Tumour immune rejection triggered by activation of α2-adrenergic receptors.

Immunotherapy based on immunecheckpoint blockade (ICB) using antibodies induces rejection of tumours and brings clinical benefit in patients with various cancer types1. However, tumours often resist immune rejection. Ongoing efforts trying to increase tumour response rates are based on combinations of ICB with compounds that aim to reduce immunosuppression in the tumour microenvironment but usually have little effect when used as monotherapies2,3. Here we show that agonists of α2-adrenergic receptors (α2-AR) have very strong anti-tumour activity when used as monotherapies in multiple immunocompetent tumour models, including ICB-resistant models, but not in immunodeficient models. We also observed marked effects in human tumour xenografts implanted in mice reconstituted with human lymphocytes. The anti-tumour effects of α2-AR agonists were reverted by α2-AR antagonists, and were absent in Adra2a-knockout (encoding α2a-AR) mice, demonstrating on-target action exerted on host cells, not tumour cells. Tumours from treated mice contained increased infiltrating T lymphocytes and reduced myeloid suppressor cells, which were more apoptotic. Single-cell RNA-sequencing analysis revealed upregulation of innate and adaptive immune response pathways in macrophages and T cells. To exert their anti-tumour effects, α2-AR agonists required CD4+ T lymphocytes, CD8+ T lymphocytes and macrophages. Reconstitution studies in Adra2a-knockout mice indicated that the agonists acted directly on macrophages, increasing their ability to stimulate T lymphocytes. Our results indicate that α2-AR agonists, some of which are available clinically, could substantially improve the clinical efficacy of cancer immunotherapy.

Correction: rbpms2 functions in Balbiani body architecture and ovary fate.

[This corrects the article DOI: 10.1371/journal.pgen.1007489.].

rbpms2 functions in Balbiani body architecture and ovary fate.

The most prominent developmental regulators in oocytes are RNA-binding proteins (RNAbps) that assemble their targets into ribonucleoprotein granules where they are stored, transported and translationally regulated. RNA-binding protein of multiple splice forms 2, or Rbpms2, interacts with molecules that are essential to reproduction and egg patterning, including bucky ball, a key factor for Bb formation. Rbpms2 is localized to germ granules in primordial germ cells (PGCs) and to the Balbiani body (Bb) of oocytes, although the mechanisms regulating Rbpms2 localization to these structures are unknown. Using mutant Rbpms2 proteins, we show that Rbpms2 requires distinct protein domains to localize within germ cells and somatic cells. Accumulation and localization to subcellular compartments in the germline requires an intact RNA binding domain. Whereas in zebrafish somatic blastula cells, the conserved C-terminal domain promotes localization to the bipolar centrosomes/spindle. To investigate Rbpms2 functions, we mutated the duplicated and functionally redundant zebrafish rbpms2 genes. The gonads of rbpms2a;2b (rbpms2) mutants initially contain early oocytes, however definitive oogenesis ultimately fails during sexual differentiation and, rbpms2 mutants develop as fertile males. Unlike other genes that promote oogenesis, failure to maintain oocytes in rbpms2 mutants was not suppressed by mutation of Tp53. These findings reveal a novel and essential role for rbpms2 in oogenesis. Ultrastructural and immunohistochemical analyses revealed that rbpms2 is not required for the asymmetric accumulation of mitochondria and Buc protein in oocytes, however its absence resulted in formation of abnormal Buc aggregates and atypical electron-dense cytoplasmic inclusions. Our findings reveal novel and essential roles for rbpms2 in Buc organization and oocyte differentiation.

Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells.

