NSCLC Digital PCR Panel Returns Low-Input Sample Results Where Sequencing Fails.

Molecular diagnostics has drastically improved the survival rate of patients diagnosed with non-small cell lung cancer (NSCLC) over the last 10 years. Despite advancements in molecular testing, targeted therapies, and national guideline recommendations, more than half of NSCLC patients in the United States either never receive testing or patient care is not informed via molecular testing. Here, we sought to explore the relationship between DNA/RNA input, the molecular testing method, and test success rates. On a shared set of low-input reference test materials (n = 3), we ran both a hybrid capture-based, next-generation sequencing (NGS) assay and a multiplexed digital PCR (dPCR) panel. The dPCR panel was highly sensitive and specific for low-input samples in dilution studies ranging from 40 to 1 ng DNA and from 20 to 2.5 ng RNA, while NGS had up to an 86% loss in sensitivity as contrived sample inputs were serially diluted. The dPCR panel also demonstrated a high PPA (>95%) at diluted inputs as low as 15/7.5 ng DNA/RNA on 23 banked clinical samples with the same NGS hybrid capture assay at a high input. These data suggest that digital PCR is an accurate and effective way of identifying clinically relevant NSCLC mutations at low nucleotide input and quality.

Analytical Performance and Concordance with Next-Generation Sequencing of a Rapid, Multiplexed dPCR Panel for the Detection of DNA and RNA Biomarkers in Non-Small-Cell Lung Cancer.

FDA approval of targeted therapies for lung cancer has significantly improved patient survival rates. Despite these improvements, barriers to timely access to biomarker information, such as nucleic acid input, still exist. Here, we report the analytical performance and concordance with next-generation sequencing (NGS) of a highly multiplexed research-use-only (RUO) panel using digital PCR (dPCR). The panel's analytical sensitivity and reactivity were determined using contrived DNA and RNA mixes. The limit of blank was established by testing FFPE curls classified as negative by pathology. Concordance was established on 77 FFPE samples previously characterized using the Oncomine Precision Assay®, and any discordant results were resolved with Archer Fusionplex® and Variantplex® panels. The analytical sensitivity, reported as the estimated mutant allele fraction (MAF), for DNA targets ranged from 0.1 to 0.9%. For RNA targets (ALK, RET, ROS, NTRK 1/2/3 Fusions, and MET Exon 14 skipping alteration), the analytical sensitivity ranged from 23 to 101 detected counts with 5 ng of total RNA input. The population prevalence-based coverage ranged from 89.2% to 100.0% across targets and exceeded 99.0% in aggregate. The assay demonstrated >97% concordance with respect to the comparator method.

Social anxiety and interpersonal risk for suicidal ideation: A longitudinal daily diary analysis.

INTRODUCTION: Social anxiety is associated with elevated suicidal ideation (SI). One potential explanation is that socially anxious persons experience frequent interpersonal stressors that elicit SI. Longitudinal designs with temporal ordering are needed to adequately test this hypothesis. Therefore, this study leveraged a longitudinal design combining trait and daily reports. METHODS: Two hundred eleven community adult participants with elevated levels of depression and/or social anxiety completed social anxiety and SI measures at baseline and again at a 1.5-month follow-up. Between these assessments, participants completed a 14-day diary study that assessed three forms of interpersonal distress: unfavorable social comparisons, perceived barriers to seeking social support, and loneliness. RESULTS: As predicted, simple mediation models revealed that baseline social anxiety had a significant indirect effect on SI severity at 1.5 months postbaseline via unfavorable social comparisons (indirect effect: ß = 0.07, p < 0.05) and barriers to seeking support (indirect effect: ß = 0.08, p < 0.05); however, social anxiety did not have a significant indirect effect on SI severity through loneliness. CONCLUSION: Study results are consistent with the proposition that increases in interpersonal distress may explain socially anxious persons' vulnerability to SI. Implications of these findings for the research, assessment, and treatment of suicidality in social anxiety are discussed.

Psychological well-being in US veterans with non-fatal suicide attempts: A multi-cohort population-based study.

