An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.

Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity.

Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination.

A resilient research approach: Using community-based participatory action research in a rural area of India.

This community-based participatory action research (CBPAR) study describes a method for evaluating an after-school resilience-focused intervention in a low-resource rural area of southern India. Communities Rising, a locally developed resilience and academic program, was evaluated in a cross-continent collaboration between a research team at a U.S. university and the local community. The CBPAR literature highlights the importance of cultural considerations, community considerations, and community participation in the research process. The present case study describes the CBPAR research process and considerations at every phase of the research project, providing a road map of how community engagement can strengthen research, empower the community, and provide valuable knowledge. This study was conducted in three phases that focused on inclusion of local voices in the development both of the resilience program and the evaluation data collection process. Youth surveyors were particularly key to the research process. Data on participant demographics, satisfaction with the program, and qualitative contributions are also provided. Strengths and limitations of this study process in a rural community are discussed.

Ccdc13 is a novel human centriolar satellite protein required for ciliogenesis and genome stability.

Here, we identify coiled-coil domain-containing protein 13 (Ccdc13) in a genome-wide RNA interference screen for regulators of genome stability. We establish that Ccdc13 is a newly identified centriolar satellite protein that interacts with PCM1, Cep290 and pericentrin and prevents the accumulation of DNA damage during mitotic transit. Depletion of Ccdc13 results in the loss of microtubule organisation in a manner similar to PCM1 and Cep290 depletion, although Ccdc13 is not required for satellite integrity. We show that microtubule regrowth is enhanced in Ccdc13-depleted cells, but slowed in cells that overexpress Ccdc13. Furthermore, in serum-starved cells, Ccdc13 localises to the basal body, is required for primary cilia formation and promotes the localisation of the ciliopathy protein BBS4 to both centriolar satellites and cilia. These data highlight the emerging link between DNA damage response factors, centriolar and peri-centriolar satellites and cilia-associated proteins and implicate Ccdc13 as a centriolar satellite protein that functions to promote both genome stability and cilia formation.

The centriolar satellite protein Cep131 is important for genome stability.

The centrosome acts as a centre for microtubule organisation and plays crucial roles in cell polarity, migration, growth and division. Cep131 has recently been described as a basal body component essential for cilium formation, but its function in non-ciliogenic cells is unknown. We identified human Cep131 (also known as AZI1) in a screen for regulators of genome stability. We show that centrosomal localisation of Cep131 is cell-cycle-regulated and requires both an intact microtubule network and a functional dynein-dynactin transport system. Cep131 is recruited to centriolar satellites by PCM1, and localised to the centriolar core region by both pericentrin and Cep290. Depletion of Cep131 results in a reduction in proliferation rate, centriole amplification, an increased frequency of multipolar mitosis, chromosomal instability and an increase in post-mitotic DNA damage. These data therefore highlight the importance of human Cep131 for maintaining genomic integrity.

Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells.

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.

Early intervention in youth psychosis: Novel approaches to understanding change.

Untreated psychosis in adolescents and young adults is associated with significant and progressive impairment. Early intervention to provide support and treatment for those at risk of psychosis is essential. Several early intervention models have been developed for those at-risk and those who are victims of a recent episode - including the Portland Identification and Early Referral model (PIER; McFarlane, 2001). This study extends previous work demonstrating a variety of positive treatment outcomes achieved by PIER in the context of a large-scale implementation across the state of Delaware. The sample included 108 youth and young adults who were either at risk for psychosis or had already experienced a first episode within the past two years. Participants received the PIER treatment model and were followed from baseline to six months after they were discharged from treatment. Researchers predicted that PIER participants would experience an increase in functioning and a decrease in positive psychosis symptoms. Change over time was examined through the lens of two analytic techniques: the Reliable Change Index (RCI) analyses and Growth Curve Modeling (GCM). Results show improvement on a number of outcomes over the course of the intervention as expected. Clinical implications, limitations, and suggestions for further research are discussed.

SARS-CoV-2 mutations affect antigen processing by the proteasome to alter CD8+ T cell responses.

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.

CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH.

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.

Affiliation and control during marital disagreement, history of divorce, and asymptomatic coronary artery calcification in older couples.

OBJECTIVE: To examine behavioral observations of affiliation (ie, warmth versus hostility) and control (ie, dominance versus submissiveness) and prior divorce as predictors of coronary artery calcification (CAC) in older couples. In some but not all studies, marital disruption and low marital quality have been shown to confer risk of coronary artery disease (CAD). Inconsistencies might reflect limitations of self-reports of marital quality compared with behavioral observations. Also, aspects of marital quality related to CAD might differ for men and women. METHODS: Couples underwent computed tomography scans for CAC and marital assessments, including observations of laboratory-based disagreement. Participants were 154 couples (mean age, 63.5 years; mean length of marriage, 36.4 years) free of prior diagnosis of CAD. RESULTS: Controlling traditional risk factors, we found behavioral measures of affiliation (low warmth) accounted for 6.2% of variance in CAC for women, p < .01, but not for men. Controlling behavior (dominance) accounted for 6.0% of variance in CAC for men, p < .02, but not for women. Behavioral measures were related to self-reports of marital quality, but the latter were unrelated to CAC. History of divorce predicted CAC for men and women. CONCLUSIONS: History of divorce and behavioral--but not self-report--measures of marital quality were related to CAD, such that low warmth and high dominance conferred risk for women and men, respectively. Prior research might underestimate the role of marital quality in CAD by relying on global self-reports of this risk factor.

C/EBPα and GATA-2 Mutations Induce Bilineage Acute Erythroid Leukemia through Transformation of a Neomorphic Neutrophil-Erythroid Progenitor.

Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.

Conflict and collaboration in middle-aged and older couples: II. Cardiovascular reactivity during marital interaction.

