using robust quantitative multi-antigen assessments, and compare patients’ exposure to that of hospital staff. MethodsThis multi-centric, cross-sectional study compared SARS-CoV-2 seroprevalence and titers of 285 patients (University Psychiatric Centre Duffel [UPCD] N = 194; Assistance-Publique-Hopitaux de Paris [AP-HP] N = 91), and 192 hospital caregivers (UPCD N = 130; AP-HP N = 62) at two large psychiatric care facilities between January 1st and the May 30th 2021. Serum levels of SARS-CoV-2 antibodies against Spike proteins (full length), spike subunit 1 (S1), spike subunit 2 (S2), spike subunit 1 receptor binding domain (S1-RBD) and Nucleocapsid proteins were quantitatively determined using an advanced capillary Western Blot technique. To assess the robustness of the between-group seroprevalence differences, we performed sensitivity analyses with stringent cut-offs for seropositivity. We also assessed peripheral concentrations of IL-6, IL-8 and TNF-a using ELLA assays. Secondary analyses included comparisons of SARS-CoV-2 seroprevalence and titers between patient diagnostic subgroups, and between newly admitted (hospitalization ≤ 7 days) and hospitalized patients (hospitalization > 7 days) and correlations between serological and cytokines. ResultsPatients had a significantly higher SARS-CoV-2 seroprevalence (67.85 % [95% CI 62.20–73.02]) than hospital caregivers (27.08% [95% CI 21.29–33.77]), and had significantly higher global SARS-CoV-2 titers (F = 29.40, df = 2, p < 0.0001). Moreover, patients had a 2.51-fold (95% CI 1.95–3.20) higher SARS-CoV-2 exposure risk compared to hospital caregivers (Fisher’s exact test, P < 0.0001). No difference was found in SARS-CoV-2 seroprevalence and titers between patient subgroups. Patients could be differentiated most accurately from hospital caregivers by their higher Spike protein titers (OR 136.54 [95% CI 43.08–481.98], P < 0.0001), lower S1 (OR 0.06 [95% CI 0.02–0.15], P < 0.0001) titers and higher IL-6 (OR 3.41 [95% CI 1.73–7.24], P < 0.0001) and TNF-α (OR 34.29 [95% CI 5.00–258.87], P < 0.0001) and lower titers of IL-8 (OR 0.13 [95% CI 0.05–0.30], P < 0.0001). Seropositive patients had significantly higher SARS-COV-2 antibody titers compared to seropositive hospital caregivers (F = 19.53, df = 2, P < 0.0001), while titers were not different in seronegative individuals. Pro-inflammatory cytokine concentrations were not associated with serological status. ConclusionOur work demonstrated a very high unrecognized exposure to SARS-CoV-2 among newly admitted and hospitalized psychiatric inpatients, which is cause for concern in the context of highly robust evidence of adverse outcomes following COVID-19 in psychiatric patients. Attention should be directed toward monitoring and mitigating exposure to infectious agents within psychiatric hospitals.

Circulating microRNA Profiles for Premature Cardiovascular Death in Patients with Kidney Failure with Replacement Therapy.