Ferroptosis is a cell death pathway that can be promoted by peroxidizable polyunsaturated fatty acids in cancer cells. Here, we investigated the mechanisms underlying the toxicity of punicic acid (PunA), an isomer of conjugated linolenic acids (CLnAs) bearing three conjugated double bonds highly prone to peroxidation, on prostate cancer (PCa) cells. PunA induced ferroptosis in PCa cells and triggered massive lipidome remodeling, more strongly in PC3 androgen-negative cells than in androgen-positive cells. The greater sensitivity of androgen-negative cells to PunA was associated with lower expression of glutathione peroxidase 4 (GPX4). We then identified the phospholipase PLA2G7 as a PunA-induced ferroptosis suppressor in PCa cells. Overexpressing PLA2G7 decreased lipid peroxidation levels, suggesting that PLA2G7 hydrolyzes hydroperoxide-containing phospholipids, thus preventing ferroptosis. Importantly, overexpressing both PLA2G7 and GPX4 strongly prevented PunA-induced ferroptosis in androgen-negative PCa cells. This study shows that PLA2G7 acts complementary to GPX4 to protect PCa cells from CLnA-induced ferroptosis.

Arid3c identifies an uncharacterized subpopulation of V2 interneurons during embryonic spinal cord development.

Motor activity is organized by neuronal networks composed of motor neurons and a wide variety of pre-motor interneuron populations located in the brainstem and spinal cord. Differential expression and single-cell RNA sequencing studies recently unveiled that these populations subdivide into multiple subsets. However, some interneuron subsets have not been described yet, and the mechanisms contributing to this neuronal diversification have only been partly deciphered. In this study, we aimed to identify additional markers to further describe the diversity of spinal V2 interneuron populations. Here, we compared the transcriptome of V2 interneurons with that of the other cells of the embryonic spinal cord and extracted a list of genes enriched in V2 interneurons, including Arid3c. Arid3c identifies an uncharacterized subset of V2 that partially overlaps with V2c interneurons. These two populations are characterized by the production of Onecut factors and Sox2, suggesting that they could represent a single functional V2 unit. Furthermore, we show that the overexpression or inactivation of Arid3c does not alter V2 production, but its absence results in minor defects in locomotor execution, suggesting a possible function in subtle aspects of spinal locomotor circuit formation.

Plasma proteomics in children with new-onset type 1 diabetes identifies new potential biomarkers of partial remission.

Partial remission (PR) occurs in only half of people with new-onset type 1 diabetes (T1D) and corresponds to a transient period characterized by low daily insulin needs, low glycemic fluctuations and increased endogenous insulin secretion. While identification of people with newly-onset T1D and significant residual beta-cell function may foster patient-specific interventions, reliable predictive biomarkers of PR occurrence currently lack. We analyzed the plasma of children with new-onset T1D to identify biomarkers present at diagnosis that predicted PR at 3 months post-diagnosis. We first performed an extensive shotgun proteomic analysis using Liquid Chromatography-Tandem-Mass-Spectrometry (LCMS/MS) on the plasma of 16 children with new-onset T1D and quantified 98 proteins significantly correlating with Insulin-Dose Adjusted glycated hemoglobin A1c score (IDAA1C). We next applied a series of both qualitative and statistical filters and selected protein candidates that were associated to pathophysiological mechanisms related to T1D. Finally, we translationally verified several of the candidates using single-shot targeted proteomic (PRM method) on raw plasma. Taken together, we identified plasma biomarkers present at diagnosis that may predict the occurrence of PR in a single mass-spectrometry run. We believe that the identification of new predictive biomarkers of PR and ß-cell function is key to stratify people with new-onset T1D for ß-cell preservation therapies.

A New Tool to Identify Pediatric Patients with Atypical Diabetes Associated with Gene Polymorphisms.

