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INTRODUCTION: To support shared decision-making (SDM) between patients and providers surrounding biologic treatments, we created IBD&me ( ibdandme.org )-a freely available, unbranded, interactive decision aid. We performed a multicenter comparative effectiveness trial comparing the impact of IBD&me on SDM vs a biologics fact sheet developed by the Crohn's & Colitis Foundation. METHODS: We enrolled patients with inflammatory bowel disease (IBD) being seen at a clinic within IBD Qorus-a multicenter adult IBD learning health system-between March 5, 2019, and May 14, 2021. Eligible patients included those with recent IBD-related symptoms who reported that they wanted to discuss biologics with their provider during their upcoming visit. Patients were randomized 1:1 using stratified block randomization and received an e-mail 1 week before their visit inviting them to review either IBD&me or a fact sheet. The primary outcome was patient perception of SDM as measured by the 9-Item SDM Questionnaire (0-100 scale; higher = better); the Student t test was used to compare outcomes between arms. RESULTS: Overall, 152 patients were randomized (biologics fact sheet 75, IBD&me 77); most patients had Crohn's disease (66.4%) and were biologic-experienced (82.9%). No differences were seen between groups regarding SDM (fact sheet 72.6 ± 25.6, IBD&me 75.0 ± 20.8; P = .57). Most patients stated they would be likely to recommend the fact sheet (79.6%) or IBD&me (84.9%; P = .48) to another patient with IBD. DISCUSSION: No differences in outcomes were seen between IBD&me and the biologics fact sheet in this comparative effectiveness study; patients reported high satisfaction with both resources. Further study, particularly among biologic naïve patients, is needed to determine the utility of interactive components to IBD decision aids.

Objective: Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system, resulting in a range of potential motor and cognitive impairments. The latter can affect both executive functions that orchestrate general goal-directed behaviour, and social cognitive processes that support our ability to interact with others and maintain healthy interpersonal relationships. Despite a long history of research into the cognitive symptoms of MS, it remains uncertain if social cognitive disruptions occur independently of, or reflect underlying disturbances to, more foundational executive functions. The present preregistered study investigated this directly. Method: Employing an experimental design, we administered a battery of computerised tasks online to a large sample comprising 134 individuals with MS and 134 age- and sex-matched healthy controls (HCs). Three tasks measured elements of executive function (working memory, response inhibition, and switching) and two assessed components of social cognition disrupted most commonly in MS (emotion perception and theory of mind). Results: Individuals with MS exhibited poorer working memory (d = .31), response inhibition (d = -.26), emotion perception (d = .32) and theory of mind (d = .35) compared with matched HCs. Furthermore, exploratory mediation analyses revealed that working memory performance accounted for approximately 20% of the group differences in both measures of social cognition. Conclusions: Disruptions to working memory appear to serve as one of the mechanisms underpinning disturbances to social cognition in MS. Future research should therefore examine if the benefits of cognitive rehabilitation programs that incorporate working memory training transfer to these social cognitive processes.

Supplementary Figures 1-6 from The miR-17-92 Cluster of MicroRNAs Confers Tumorigenicity by Inhibiting Oncogene-Induced Senescence

In mammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, including apoptosis and senescence, which are compromised by additional mutations before cancers are developed. The miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed in human cancers and has been shown to promote several aspects of oncogenic transformation, including evasion of apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced senescence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring resistance to ras-induced senescence by directly targeting p21WAF1, a key effector of senescence. By contrast, these components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, disruption of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transformation by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits apoptosis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster promotes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster. Cancer Res; 70(21); 8547–57. ©2010 AACR.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