Patients with kidney failure with replacement therapy (KFRT) suffer from a disproportionately high cardiovascular disease burden. Circulating small non-coding RNAs (c-sncRNAs) have emerged as novel epigenetic regulators and are suggested as novel biomarkers and therapeutic targets for cardiovascular disease; however, little is known about the associations of c-sncRNAs with premature cardiovascular death in KFRT. In a pilot case-control study of 50 hemodialysis patients who died of cardiovascular events as cases, and 50 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we performed c-sncRNAs profiles using next-generation sequencing to identify differentially expressed circulating microRNAs (c-miRNAs) between the plasma of cases and that of controls. mRNA target prediction and pathway enrichment analysis were performed to examine the functional relevance of differentially expressed c-miRNAs to cardiovascular pathophysiology. The association of differentially expressed c-miRNAs with cardiovascular mortality was examined using multivariable conditional logistic regression. The patient characteristics were similar between cases and controls, with a mean age of 63 years, 48% male, and 54% African American in both groups. We detected a total of 613 miRNAs in the plasma, among which five miRNAs (i.e., miR-129-1-5p, miR-500b-3p, miR-125b-1-3p, miR-3648-2-5p, and miR-3150b-3p) were identified to be differentially expressed between cases and controls with cut-offs of p < 0.05 and log2 fold-change (log2FC) > 1. When using more stringent cut-offs of p-adjusted < 0.05 and log2FC > 1, only miR-129-1-5p remained significantly differentially expressed, with higher levels of miR-129-1-5p in the cases than in the controls. The pathway enrichment analysis using predicted miR-129-1-5p mRNA targets demonstrated enrichment in adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy, and oxytocin signaling pathways. In parallel, the circulating miR-129-1-5p levels were significantly associated with the risk of cardiovascular death (adjusted OR [95% CI], 1.68 [1.01-2.81] for one increase in log-transformed miR-129-1-5p counts), independent of potential confounders. Circulating miR-129-1-5p may serve as a novel biomarker for premature cardiovascular death in KFRT.

Body Mass Index (BMI) Cutoffs and Racial, Ethnic, Sex, or Age Disparities in Patients Treated With Total Ankle Arthroplasty.

The rising prevalence of obesity among American adults has disproportionately affected Black adults and women. Furthermore, body mass index (BMI) has historically been used as a relative contraindication to many total joint arthroplasty (TJA) procedures, including total ankle arthroplasty. The purpose of this study was to investigate potential disparities in patient eligibility for total ankle arthroplasty based on race, ethnicity, sex, and age by applying commonly used BMI cutoffs to the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. Patients in the ACS-NSQIP database who underwent TAA from 2011 to 2020 were retrospectively reviewed in a cross-sectional analysis. BMI cutoffs of <50, <45, <40, and <35 were then applied. The eligibility rate for TAA was examined for each BMI cutoff, and findings were stratified by race, ethnicity, sex, and age. Independent t tests, chi-squared tests, and Fisher exact tests were performed to compare differences at an α = 0.05. A total of 1215 of 1865 TAA patients (65.1%) were included after applying the exclusion criteria. Black patients had disproportionately lower rates of eligibility at the most stringent BMI cutoff of <35 (P = .004). Hispanic patients had generally lower rates of eligibility across all BMI cutoffs. In contrast, Asian American and Pacific Islander patients had higher rates of eligibility at the BMI cutoffs of <35 (P = .033) and <40 (P = .039), and White non-Hispanic patients had higher rates of eligibility across all BMI cutoffs. Females had lower eligibility rates across all BMI cutoffs. Ineligible patients were also younger compared to eligible patients across all BMI cutoffs. Stringent BMI cutoffs may disproportionately disqualify Black, female, and younger patients from receiving total ankle arthroplasty. Level III, retrospective cross-sectional study.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Species-Deconvolved Proteomics for In Situ Investigation of Tumor-Stroma Interactions after Treatment of Pancreatic Cancer Patient-Derived Xenografts with Combined Gemcitabine and Paclitaxel.

Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.

The Immunopeptidome from a Genomic Perspective: Establishing the Noncanonical Landscape of MHC Class I-Associated Peptides.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

Antibiotic Adjuvants: A Versatile Approach to Combat Antibiotic Resistance.