BACKGROUND: Most people who survive suicide attempts neither re-attempt suicide nor die by suicide. Research on suicide attempt survivors has primarily focused on negative endpoints (e.g., increased suicide risk) rather than positive outcomes. One important outcome is psychological well-being (PWB), defined as positive functioning across emotional, intrapersonal, and interpersonal domains. We compared PWB among US military veterans with (i.e., attempt survivors) and without (i.e., non-attempters) a history of suicide attempt(s) using data from three nationally representative cohorts. METHODS: Each US veteran cohort (Cohort1: N = 3148; Cohort2: N = 1474; Cohort3: N = 4042) completed measures of suicidality (e.g., attempt history), character strengths (e.g., curiosity, optimism), psychological symptoms (e.g., depression), and indicators of PWB (e.g., happiness). t-Tests were conducted to examine group differences in PWB; hierarchical regressions were conducted to examine suicide attempt status as a predictor of PWB controlling for symptoms and demographics. Multivariable regressions were conducted to identify predictors of PWB among attempt survivors. RESULTS: In each cohort, reported PWB was markedly lower among suicide attempt survivors than non-attempters (ds = 0.9-1.2), even after adjusting for mental health symptoms. Individual differences in PWB were observed, with a subset of suicide attempt survivors reporting higher PWB levels than non-attempters (1.4-7.4 %). Curiosity and optimism were positively associated with PWB among suicide attempt survivors (rs = 0.60-0.78). LIMITATIONS: Data were cross-sectional, limiting inferences about causation and directionality of associations. CONCLUSIONS: Findings highlight diminished PWB as an important and understudied concern among veteran attempt survivors. Collectively, our findings underscore the importance of considering PWB in the research, assessment, and treatment of suicidality.

Motives and Consequences of Alcohol Use in People With Social Anxiety Disorder: A Daily Diary Study.

People with social anxiety disorder (SAD) are at increased risk for alcohol-related problems. Most research exploring social anxiety and alcohol use has examined negative drinking consequences, with less consideration of positive consequences-namely positive social experiences-that may reinforce alcohol use. In this daily diary study, we examined how adults diagnosed with SAD (N = 26) and a psychologically healthy control group (N = 28) experienced positive drinking consequences in naturally occurring drinking episodes during the study period. For 14 consecutive days, participants answered questions about alcohol use, motives for drinking, and positive consequences of drinking. On days when participants drank, those with SAD were more likely than healthy controls to perceive a reduction in anxiety, but the two groups did not differ in their likelihood of experiencing positive social drinking consequences. For both groups, on days when they were more motivated to drink to enhance social experiences (affiliation motives) or cope with distress (coping motives), they were more likely to obtain positive consequences from drinking. Compared to controls, participants with SAD endorsed stronger trait and daily coping motives (anxiety-coping, social anxiety-coping, and depression-coping). Results are discussed in the context of reinforcement mechanisms that may maintain social anxiety and alcohol use.

Does negative emotion differentiation influence how people choose to regulate their distress after stressful events? A four-year daily diary study.

Much is known about the types of strategies people use to regulate emotions. Less is known about individual differences that influence emotion regulation strategy selection. In this study, we tested the moderating role of negative emotion differentiation (NED; i.e., the ability to label and describe subtle differences among negative emotions) on the relationship between the intensity of stressful daily events and the strategies used to regulate distress arising from these events. Prior research shows that NED is associated with low endorsement of disengagement emotion regulation (e.g., substance use), but less is known about the link to engagement regulation (e.g., problem-solving, seeking social support). Participants were college students (N = 502) completing a 30-day daily diary survey for each of four college years. We preregistered hypotheses that 1) the intensity of each day's most stressful event would be associated with greater use of disengagement and engagement regulation strategies, and 2) people higher in NED would be less likely to use disengagement and more likely to use engagement strategies when highly stressed. Results suggest that higher stress intensity is associated with greater use of all regulation strategies. Greater NED is associated with less use of disengagement regulation strategies, whereas NED was unrelated to engagement regulation strategies and did not moderate the relationship between stress and engagement strategies. The majority of hypothesized moderation effects of NED were nonsignificant, prompting a reconsideration of whether, when, and how NED plays a role in stress responding. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

UNLABELLED: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 µM. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations. IMPORTANCE: Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site.

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C(pro). Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

Identification of a region of hantavirus nucleocapsid protein required for RNA chaperone activity.

Sin Nombre hantavirus (SNV) is a New World hantavirus and causes hantavirus cardiopulmonary syndrome. The viral nucleocapsid protein (N) is an RNA chaperone and has multiple functions important in virus replication. The three negative sense RNA segments of hantaviruses form panhandle structures through imperfect hydrogen bonding of the 5' and 3' termini, and the chaperone activity of N can mediate correct panhandle formation. N also functions during transcription and translation initiation and the chaperone activity of N is likely to be involved in aspects of these processes. Using a series of mutations in the N gene we identified a region of N required for chaperone activity. The N-terminal 100 amino acids of N contain a domain that is both necessary and sufficient for RNA chaperone activity. We propose that this region of N may reside in one of two potential states. First, the region may be highly disordered and function in N-mediated RNA chaperone activity. Alternatively, in trimeric form, the region likely becomes ordered and serves in high affinity vRNA panhandle recognition.