Marital strain confers risk of cardiovascular disease (CVD), perhaps though cardiovascular reactivity (CVR) to stressful marital interactions. CVR to marital stressors may differ between middle-age and older adults, and types of marital interactions that evoke CVR may also differ across these age groups, as relationship contexts and stressors differ with age. The authors examined cardiovascular responses to a marital conflict discussion and collaborative problem solving in 300 middle-aged and older married couples. Marital conflict evoked greater increases in blood pressure, cardiac output, and cardiac sympathetic activation than did collaboration. Older couples displayed smaller heart rate responses to conflict than did middle-aged couples but larger blood pressure responses to collaboration-especially in older men. These effects were maintained during a posttask recovery period. Women did not display greater CVR than men on any measure or in either interaction context, though they did display greater parasympathetic withdrawal. CVR to marital conflict could contribute to the association of marital strain with CVD for middle-aged and older men and women, but other age-related marital contexts (e.g., collaboration among older couples) may also contribute to this mechanism.

Conflict and collaboration in middle-aged and older couples: I. Age differences in agency and communion during marital interaction.

Prior theory and research regarding age differences in marital interaction suggest that older couples display and experience more positivity and less negativity than middle-aged couples. However, studies of overt behavior in older couples are relatively rare and have emphasized disagreement, neglecting other important contexts for older couples such as collaboration during everyday problem solving. Further, the affiliation or communion dimension of social interaction (i.e., warmth vs. hostility) is commonly assessed but not the control or agency dimension (e.g., dominance vs. submissiveness). The present study examined affect, cognitive appraisals, and overt behavior during disagreement (i.e., discussing a current conflict) and collaboration (i.e., planning errands) in 300 middle-aged and older married couples. Older couples reported less negative affect during disagreement and rated spouses as warmer than did middle-aged couples. However, these effects were eliminated when older couples' greater marital satisfaction was controlled. For observed behavior, older couples displayed little evidence of greater positivity and reduced negativity-especially women. During collaboration, older couples displayed a unique blend of warmth and control, suggesting a greater focus on emotional and social concerns during problem solving.

Neurogaming With Motion-Onset Visual Evoked Potentials (mVEPs): Adults Versus Teenagers.

Motion-onset visually evoked potentials (mVEPs) are neural potentials that are time-locked to the onset of motion of evoking stimuli. Due to their visually elegant properties, mVEP stimuli may be suited to video game control given gaming's inherent demand on the users' visual attention and the requirement to process rapidly changing visual information. Here, we investigate mVEPs associated with five different stimuli to control the position of a car in a visually rich 3D racing game in a group of 15 BCI naïve teenagers and compared with 19 BCI naive adults. Results from an additional 14 BCI experienced adults were compared with BCI naïve adults. Our results demonstrate that the game control accuracy is related to the number of trials used to make a decision on the users' chosen button/stimulus (76%, 62%, and 35% for 5, 3, and 1 trials, respectively) and information transfer rate (ITR) (13.4, 13.9, and 6.6 bits per minute (BPM)), although, even though accuracy decreases when using three compared to the commonly used five trial repetitions, ITR is maintained. A Kruskal-Wallis test suggests that BCI naïve adults do not outperform BCI naïve teenagers in the 3D racing game in the first and seconds laps (p > 0.05), but do outperform in the third lap (p < 0.05). A comparison between BCI naïve and BCI experienced adults indicates BCI experienced adults do not perform better than BCI naïve adults (p > 0.05).

Associations of self-reports versus spouse ratings of negative affectivity, dominance, and affiliation with coronary artery disease: where should we look and who should we ask when studying personality and health?

OBJECTIVE: Aspects of negative affect and social behavior studied as risk factors for coronary heart disease are usually examined separately and through self-reports. Using structural models of these personality domains, we tested associations of self-reports and spouse ratings of anxiety, depressive symptoms, anger, affiliation and dominance with coronary artery disease (CAD). DESIGN: In 154 healthy older couples, the authors tested cross-sectional associations with CAD of three facets of negative affectivity and two dimensions of the Interpersonal Circumplex, (IPC) using scales derived from the NEO-PI-R. MAIN OUTCOME MEASURES: CAD was quantified as Agatston scores from CT scans of coronary artery calcification (CAC). RESULTS: Self-reports were generally unrelated to CAC, whereas spouse ratings were consistently associated, largely independent of potential confounds. When considered simultaneously, anxiety and anger were related to CAC but depression was not. When considered together, both dominance and (low) affiliation were related to CAC. CONCLUSIONS: Structural models of negative affectivity and social behavior can facilitate integrative study of psychosocial risk factors. Further, self-report measures of these traits might under-estimate related CHD risk.

UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis.

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair.

Age, marital satisfaction, and optimism as predictors of positive sentiment override in middle-aged and older married couples.

This study examined whether positive sentiment override (greater positive appraisal of spouse's affiliative behavior than is warranted by observed behavior) occurred more frequently in older compared with middle-aged married couples and whether age differences were mediated by older adults' greater marital satisfaction when controlling for optimism. Participants included 270 middle-aged (40-50 years old) and older (60-70 years old) couples who discussed a marital disagreement and completed an errand task. Couples provided appraisals of their spouse's affiliation, and the authors coded affiliative interactions using the structural analysis of social behavior. Hierarchical multivariate linear modeling indicated that older husbands and wives viewed their spouse's behavior as more positive during disagreement interactions than did independent observers; in the errand task, only older wives demonstrated positive sentiment override. Age differences in positive sentiment override were mediated by marital satisfaction, even when controlling for optimism. The results are consistent with theories of emotion regulation, such as socioemotional selectivity theory, that suggest that older adults are biased toward the positive aspects of close relationships.