We describe our institutional experience of developing a liquid biopsy approach using circulating tumor DNA (ctDNA) analysis for personalized medicine in cancer patients, focusing on the hurdles encountered during the multistep process in order to benefit other investigators wishing to set up this type of study in their institution. Blood samples were collected at the time of cancer surgery from 209 patients with one of nine different cancer types. Extracted tumor DNA and circulating cell-free DNA were sequenced using cancer-specific panels and the Illumina MiSeq machine. Almost half of the pairs investigated were uninformative, mostly because there was no trackable pathogenic mutation detected in the original tumor. The pairs with interpretable data corresponded to 107 patients. Analysis of 48 gene sequences common to both panels was performed and revealed that about 40% of these pairs contained at least one driver mutation detected in the DNA extracted from plasma. Here, we describe the choice of our overall approach, the selection of the cancer panels, and the difficulties encountered during the multistep process, including the use of several tumor types and in the data analysis. We also describe some case reports using longitudinal samples, illustrating the potential advantages and rewards in performing ctDNA sequencing to monitor tumor burden or guide treatment for cancer patients.

BackgroundLung cancer screening programs provide an opportunity to support smokers to quit, but the most appropriate model for delivery remains to be determined. Immediate face to face smoking cessation support for people undergoing screening can increase quit rates, but it is not known whether remote delivery of immediate smoking cessation counselling and pharmacotherapy in this context is also effective.Materials and MethodsIn a single-blind randomised controlled trial, smokers aged 55-75 years attending a Targeted Lung Health Check (TLHC) were allocated by day of attendance to receive either immediate telephone smoking cessation support (TSI) (starting immediately and lasting for 6 weeks) with appropriate pharmacotherapy, or usual care (very brief advice to quit and signposting to smoking cessation services) (UC). The primary outcome was self-reported 7-day point prevalence smoking abstinence at three months. Differences between groups were assessed using logistic regression.Results315 current smokers taking part in the screening programme, mean (SD) age 63(5.4) years, 48% female, were randomised to telephone smoking cessation (n=152) or usual care (n=163). The two groups were well-matched at baseline. Self-reported quit rates were higher in the intervention arm, 21.1% vs 8.9% (odds ratio [OR]: 2.83, 95% CI 1.44-5.61, p=0.002). Controlling for participant demographics, baseline smoking characteristics or the discovery of abnormalities on low dose CT scanning did not modify the effect of the intervention.ConclusionImmediate provision of an intensive telephone-based smoking cessation intervention, delivered within a targeted lung screening context, is associated with increased smoking abstinence at three months.Trial registrationThis study is registered online: ISRCTN12455871.Take home messageProviding immediate, telephone smoking cessation support with pharmacotherapy to smokers enrolled in a TLHC program increases quit rates in this population by almost a third.

AimsTo investigate whether ‘low sugar’ and related claims influence consumers’ perceptions of the healthiness or other attributes of alcoholic drinks, promote greater consumption or impact diet and activity behaviour intentions.MethodN = 501 Australian women aged 18–35 viewed and rated six images of alcoholic drinks in a randomized online experiment with a 2 (claim: low sugar claim, no claim control) × 2 (drink type: cider, ready-to-drink spirits) between-subjects design.ResultsParticipants who viewed drinks with low sugar claims rated them as healthier, less harmful to health, lower in sugar and kilojoules, and more suitable for weight management and a healthy diet than participants who viewed identical drinks with no claim (P ConclusionsLow sugar and related claims on alcoholic drinks can generate a health halo: consumers generalise from a specific favourable attribute (low sugar) to misperceive other favourable health- and nutrition-related attributes, including lower alcohol content. Findings support calls to reconsider the permissibility of low sugar claims on alcoholic drinks as they may mislead consumers.