BACKGRUOUND: Recent diabetes subclassifications have improved the differentiation between patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus despite several overlapping features, yet without considering genetic forms of diabetes. We sought to facilitate the identification of monogenic diabetes by creating a new tool that we validated in a pediatric maturity-onset diabetes of the young (MODY) cohort. METHODS: We first created the DIAgnose MOnogenic DIAbetes (DIAMODIA) criteria based on the pre-existing, but incomplete, MODY calculator. This new score is composed of four strong and five weak criteria, with patients having to display at least one weak and one strong criterion. RESULTS: The effectiveness of the DIAMODIA criteria was evaluated in two patient cohorts, the first consisting of patients with confirmed MODY diabetes (n=34) and the second of patients with T1DM (n=390). These DIAMODIA criteria successfully detected 100% of MODY patients. Multiple correspondence analysis performed on the MODY and T1DM cohorts enabled us to differentiate MODY patients from T1DM. The three most relevant variables to distinguish a MODY from T1DM profile were: lower insulin-dose adjusted A1c score ≤9, glycemic target-adjusted A1c score ≤4.5, and absence of three anti-islet cell autoantibodies. CONCLUSION: We validated the DIAMODIA criteria, as it effectively identified all monogenic diabetes patients (MODY cohort) and succeeded to differentiate T1DM from MODY patients. The creation of this new and effective tool is likely to facilitate the characterization and therapeutic management of patients with atypical diabetes, and promptly referring them for genetic testing which would markedly improve clinical care and counseling, as well.

Post-Hypoglycemic hyperglycemia are highly relevant markers for stratification of glycemic variability and partial remission status of pediatric patients with new-onset type 1 diabetes.

AIMS: To evaluate whether parameters of post-hypoglycemic hyperglycemia (PHH) correlated with glucose homeostasis during the first year after type 1 diabetes onset and helped to distinguish pediatric patients undergoing partial remission or not. METHODS: In the GLUREDIA (GLUcagon Response to hypoglycemia in children and adolescents with new-onset type 1 DIAbetes) study, longitudinal values of clinical parameters, continuous glucose monitoring metrics and residual ß-cell secretion from children with new-onset type 1 diabetes were analyzed during the first year after disease onset. PHH parameters were calculated using an in-house algorithm. Correlations between PHH parameters (i.e., PHH frequency, PHH duration, PHH area under the curve [PHHAUC]) and glycemic homeostasis markers were studied using adjusted mixed-effects models. RESULTS: PHH parameters were strong markers to differentiate remitters from non-remitters with PHH/Hyperglycemia duration ratio being the most sensitive (ratio<0.02; sensitivity = 86% and specificity = 68%). PHHAUC moderately correlated with parameters of glucose homeostasis including TIR (R2 = 0.35, p-value < 0.05), coefficient of variation (R2 = 0.22, p-value < 0.05) and Insulin-Dose Adjusted A1c (IDAA1C) (R2 = 0.32, p-value < 0.05) and with residual ß-cell secretion (R2 = 0.17, p-value < 0.05). Classification of patients into four previously described glucotypes independently validated PHH parameters as reliable markers of glucose homeostasis and improved the segregation of patients with intermediate values of IDAA1C and estimated C-peptide (CPEPEST). Finally, a combination of PHH parameters identified groups of patients with specific patterns of hypoglycemia. CONCLUSION: PHH parameters are new minimal-invasive markers to discriminate remitters from non-remitters and evaluate glycemic homeostasis during the first year of type 1 diabetes. PHH parameters may also allow patient-targeted therapeutic management of hypoglycemic episodes.

The experience of being a caregiver of patients with digestive cancer, from patients and caregivers' perception: A mixed study.

