RESUMO
Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.
Assuntos
Receptores de Antígenos Quiméricos , Animais , Camundongos , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Movimento Celular , Imunoterapia Adotiva , Antígeno-1 Associado à Função Linfocitária , Espectrina , Humanos , FemininoRESUMO
Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.
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Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Fagócitos/imunologia , Receptores CCR2/metabolismo , Animais , Apresentação de Antígeno , Apoptose , Movimento Celular , Células Cultivadas , Humanos , Imunidade Inata , Microscopia Intravital , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Receptores CCR2/genéticaRESUMO
Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Neutrófilos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/imunologia , Artrite Experimental/diagnóstico , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Quimiotaxia/genética , Quimiotaxia/imunologia , Colágeno/imunologia , Complemento C5a/imunologia , Complemento C5a/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Microscopia Intravital , Articulações/citologia , Articulações/imunologia , Leucotrieno B4/imunologia , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Polimorfismo de Nucleotídeo Único , Proteômica , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Imagem com Lapso de TempoRESUMO
Extensive efforts have been undertaken to combine superconductivity and the quantum Hall effect so that Cooper-pair transport between superconducting electrodes in Josephson junctions is mediated by one-dimensional edge states1-6. This interest has been motivated by prospects of finding new physics, including topologically protected quasiparticles7-9, but also extends into metrology and device applications10-13. So far it has proven challenging to achieve detectable supercurrents through quantum Hall conductors2,3,6. Here we show that domain walls in minimally twisted bilayer graphene14-18 support exceptionally robust proximity superconductivity in the quantum Hall regime, allowing Josephson junctions to operate in fields close to the upper critical field of superconducting electrodes. The critical current is found to be non-oscillatory and practically unchanging over the entire range of quantizing fields, with its value being limited by the quantum conductance of ballistic, strictly one-dimensional, electronic channels residing within the domain walls. The system described is unique in its ability to support Andreev bound states at quantizing fields and offers many interesting directions for further exploration.
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T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.
Assuntos
Inflamação/imunologia , Células Th1/imunologia , Células Th2/imunologia , Transferência Adotiva , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Quimiocinas/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT6/metabolismoRESUMO
Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.
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Estudo de Associação Genômica Ampla , Esquizofrenia , Alelos , Predisposição Genética para Doença/genética , Genômica , Humanos , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/genéticaRESUMO
Chronic inflammatory milieu in the tumor microenvironment (TME) leads to the recruitment and differentiation of myeloid-derived suppressor cells (MDSCs). Polymorphonuclear (PMN)-MDSCs, which are phenotypically and morphologically defined as a subset of neutrophils, cause major immune suppression in the TME, posing a significant challenge in the development of effective immunotherapies. Despite recent advances in our understanding of PMN-MDSC functions, the mechanism that gives rise to immunosuppressive neutrophils within the TME remains elusive. Both in vivo and in vitro, newly recruited neutrophils into the tumor sites remained activated and highly motile for several days and developed immunosuppressive phenotypes, as indicated by increased arginase 1 (Arg1) and dcTrail-R1 expression and suppressed anticancer CD8 T cell cytotoxicity. The strong suppressive function was successfully recapitulated by incubating naive neutrophils with cancer cell culture supernatant in vitro. Cancer metabolite secretome analyses of the culture supernatant revealed that both murine and human cancers released lipid mediators to induce the differentiation of immunosuppressive neutrophils. Liquid chromatography-mass spectrometry (LC-MS) lipidomic analysis identified platelet-activation factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) as a common tumor-derived lipid mediator that induces neutrophil differentiation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2), the PAF biosynthetic enzyme, is up-regulated in human pancreatic ductal adenocarcinoma (PDAC) and shows an unfavorable correlation with patient survival across multiple cancer types. Our study identifies PAF as a lipid-driven mechanism of MDSC differentiation in the TME, providing a potential target for cancer immunotherapy.