INTRODUCTION: Although the genome is often called the blueprint of an organism, it is perhaps more accurate to describe it as a parts list composed of the various genes that may or may not be used in the different cell types of a multicellular organism. Although nearly every cell in the body has essentially the same genome, each cell type makes different use of that genome and expresses a subset of all possible genes. This has motivated efforts to characterize the molecular composition of various cell types within humans and multiple model organisms, both by transcriptional and proteomic approaches. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. RATIONALE: One caveat to current approaches to make cell atlases is that individual organs are often collected at different locations, collected from different donors, and processed using different protocols. Controlled comparisons of cell types between different tissues and organs are especially difficult when donors differ in genetic background, age, environmental exposure, and epigenetic effects. To address this, we developed an approach to analyzing large numbers of organs from the same individual. RESULTS: We collected multiple tissues from individual human donors and performed coordinated single-cell transcriptome analyses on live cells. The donors come from a range of ethnicities, are balanced by gender, have a mean age of 51 years, and have a variety of medical backgrounds. Tissue experts used a defined cell ontology terminology to annotate cell types consistently across the different tissues, leading to a total of 475 distinct cell types with reference transcriptome profiles. The full dataset can be explored online with the cellxgene tool. Data were collected for the bladder, blood, bone marrow, eye, fat, heart, kidney, large intestine, liver, lung, lymph node, mammary, muscle, pancreas, prostate, salivary gland, skin, small intestine, spleen, thymus, tongue, trachea, uterus, and vasculature. Fifty-nine separate specimens in total were collected, processed, and analyzed, and 483,152 cells passed quality control filtering. On a per-compartment basis, the dataset includes 264,824 immune cells, 104,148 epithelial cells, 31,691 endothelial cells, and 82,478 stromal cells. Working with live cells, as opposed to isolated nuclei, ensured that the dataset includes all mRNA transcripts within the cell, including transcripts that have been processed by the cell’s splicing machinery, thereby enabling insight into variation in alternative splicing. The Tabula Sapiens also provided an opportunity to densely and directly sample the human microbiome throughout the gastrointestinal tract. The intestines from two donors were sectioned into five regions: the duodenum, jejunum, ileum, and ascending and sigmoid colon. Each section was transected, and three to nine samples were collected from each location, followed by amplification and sequencing of the 16S ribosomal RNA gene. CONCLUSION: The Tabula Sapiens has revealed discoveries relating to shared behavior and subtle, organ-specific differences across cell types. We found T cell clones shared between organs and characterized organ-dependent hypermutation rates among B cells. Endothelial cells and macrophages are shared across tissues, often showing subtle but clear differences in gene expression. We found an unexpectedly large and diverse amount of cell type–specific RNA splice variant usage and discovered and validated many previously undefined splices. The intestinal microbiome was revealed to have nonuniform species distributions down to the 3-inch (7.62-cm) length scale. These are but a few examples of how the Tabula Sapiens represents a broadly useful reference to deeply understand and explore human biology at cellular resolution.

ObjectivesLung cancer screening programmes offer an opportunity to address tobacco dependence in current smokers. The effectiveness of different approaches to smoking cessation in this context has not yet been established. We investigated if immediate smoking cessation support, including pharmacotherapy, offered as part of a lung cancer screening programme, increases quit rates compared to usual care (Very Brief Advice to quit and signposting to smoking cessation services).Materials and methodsWe conducted a single-blind randomised controlled trial of current smokers aged 55–75 years attending a Targeted Lung Health Check. On randomly allocated days smokers received either (1) immediate support from a trained smoking cessation counsellor with appropriate pharmacotherapy or (2) usual care. The primary outcome was self-reported quit rate at 3 months. We performed thematic analysis of participant interview responses.ResultsOf 412 people attending between January and March 2020, 115 (27.9%) were current smokers; 46% female, mean (SD) 62.4 (5.3) years. Follow-up data were available for 84 smokers. At 3 months, quit rates in the intervention group were higher 14/48 (29.2%) vs 4/36 (11%) (χ23.98, p=0.04). Participant interviews revealed four smoking-cessation related themes: (1) stress and anxiety, (2) impact of the COVID-19 pandemic, (3) CT scans influencing desire to quit and (4) individual beliefs about stopping smoking.ConclusionThe provision of immediate smoking cessation support is associated with a substantial increase in quit rates at 3 months. Further research is needed to investigate longer-term outcomes and to refine future service delivery.Trial registration numberISRCTN12455871.