BACKGROUNDS: Caregivers are essential in the care of a patient with digestive cancer. Considering their experience and needs is crucial. OBJECTIVES: To explore the experience of caregivers of patients with digestive cancer and to compare the perspectives of patients and caregivers. METHODS: A mixed-methods study with a cross-sectional prospective and a comprehensive qualitative dimension was performed in a medical oncology unit in a French tertiary hospital. Dyads made of patients with digestive cancer and their caregiver were recruited. The Caregiver Reaction Assessment (CRA) and the Supportive Care Needs Survey for Partners and Caregivers (SCNS-PC) questionnaires were distributed to caregivers. The CRA was used to measure the caregiver burden and the SCNS-PC was used to identify the unmet supportive care needs of caregivers. Semi-structured interviews with the dyads were conducted. Qualitative interviews addressed various dimensions of the caregiver's experience from each dyad's member perspective. RESULTS: Thirty-two caregivers completed the questionnaires. Responses showed high self-esteem, schedule burden, and a need for care and information services. Ten dyads participated in the interviews. Three themes emerged from the caregiver's interviews: illness is an upheaval; loneliness and helplessness are experienced; caring is a natural role with positive outcomes. Four themes emerged from patient's interviews: the caregiver naturally assumes the role and gets closer; he is the patient's anchor; his life is disrupted; anxiety and guilt accompany the desire to protect him. In comparing patient and caregiver data, the main theme of disagreement was their relationship. CONCLUSIONS: Caregiver care does not appear to be optimal, particularly in terms of their need for information. Patients have a fairly good representation of their experience, but the caregivers' opinion need to be considered.

Splenectomy improves erythrocyte functionality in spherocytosis based on septin abundance, but not maturation defects.

Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the ß-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in ß-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases.

Glycemic Variability Patterns Strongly Correlate With Partial Remission Status in Children With Newly Diagnosed Type 1 Diabetes.

OBJECTIVE: To evaluate whether indexes of glycemic variability may overcome residual ß-cell secretion estimates in the longitudinal evaluation of partial remission in a cohort of pediatric patients with new-onset type 1 diabetes. RESEARCH DESIGN AND METHODS: Values of residual ß-cell secretion estimates, clinical parameters (e.g., HbA1c or insulin daily dose), and continuous glucose monitoring (CGM) from 78 pediatric patients with new-onset type 1 diabetes were longitudinally collected during 1 year and cross-sectionally compared. Circadian patterns of CGM metrics were characterized and correlated to remission status using an adjusted mixed-effects model. Patients were clustered based on 46 CGM metrics and clinical parameters and compared using nonparametric ANOVA. RESULTS: Study participants had a mean (± SD) age of 10.4 (± 3.6) years at diabetes onset, and 65% underwent partial remission at 3 months. ß-Cell residual secretion estimates demonstrated weak-to-moderate correlations with clinical parameters and CGM metrics (r2 = 0.05-0.25; P < 0.05). However, CGM metrics strongly correlated with clinical parameters (r2 >0.52; P < 0.05) and were sufficient to distinguish remitters from nonremitters. Also, CGM metrics from remitters displayed specific early morning circadian patterns characterized by increased glycemic stability across days (within 63-140 mg/dL range) and decreased rate of grade II hypoglycemia (P < 0.0001) compared with nonremitters. Thorough CGM analysis allowed the identification of four novel glucotypes (P < 0.001) that segregate patients into subgroups and mirror the evolution of remission after diabetes onset. CONCLUSIONS: In our pediatric cohort, combination of CGM metrics and clinical parameters unraveled key clinical milestones of glucose homeostasis and remission status during the first year of type 1 diabetes.

Urine metabolomics links dysregulation of the tryptophan-kynurenine pathway to inflammation and severity of COVID-19.

SARS-CoV-2 causes major disturbances in serum metabolite levels, associated with severity of the immune response. Despite the numerous advantages of urine for biomarker discovery, the potential association between urine metabolites and disease severity has not been investigated in coronavirus disease 2019 (COVID-19). In a proof-of-concept study, we performed quantitative urine metabolomics in patients hospitalized with COVID-19 and controls using LC-MS/MS. We assessed whether metabolites alterations were associated with COVID-19, disease severity, and inflammation. The study included 56 patients hospitalized with COVID-19 (26 non-critical and 30 critical disease); 16 healthy controls; and 3 controls with proximal tubule dysfunction unrelated to SARS-CoV-2. Metabolomic profiling revealed a major urinary increase of tryptophan metabolites kynurenine (P < 0.001), 3-hydroxykynurenine (P < 0.001) and 3-hydroxyanthranilate (P < 0.001) in SARS-CoV-2 infected patients. Urine levels of kynurenines were associated with disease severity and systemic inflammation (kynurenine, r 0.43, P = 0.001; 3-hydroxykynurenine, r 0.44, P < 0.001). Increased urinary levels of neutral amino acids and imino acid proline were also common in COVID-19, suggesting specific transport defects. Urine metabolomics identified major alterations in the tryptophan-kynurenine pathway, consistent with changes in host metabolism during SARS-CoV-2 infection. The association between increased urinary levels of kynurenines, inflammation and COVID-19 severity supports further evaluation of these easily available biomarkers.

The intra-mitochondrial O-GlcNAcylation system rapidly modulates OXPHOS function and ROS release in the heart.

Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we use an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc system without potential cytoplasmic confounding effects. We compare the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific inhibitor of glycoside hydrolase. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 display increased O-GlcNAcylation in response to NButGT. This is associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of pyruvate-malate, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of specific subunits of ETC complexes (CI, CIII) and TCA cycle enzymes. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

Inflammation-Induced Coagulopathy Substantially Differs Between COVID-19 and Septic Shock: A Prospective Observational Study.

Critical COVID-19, like septic shock, is related to a dysregulated systemic inflammatory reaction and is associated with a high incidence of thrombosis and microthrombosis. Improving the understanding of the underlying pathophysiology of critical COVID-19 could help in finding new therapeutic targets already explored in the treatment of septic shock. The current study prospectively compared 48 patients with septic shock and 22 patients with critical COVID-19 regarding their clinical characteristics and outcomes, as well as key plasmatic soluble biomarkers of inflammation, coagulation, endothelial activation, platelet activation, and NETosis. Forty-eight patients with matched age, gender, and co-morbidities were used as controls. Critical COVID-19 patients exhibited less organ failure but a prolonged ICU length-of-stay due to a prolonged respiratory failure. Inflammatory reaction of critical COVID-19 was distinguished by very high levels of interleukin (IL)-1ß and T lymphocyte activation (including IL-7 and CD40L), whereas septic shock displays higher levels of IL-6, IL-8, and a more significant elevation of myeloid response biomarkers, including Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) and IL-1ra. Subsequent inflammation-induced coagulopathy of COVID-19 also differed from sepsis-induced coagulopathy (SIC) and was characterized by a marked increase in soluble tissue factor (TF) but less platelets, antithrombin, and fibrinogen consumption, and less fibrinolysis alteration. In conclusion, COVID-19 inflammation-induced coagulopathy substantially differs from SIC. Modulating TF release and activity should be evaluated in critical COVID-19 patients.

PepsNMR for 1H NMR metabolomic data pre-processing.

In the analysis of biological samples, control over experimental design and data acquisition procedures alone cannot ensure well-conditioned 1H NMR spectra with maximal information recovery for data analysis. A third major element affects the accuracy and robustness of results: the data pre-processing/pre-treatment for which not enough attention is usually devoted, in particular in metabolomic studies. The usual approach is to use proprietary software provided by the analytical instruments' manufacturers to conduct the entire pre-processing strategy. This widespread practice has a number of advantages such as a user-friendly interface with graphical facilities, but it involves non-negligible drawbacks: a lack of methodological information and automation, a dependency of subjective human choices, only standard processing possibilities and an absence of objective quality criteria to evaluate pre-processing quality. This paper introduces PepsNMR to meet these needs, an R package dedicated to the whole processing chain prior to multivariate data analysis, including, among other tools, solvent signal suppression, internal calibration, phase, baseline and misalignment corrections, bucketing and normalisation. Methodological aspects are discussed and the package is compared to the gold standard procedure with two metabolomic case studies. The use of PepsNMR on these data shows better information recovery and predictive power based on objective and quantitative quality criteria. Other key assets of the package are workflow processing speed, reproducibility, reporting and flexibility, graphical outputs and documented routines.