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Diferenciação Celular , Células Supressoras Mieloides , Neutrófilos , Fator de Ativação de Plaquetas , Microambiente Tumoral , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , Animais , Camundongos , Microambiente Tumoral/imunologia , Fator de Ativação de Plaquetas/metabolismo , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/imunologia , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Heat shock protein 101 (HSP101) in plants, and bacterial and yeast orthologs, is essential for thermotolerance. To investigate thermotolerance mechanisms involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a missense HSP101 allele (hot1-4). hot1-4 plants are sensitive to acclimation heat treatments that are otherwise permissive for HSP101 null mutants, indicating that the hot1-4 protein is toxic. We report one suppressor (shot2, suppressor of hot1-4 2) has a missense mutation of a conserved residue in CLEAVAGE STIMULATION FACTOR77 (CstF77), a subunit of the polyadenylation complex critical for mRNA 3' end maturation. We performed ribosomal RNA depletion RNA-Seq and captured transcriptional readthrough with a custom bioinformatics pipeline. Acclimation heat treatment caused transcriptional readthrough in hot1-4 shot2, with more readthrough in heat-induced genes, reducing the levels of toxic hot1-4 protein and suppressing hot1-4 heat sensitivity. Although shot2 mutants develop like the wild type in the absence of stress and survive mild heat stress, reduction of heat-induced genes and decreased HSP accumulation makes shot2 in HSP101 null and wild-type backgrounds sensitive to severe heat stress. Our study reveals the critical function of CstF77 for 3' end formation of mRNA and the dominant role of HSP101 in dictating the outcome of severe heat stress.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Poliadenilação/genética , Temperatura Alta , Resposta ao Choque Térmico/genética , Mutação/genética , Arabidopsis/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator Estimulador de Clivagem/genética , Fator Estimulador de Clivagem/metabolismoRESUMO
BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.
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Sepse , Trombocitopenia , Camundongos , Animais , Humanos , Monócitos/metabolismo , Trombocitopenia/metabolismo , Plaquetas/metabolismo , Imunidade , Sepse/metabolismo , Ativação PlaquetáriaRESUMO
Single-cell RNA-seq enabled in-depth study on tissue micro-environment and immune-profiling, where a crucial step is to annotate cell identity. Immune cells play key roles in many diseases, whereas their activities are hard to track due to their diverse and highly variable nature. Existing cell-type identifiers had limited performance for this purpose. We present HiCAT, a hierarchical, marker-based cell-type identifier utilising gene set analysis for statistical scoring for given markers. It features successive identification of major-type, minor-type and subsets utilising subset markers structured in a three-level taxonomy tree. Comparison with manual annotation and pairwise match test showed HiCAT outperforms others in major- and minor-type identification. For subsets, we qualitatively evaluated the marker expression profile demonstrating that HiCAT provide the clearest immune-cell landscape. HiCAT was also used for immune-cell profiling in ulcerative colitis and discovered distinct features of the disease in macrophage and T-cell subsets that could not be identified previously.
Assuntos
Perfilação da Expressão Gênica , Macrófagos , RNARESUMO
Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Glicocálix/metabolismo , Mucinas/metabolismo , Antineoplásicos/metabolismo , Neoplasias/terapiaRESUMO
Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues seems to be integrin independent and based on actomyosin-mediated protrusion and contraction, during inflammation, changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that the interstitial motility of T cells was critically dependent on Arg-Gly-Asp (RGD)-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to higher expression of integrin αV on effector CD4⺠T cells. By intravital multiphoton imaging, we found that the motility of CD4⺠T cells was dependent on αV expression. Selective blockade or knockdown of αV arrested T helper type 1 (TH1) cells in the inflamed tissue and attenuated local effector function. Our data demonstrate context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Movimento Celular/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Integrina alfaV/metabolismo , Animais , Derme/imunologia , Derme/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Inflamação/genética , Integrina alfaV/genética , Linfonodos/imunologia , Camundongos , Oligopeptídeos/metabolismo , Ligação Proteica , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Metagenomic studies show that diverse resident viruses inhabit the healthy gut; however, little is known about the role of these viruses in the maintenance of gut homeostasis. We found that mice treated with antiviral cocktail displayed more severe dextran sulfate sodium (DSS)-induced colitis compared with untreated mice. DSS-induced colitis was associated with altered enteric viral abundance and composition. When wild-type mice were reconstituted with Toll-like receptor 3 (TLR3) or TLR7 agonists or inactivated rotavirus, colitis symptoms were significantly ameliorated. Mice deficient in both TLR3 and TLR7 were more susceptible to DSS-induced experimental colitis. In humans, combined TLR3 and TLR7 genetic variations significantly influenced the severity of ulcerative colitis. Plasmacytoid dendritic cells isolated from inflamed mouse colon produced interferon-ß in a TLR3 and TLR7-dependent manner. These results imply that recognition of resident viruses by TLR3 and TLR7 is required for protective immunity during gut inflammation.
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Colite/imunologia , Trato Gastrointestinal/virologia , Interferon beta/imunologia , Glicoproteínas de Membrana/imunologia , Rotavirus/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Antivirais/farmacologia , Colite/induzido quimicamente , Células Dendríticas/imunologia , Sulfato de Dextrana , Microbioma Gastrointestinal , Trato Gastrointestinal/imunologia , Humanos , Inflamação/imunologia , Interferon beta/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Ribossômico 16S/genética , Receptor 3 Toll-Like/genética , Receptor 7 Toll-Like/genéticaRESUMO
A psychotherapeutic regimen that uses alternating bilateral sensory stimulation (ABS) has been used to treat post-traumatic stress disorder. However, the neural basis that underlies the long-lasting effect of this treatment-described as eye movement desensitization and reprocessing-has not been identified. Here we describe a neuronal pathway driven by the superior colliculus (SC) that mediates persistent attenuation of fear. We successfully induced a lasting reduction in fear in mice by pairing visual ABS with conditioned stimuli during fear extinction. Among the types of visual stimulation tested, ABS provided the strongest fear-reducing effect and yielded sustained increases in the activities of the SC and mediodorsal thalamus (MD). Optogenetic manipulation revealed that the SC-MD circuit was necessary and sufficient to prevent the return of fear. ABS suppressed the activity of fear-encoding cells and stabilized inhibitory neurotransmission in the basolateral amygdala through a feedforward inhibitory circuit from the MD. Together, these results reveal the neural circuit that underlies an effective strategy for sustainably attenuating traumatic memories.
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Ansiedade/psicologia , Ansiedade/terapia , Extinção Psicológica/fisiologia , Medo/fisiologia , Medo/psicologia , Vias Neurais/fisiologia , Colículos Superiores/citologia , Colículos Superiores/fisiologia , Animais , Ansiedade/fisiopatologia , Complexo Nuclear Basolateral da Amígdala/citologia , Complexo Nuclear Basolateral da Amígdala/fisiologia , Condicionamento Clássico/fisiologia , Retroalimentação Fisiológica , Masculino , Núcleo Mediodorsal do Tálamo/citologia , Núcleo Mediodorsal do Tálamo/fisiologia , Camundongos , Inibição Neural , Optogenética , Estimulação Luminosa , Transtornos de Estresse Pós-Traumáticos , Fatores de TempoRESUMO
Electric vehicles (EVs) are imposing ever-challenging standards on the lifetime and safety of lithium-ion batteries (LIBs); consequently, real-time nondestructive monitoring of battery cell degradation is highly desired. Unfortunately, high-nickel (Ni) layered oxides, the preferred LIB cathodes for EVs, undergo performance degradation originating from microcrack formation during cycling. Entropymetry is introduced as a real-time analytic tool for monitoring the evolution of microcracks in these cathodes along the state of charge. The entropy change of the layered cathode is associated with the lattice configuration and reflects the structural heterogeneity relevant to the evolution of these microcracks. The structural heterogeneity was correlated with peak broadening in in-situ X-ray diffractometry while varying the experimental conditions that affect crack formation such as the upper cutoff voltage during charging and the Ni-content of the active material. Entropymetry, proposed here as a nondestructive diagnostic tool, can contribute greatly to the safe and reliable operation of LIBs for EVs.
RESUMO
The selenium-binding protein 1 (SELENBP1) has been reported to be up-regulated in the prefrontal cortex (PFC) of schizophrenia patients in postmortem reports. However, no causative link between SELENBP1 and schizophrenia has yet been established. Here, we provide evidence linking the upregulation of SELENBP1 in the PFC of mice with the negative symptoms of schizophrenia. We verified the levels of SELENBP1 transcripts in postmortem PFC brain tissues from patients with schizophrenia and matched healthy controls. We also generated transgenic mice expressing human SELENBP1 (hSELENBP1 Tg) and examined their neuropathological features, intrinsic firing properties of PFC 2/3-layer pyramidal neurons, and frontal cortex (FC) electroencephalographic (EEG) responses to auditory stimuli. Schizophrenia-like behaviors in hSELENBP1 Tg mice and mice expressing Selenbp1 in the FC were assessed. SELENBP1 transcript levels were higher in the brains of patients with schizophrenia than in those of matched healthy controls. The hSELENBP1 Tg mice displayed negative endophenotype behaviors, including heterotopias- and ectopias-like anatomical deformities in upper-layer cortical neurons and social withdrawal, deficits in nesting, and anhedonia-like behavior. Additionally, hSELENBP1 Tg mice exhibited reduced excitabilities of PFC 2/3-layer pyramidal neurons and abnormalities in EEG biomarkers observed in schizophrenia. Furthermore, mice overexpressing Selenbp1 in FC showed deficits in sociability. These results suggest that upregulation of SELENBP1 in the PFC causes asociality, a negative symptom of schizophrenia.
Assuntos
Esquizofrenia , Humanos , Animais , Camundongos , Esquizofrenia/genética , Esquizofrenia/metabolismo , Córtex Pré-Frontal/metabolismo , Células Piramidais/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Proteínas de Ligação a Selênio/genética , Proteínas de Ligação a Selênio/metabolismoRESUMO
BACKGROUND: Deficiency of adenosine deaminase 1 (ADA) has broad clinical and genetic heterogeneity. Screening techniques can identify asymptomatic infants whose phenotype and prognosis are indeterminate, and who may carry ADA variants of unknown significance. OBJECTIVE: To systematically assess the pathogenic potential of rare ADA missense variants, and to better define the relationship of red cell deoxyadenosine nucleotide (dAXP) content to phenotype. METHODS: We expressed 46 ADA missense variants in the ADA-deficient SØ3834 strain of E. coli and defined Genotype Categories (GC) ranked I - IV by increasing expressed ADA activity. We assessed relationships among GC rank, red cell dAXP, and phenotype in 58 reference patients with 50 different genotypes. We used our GC ranking system to benchmark AlphaMissense for predicting variant pathogenicity, and a minigene assay to identify an exonic splicing variant in ADA exon 9. RESULTS: The 46 missense variants expressed â¼0.001% to â¼70% of WT ADA activity (40% had <0.05% of WT ADA activity and 50% expressed >1%). Red cell dAXP ranged from undetectable to >75% of total adenine nucleotides and correlated well with phenotype. Both red cell dAXP and clinical severity were inversely related to "txADA" (total ADA activity expressed by both inherited variants). Our GC scoring system performed better than AlphaMissense in assessing variant pathogenicity, particularly for less deleterious variants. CONCLUSION: For ADA deficiency, pathogenicity is a continuum and conditional, depending on the total ADA activity contributed by both inherited variants as indicated by GC rank. However, in patients with indeterminate phenotype identified by screening, red cell dAXP measured at diagnosis may have greater prognostic value than GC rank .
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Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Variação Genética , Pirazóis , Humanos , Benzodioxóis/farmacologia , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Mutação , Biossíntese de Proteínas/genética , Proteostase/efeitos dos fármacos , Pirazóis/farmacologia , Proteínas Ribossômicas/genéticaRESUMO
BACKGROUND: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. METHODS: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. RESULTS: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. CONCLUSIONS: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.
Assuntos
Diabetes Mellitus , Insuficiência Cardíaca , Humanos , Insuficiência Cardíaca/metabolismo , Volume Sistólico , Miocárdio/metabolismo , Diabetes Mellitus/patologia , Obesidade/patologia , Ácidos GraxosRESUMO
SUMMARY: With the continued deluge of results from genome-wide association and functional genomic studies, it has become increasingly imperative to quickly combine and visualize different layers of genetic and genomic data within a given locus to facilitate exploratory and integrative data analyses. While several tools have been developed to visualize locus-level genetic results, the limited speed, scalability and flexibility of current approaches remain a significant bottleneck. Here, we present a Julia package for high-performance genetics and genomics-related data visualization that enables fast, simultaneous plotting of hundreds of association results along with multiple relevant genomic annotations. Leveraging the powerful plotting and layout utilities from Makie.jl facilitates the customization and extensibility of every component of a plot, enabling generation of publication-ready figures. AVAILABILITY AND IMPLEMENTATION: The GeneticsMakie.jl package is open source and distributed under the MIT license via GitHub (https://github.com/mmkim1210/GeneticsMakie.jl). The GitHub repository contains installation instructions as well as examples and documentation for built-in functions. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.