Significance Durable protection against COVID-19 infection may be achieved by generating robust T cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emerging SARS-CoV-2 variants; for those infected, effective treatments are urgently needed. For these strategies to be successful, accurate identification of T cell epitopes is critical. In this study, we used major histocompatibility complex immune precipitation, acid elution, and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycoprotein (MGP) and the nonstructural protein. Furthermore, taking advantage of a highly robust endogenous T cell workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID-19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally if not more important than humoral responses in mediating recovery and immune protection. One major challenge in developing T cell–based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities. Our studies find that, in contrast to current convention, “immunodominant” SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from the naturally processed peptide–major histocompatibility complex (MHC) and then validating immunogenicity by determining whether such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes derived from not only structural but also nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, there are no reported T cell receptor–engineered T cell technology that can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here several SARS-CoV-2 epitopes defined by mass spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity, and demonstrate engineered TCR-redirected killing.

Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by pro-inflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin’s B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin’s enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.

Singlet fission (SF) is a photophysical process in which one of two adjacent organic molecules absorbs a single photon, resulting in rapid formation of a correlated triplet pair (T(1)T(1)) state whose spin dynamics influence the successful generation of uncorrelated triplets (T(1)). Femtosecond transient visible and near-infrared absorption spectroscopy of a linear terrylene-3,4:11,12-bis(dicarboximide) dimer (TDI(2)), in which the two TDI molecules are directly linked at one of their imide positions, reveals ultrafast formation of the (T(1)T(1)) state. The spin dynamics of the (T(1)T(1)) state and the processes leading to uncoupled triplets (T(1)) were studied at room temperature for TDI(2) aligned in 4-cyano-4′-pentylbiphenyl (5CB), a nematic liquid crystal. Time-resolved electron paramagnetic resonance spectroscopy shows that the (T(1)T(1)) state has mixed (5)(T(1)T(1)) and (3)(T(1)T(1)) character at room temperature. This mixing is magnetic field dependent, resulting in a maximum triplet yield at ∼200 mT. The accessibility of the (3)(T(1)T(1)) state opens a pathway for triplet–triplet annihilation that produces a single uncorrelated T(1) state. The presence of the (5)(T(1)T(1)) state at room temperature and its relationship with the (1)(T(1)T(1)) and (3)(T(1)T(1)) states emphasize that understanding the relationship among different (T(1)T(1)) spin states is critical for ensuring high-yield T(1) formation from singlet fission.

Technology-enhanced learning strategies have proliferated and/or re-purposed since the COVID-19 pandemic as education around the world transitioned to online learning. A review of existing literature has identified that many technologies used during the pandemic for remote emergency education were developed or utilised as a crisis management tool, without an emphasis on the student’s engagement or learning experience. Hence, it is worthwhile studying the effects of technology-enhanced learning strategies on students’ engagement to guide educators’ decisions in retaining or utilising certain technologies as the world returns to face-to-face teaching and hybrid learning. There is little attention within the literature on the effects of technology on student engagement post pandemic, and highly limited research that is supported by theoretical frameworks. As such, this study uses constructivism and connectivism to unpack and understand student engagement in this transforming education space. These frameworks are two of many learning theories that have been central to understanding how students learn. Connectivism and constructivism have been selected here for their relevance in the digital age where technological change is fast and ubiquitous, and for their emphasis on student autonomy and ability to connect information. This critical research is aiming at identifying successfully engaging technologies for healthcare and biomedical students at the University of Sydney and diagnose areas for improvement in the digital learning space. This study will look at all technologies, including those used prior to COVID-19 as well as crisis management tools. A hybrid student engagement scale, customised from existing scales in the literature, namely the Student Engagement in e-Learning Environment Scale (SELES) and Distance Education Learning Environment Scale (DELES), is used to measure student engagement across varying technologies in different class delivery modalities. A mixed quantitative and qualitative methods survey for healthcare and biomedical students is conducted utilising this customized hybrid scale to investigate how different technological strategies have enhanced their engagement during and post COVID-19 restrictions. Furthermore, interviews with both students and educators are conducted to understand in greater depth the factors behind student engagement with technologies. The quantitative data are analysed using SPSS for statistical interpretations while the qualitative outcomes are thematically analysed using NVivo. The presented findings of this study will inform future practices in using technology-enhanced strategies for better student engagement and their wider learning experience. Preliminary findings of the study indicate that whilst students are engaged in online learning using various technological tools, they prefer to experience face-to-face teaching supplemented by technologies that are used in an effective manner. Both students and educators have identified the need for learning to be focused on acquiring skills and graduate qualities, as opposed to traditional knowledge focused teaching methods, which is consistent with connectivism and constructivism frameworks. A full set of study results and their interpretations, as well as consolidated recommendations of a range of effective technologies and their practical features will be presented in the conference.