The problem of antibiotic resistance is on the rise, with multidrug-resistant strains emerging even to the last resort antibiotics. The drug discovery process is often stalled by stringent cut-offs required for effective drug design. In such a scenario, it is prudent to delve into the varying mechanisms of resistance to existing antibiotics and target them to improve antibiotic efficacy. Nonantibiotic compounds called antibiotic adjuvants which target bacterial resistance can be used in combination with obsolete drugs for an improved therapeutic regime. The field of "antibiotic adjuvants" has gained significant traction in recent years where mechanisms other than β-lactamase inhibition have been explored. This review discusses the multitude of acquired and inherent resistance mechanisms employed by bacteria to resist antibiotic action. The major focus of this review is how to target these resistance mechanisms by the use of antibiotic adjuvants. Different types of direct acting and indirect resistance breakers are discussed including enzyme inhibitors, efflux pump inhibitors, inhibitors of teichoic acid synthesis, and other cellular processes. The multifaceted class of membrane-targeting compounds with poly pharmacological effects and the potential of host immune-modulating compounds have also been reviewed. We conclude with providing insights about the existing challenges preventing clinical translation of different classes of adjuvants, especially membrane-perturbing compounds, and a framework about the possible directions which can be pursued to fill this gap. Antibiotic-adjuvant combinatorial therapy indeed has immense potential to be used as an upcoming orthogonal strategy to conventional antibiotic discovery.

Systolic blood pressure, low-density lipoprotein cholesterol levels, and adverse kidney outcome: results from KNOW-CKD.

It is unknown whether intensive control of blood pressure (BP) and lipids can delay the progression of chronic kidney disease (CKD). This study examined the combined association of strict targets of systolic BP (SBP) and low-density lipoprotein cholesterol (LDL-C) levels with adverse kidney outcomes. In total, 2012 patients from the KoreaN Cohort Study for Outcomes in Patients With CKD (KNOW-CKD) were classified into four groups according to SBP of 120 mmHg and LDL-C of 70 mg/dl: group 1, <120 and <70; group 2, <120 and ≥70; group 3, ≥120 and <70; group 4, ≥120 and ≥70. We constructed time-varying models treating two variables as time-varying exposures. The primary outcome was the progression of CKD, defined as a ≥50% decrease in estimated glomerular filtration rate from the baseline or the onset of kidney failure requiring replacement therapy. The primary outcome events occurred in 27.9%, 26.7%, 40.3%, and 39.1% from groups 1 to 4. In the time-varying model, the hazard ratios (95% confidence intervals) for the primary outcome were 0.48 (0.33-0.69), 0.78 (0.63-0.96), and 0.96 (0.74-1.23) for groups 1 to 3, respectively, compared with group 4. When less stringent cut-offs of SBP of 130 mmHg and LDL-C of 100 mg/dl were used, this graded association was lost, while only SBP was associated with adverse kidney outcomes. In this study, the lower targets of SBP of <120 mmHg and LDL-C < 70 mg/dl were synergistically associated with a lower risk of adverse kidney outcomes.

Assessing the sensitivity and specificity of phosphatidylethanol (PEth) cutoffs to identify alcohol exposed pregnancies

In the literature on alcohol use biomarkers, there has been debate as to what a valid and/or utilitarian cut off level should be for various research applications. In this manuscript, we assessed the sensitivity and specificity of multiple cutoff values for phosphatidylethanol (PEth) from bloodspots relative to self-report, the Alcohol Use Disorder Identification Test (AUDIT) scores, and another alcohol use biomarker ethyl glucuronide (EtG) from fingernails in a sample of 222 pregnant women in the Western Cape Province of South Africa. Receiver operating characteristic (ROC) curves were used to assess the area under the curve (AUC) and assess PEth cutoff values of ≥2, ≥4, ≥8, ≥14, and ≥20 nanograms per milliliter (ng/ml). The highest AUC value was attained when PEth was compared to an AUDIT score of 1 or more. Depending on the cutoff used to determine alcohol consumption, PEth identified 47%–70% of the individuals as alcohol-consuming while 62.6%–75.2% were identified by self-reported measures, and 35.6% were identified by EtG. In this sample, sensitivity and accuracy were highest at less stringent PEth cutoffs when compared to self-report, AUDIT score of 1 or more, 5 or more, 8 or more, and EtG ≥ 8 picograms per milligram (pg/mg). For research purposes, less stringent cutoffs, such as PEth ≥ 8 ng/ml, may be considered a valid, positive cutoff for identifying women who consume alcohol during pregnancy in this population. A cutoff of PEth ≥ 20 ng/ml may miss individuals who reported consuming alcohol (false negatives).

PTEN in triple-negative breast carcinoma: protein expression and genomic alteration in pretreatment and posttreatment specimens.

Recent advances have been made in targeting the phosphoinositide 3-kinase pathway in breast cancer. Phosphatase and tensin homolog (PTEN) is a key component of that pathway. To understand the changes in PTEN expression over the course of the disease in patients with triple-negative breast cancer (TNBC) and whether PTEN copy number variation (CNV) by next-generation sequencing (NGS) can serve as an alternative to immunohistochemistry (IHC) to identify PTEN loss. We compared PTEN expression by IHC between pretreatment tumors and residual tumors in the breast and lymph nodes after neoadjuvant chemotherapy in 96 patients enrolled in a TNBC clinical trial. A correlative analysis between PTEN protein expression and PTEN CNV by NGS was also performed. With a stringent cutoff for PTEN IHC scoring, PTEN expression was discordant between pretreatment and posttreatment primary tumors in 5% of patients (n = 96) and between posttreatment primary tumors and lymph node metastases in 9% (n = 33). A less stringent cutoff yielded similar discordance rates. Intratumoral heterogeneity for PTEN loss was observed in 7% of the patients. Among pretreatment tumors, PTEN copy numbers by whole exome sequencing (n = 72) were significantly higher in the PTEN-positive tumors by IHC compared with the IHC PTEN-loss tumors (p < 0.0001). However, PTEN-positive and PTEN-loss tumors by IHC overlapped in copy numbers: 14 of 60 PTEN-positive samples showed decreased copy numbers in the range of those of the PTEN-loss tumors. Testing various specimens by IHC may generate different PTEN results in a small proportion of patients with TNBC; therefore, the decision of testing one versus multiple specimens in a clinical trial should be defined in the patient inclusion criteria. Although a distinct cutoff by which CNV differentiated PTEN-positive tumors from those with PTEN loss was not identified, higher copy number of PTEN may confer positive PTEN, whereas lower copy number of PTEN would necessitate additional testing by IHC to assess PTEN loss. NCT02276443.

Assessment of Carbon Monoxide in Exhaled Breath using the Smokerlyzer Handheld Machine: A Cross- Sectional Study.

Nicotine dependence is known to be a chronic remitting and relapsing addictive disorder. Among cancer patients who smoke, nicotine addiction has been found to be more when compared with smoking healthy individuals. Smokerlyzer machine can be used to test smoking substance use and de-addiction services can be provided at Preventive Oncology units. The objectives of the study include: (i) To assess eCO using a Smokerlyzer hand-held machine and correlate it with the smoking status, (ii) To assess the cut-off value for smoking use, and discusses the benefits of this method. In this cross-sectional study, healthy individuals at the workplace were tested for exhaled CO (eCO), which is used as a biological marker for monitoring the tobacco smoking. We discuss the feasibility of testing and its implications for cancer patients. The Bedfont EC50 Smokerlyzer machine was used to measure the concentration of CO in the end-tidal expired air. Among 643 study subjects, we found a statistically significant difference (P < .001) of median (IQR) eCO (measured in ppm) among smokers and non-smokers 2(1,5) vs 1(1,2). A significant and moderate positive correlation (Spearman rank correlation coefficient: .463) was observed between eCO and subjects who used cigarettes (measured in pack years). The ROC curve shows a cut off value for eCO as 2.5 with sensitivity 43.6% and 1 - specificity 2.76% (Specificity: 97.24%), which was rounded to 3. The area under the curve is 74.9%, which indicates a moderate discrimination performance of the test. The diagnostic accuracy of the test is 82.89%, which shows the proportion of correct test results. Estimating eCO in health care settings will enable monitoring the smoking substance use which has important impact on clinical outcomes. In cancer hospitals, when the goal is complete abstinence a stringent CO cutoff in the range of 3-4 ppm should be used.