Trait self-control (TSC) is a well-known predictor of well-being. TSC may reduce distraction from hedonistic pursuits and enable more effective goal-directed behavior. Attachment anxiety (AA) is a trait that predicts lower relationship quality and lower well-being; people high on AA are characterized by hyper-sensitivity, proximity-seeking and excessive rumination. In retirement, these traits may interact to predict wellbeing. Methods: 120 retired participants took part in data collection (M = 62.37 years old; SD = 10.07 years; 68% women, 70% white). Data collection included self-reported AA (Experience in Close Relationships), TSC (Brief Self-Control Scale), depressive symptoms (Center for Epidemiological Studies Depression Scale) and emotional well-being (RAND 36-Item Health Survey). We conducted hierarchical linear regressions and accounted for demographic and health-related covariates. Results: TSC was negatively related to depressive symptoms and positively related to emotional well-being. AA was positively related to depressive symptoms and negatively related to emotional well-being. We found an interaction predicting depressive symptoms such that participants with low TSC and high AA had the highest depressive symptoms (p = .036). This interaction was not significant for emotional well-being. Discussion: After retiring, maintaining a healthy social life may be more effortful, as there are fewer ‘built-in’ social interactions (i.e., with coworkers). Retirees with higher TSC may be able to maintain their social life more effectively than those with lower TSC. Similarly, people with lower AA may be able to obtain more enjoyment from their social lives. Together, lower AA and higher TSC may be protective mechanisms for depressive symptoms.

Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.

SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, T cell responses are important in mediating recovery and immune-protection. The identification of immunogenic epitopes that can elicit a meaningful T cell response can be elusive. Traditionally, this has been achieved using sophisticatedin silicomethods to predict putative epitopes; however, our previous studies find that ‘immunodominant’ SARS-CoV-2 peptides defined by suchin silicomethods often fail to elicit T cell responses recognizing SARS-CoV-2. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined by directly analyzing peptides eluted from the peptide-MHC complex and then validating immunogenicity empirically by determining if such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes of SARS-CoV-2 derived not only from structural but also non-structural genes in regions highly conserved among SARS-CoV-2 strains including recently recognized variants. We report here, for the first time, several novel SARS-CoV-2 epitopes from membrane glycol-protein (MGP) and non-structure protein-13 (NSP13) defined by mass-spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity to induce T cell response.Significance StatementCurrent state of the art uses putative epitope peptides based onin silicoprediction algorithms to evaluate the T cell response among COVID-19 patients. However, none of these peptides have been tested for immunogenicity, i.e. the ability to elicit a T cell response capable of recognizing endogenously presented peptide. In this study, we used MHC immune-precipitation, acid elution and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycol-protein and the non-structural protein. Furthermore, taking advantage of a highly robust endogenous T cell (ETC) workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines.