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1.
Gene ; 806: 145921, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34454033

RESUMO

Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a ß-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via ß-adrenergic receptors in vivo.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Anti-Inflamatórios/farmacologia , Inflamação/prevenção & controle , Interleucina-10/genética , Extratos Vegetais/farmacologia , Receptores Adrenérgicos beta/genética , Administração Oral , Animais , Modelos Animais de Doenças , Efedrina/farmacologia , Regulação da Expressão Gênica , Injeções Intraperitoneais , Interleucina-10/agonistas , Interleucina-10/imunologia , Japão , Masculino , Medicina Kampo/métodos , Camundongos Endogâmicos BALB C , Camundongos Nus , Poli I-C/administração & dosagem , Poli I-C/antagonistas & inibidores , Receptores Adrenérgicos beta/imunologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
2.
Gene ; 806: 145922, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34454032

RESUMO

Gastric cancer (GC)-derived cell lines were generally used in basic cancer research and drug screening. However, it is always concerned about the difference between cultured cells and primary tumor by oncologists. To address this question, we compared differentially expressed genes (DEGs) in primary cancers, healthy tissues, and cell lines both in vitro and in silico. Seven reported genes with decreased expression in GCs by DNA methylation were analyzed in our cohort studies and experimentally validation. Selected datasets from TCGA (The Cancer Genome Atlas), CCLE (The Broad Institute Cancer Cell Line Encyclopedia), and GTEx (The Genotype-Tissue Expression project) were used to represent GCs, GC-derived cell lines, and healthy tissues respectively in the in silico analysis. Thirty gastric tissues together with six cell lines were used for validations. Unexpectedly, we experimentally found that reported cancer-related downregulated genes were only found in cancer cell lines but not in biopsies. The unchanged gene expressions in primary GCs were generally consistent with our cohort study, using information from cancerous (TCGA) and healthy tissues (GETx). Substantial differences were also found between DEGs of cancer tissues (TGCA)/ healthy tissues (GTEx) pair and cell lines (CCLE)/ healthy tissues (GTEx) pair, which confirmed the significant differences between primary cancer and cancer cell lines. Moreover, elevated expression of YWHAQ (14-3-3 δ) and THBS1 were observed in the GC biopsies, which might be potential biomarkers for GC diagnosis, considering the increased YWHAQ and THBS1 associated with poor survival rates in gastric cancer patients. In sum, it is suggested that cautions should be taken when using GC cell lines to study genes that show great differences between cell lines and tissues.


Assuntos
Proteínas 14-3-3/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Trombospondinas/genética , Proteínas 14-3-3/metabolismo , Idoso , Atlas como Assunto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Conjuntos de Dados como Assunto , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Cultura Primária de Células , Prognóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida , Trombospondinas/metabolismo , Células Tumorais Cultivadas
3.
Gene ; 806: 145920, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34455026

RESUMO

Depression is deemed a mood disorder characterized by a high rate of relapse. Therefore, overcoming of the recurrent depression is globally expecting. Kososan, a traditional Japanese herbal medicine, has been clinically used for mild depressive mood, and our previous studies have shown some evidence for its antidepressive-like efficacy in experimental animal models of depression. However, it remains unclear whether kososan has beneficial effects on recurrent depression. Here, we examined its effect using a mouse model of modified repeated social defeat stress (SDS) paradigm. Male BALB/c mice were exposed to a 5-min SDS from unfamiliar aggressive CD-1 mice for 5 days. Kososan extract (1.0 kg/kg/day) or an antidepressant milnacipran (60 mg/kg/day) was administered orally for 26 days (days 7-32) to depression-like mice with social avoidant behaviors on day 6. Single 5 min of SDS was subjected to mice recovered from the social avoidance on day 31, and then the recurrence of depression-like behaviors was evaluated on day 32. Hippocampal gene expression patterns were also assayed by DNA microarray analysis. Water- or milnacipran-administered mice resulted in a recurrence of depression-like behaviors by re-exposure of single SDS, whereas kososan-administered mice did not recur depression-like behaviors. Distinct gene expression patterns were also found for treating kososan and milnacipran. Collectively, this finding suggests that kososan exerts a preventive effect on recurrent depression-like behaviors in mice. Pretreatment of kososan is more useful for recurrent depression than that of milnacipran.


Assuntos
Antidepressivos/farmacologia , Depressão/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Proteínas do Tecido Nervoso/genética , Derrota Social , Estresse Psicológico/tratamento farmacológico , Administração Oral , Animais , Depressão/genética , Depressão/fisiopatologia , Depressão/psicologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Japão , Masculino , Medicina Kampo/métodos , Camundongos , Camundongos Endogâmicos BALB C , Milnaciprano/farmacologia , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Recidiva , Estresse Psicológico/genética , Estresse Psicológico/fisiopatologia
4.
Gene ; 806: 145928, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34455027

RESUMO

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.


Assuntos
Proteínas Aviárias/genética , Família 19 do Citocromo P450/genética , Gansos/genética , Regulação Enzimológica da Expressão Gênica , RNA Mensageiro/genética , Transcrição Genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/metabolismo , Família 19 do Citocromo P450/metabolismo , Feminino , Gansos/classificação , Regulação da Expressão Gênica no Desenvolvimento , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Filogenia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de Transcrição
5.
Gene ; 806: 145929, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34461150

RESUMO

The body color of Neocaridina denticulate sinensis is a compelling phenotypic trait, in which a cascade of carotenoid metabolic processes plays an important role. The study was conducted to compare the transcriptome of cephalothoraxes among three pigmentation phenotypes (red, blue, and chocolate) of N. denticulate sinensis. The purpose of this study was to explore the candidate genes associated with different colors of N. denticulate sinensis. Nine cDNA libraries in three groups were constructed from the cephalothoraxes of shrimps. After assembly, 75022 unigenes were obtained in total with an average length of 1026 bp and N50 length of 1876 bp. There were 45977, 25284, 23605, 21913 unigenes annotated in the Nr, Swissprot, KOG, and KEGG databases, respectively. Differential expression analysis revealed that there were 829, 554, and 3194 differentially expressed genes (DEGs) in RD vs BL, RD vs CH, and BL vs CH, respectively. These DEGs may play roles in the absorption, transport, and metabolism of carotenoids. We also emphasized that electron transfer across the inner mitochondrial membrane (IMM) was a key process in pigment metabolism. In addition, a total of 6328 simple sequence repeats (SSRs) were also detected in N. denticulate sinensis. The results laid a solid foundation for further research on the molecular mechanism of integument pigmentation in the crustacean and contributed to developing more attractive aquatic animals.


Assuntos
Proteínas de Artrópodes/genética , Carotenoides/metabolismo , Decápodes/genética , Pigmentação/genética , Transcriptoma , Animais , Organismos Aquáticos , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/metabolismo , Transporte Biológico , Cor , Bases de Dados Genéticas , Decápodes/anatomia & histologia , Decápodes/metabolismo , Água Doce , Regulação da Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Repetições de Microssatélites , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Anotação de Sequência Molecular , Característica Quantitativa Herdável
6.
Gene ; 806: 145935, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34478821

RESUMO

Soluble molecules of programmed death ligand 1 (sPD-L1) are known to modulate T-cell depletion, an important mechanism of hepatitis B virus (HBV) persistence and liver disease progression. In addition, PD-L1 polymorphisms in the 3'-UTR can influence PD-L1 expression and have been associated with cancer risk, although not definitively. The purpose of this study was to investigate the association of PD-L1 polymorphisms and circulating levels of sPD-L1 in HBV infection and live disease progression. In this study, five hundred fifty-one HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 186), liver cirrhosis (LC, n = 142) and hepatocellular carcinoma (HCC, n = 223) and 240 healthy individuals (HC) were enrolled. PD-L1 polymorphisms (rs2297136 and rs4143815) were genotyped by in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of PD-L1 polymorphisms with HBV infection as well as with progression of related liver diseases. Plasma sPD-L1 levels were quantified by ELISA assays. The PD-L1 rs2297136 AA genotype was associated with HBV infection susceptibility (HBV vs. HC: OR = 1.6; 95%CI = 1.1-2.3; p = 0.0087) and disease progression (LC vs. CHB: OR = 1.8; 95%CI = 1.1-2.9; p = 0.018). Whereas, the rs2297136 GG genotype was a protective factor for HCC development. Plasma sPD-L1 levels were significantly high in HBV patients (p < 0.0001) and higher in the LC followed by CHB and HCC groups. High sPD-L1 levels correlated with increased liver enzymes and with advanced liver disease progression (Child-pugh C > B > A, p < 0.0001) and BCLC classification (BCLC D > C > B > A, p = 0.031). We could, for the first time, conclude that PD-L1 rs2297136 polymorphism and plasma sPD-L1 protein levels associate with HBV infection and HBV-related liver disease progression.


Assuntos
Antígeno B7-H1/genética , Carcinoma Hepatocelular/genética , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Polimorfismo Genético , Regiões 3' não Traduzidas , Adulto , Idoso , Antígeno B7-H1/sangue , Biomarcadores/sangue , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Expressão Gênica , Predisposição Genética para Doença , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B Crônica/complicações , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes
7.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34028531

RESUMO

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-resident integral membrane proteins.


Assuntos
Gotículas Lipídicas , Proteínas de Membrana , Animais , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Fosfolipídeos , Saccharomyces cerevisiae
8.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912961

RESUMO

Septins colocalize with membrane sterol-rich regions and facilitate recruitment of cell wall synthases during wall remodeling. We show that null mutants missing an Aspergillus nidulans core septin present in hexamers and octamers (ΔaspAcdc11, ΔaspBcdc3 or ΔaspCcdc12) are sensitive to multiple cell wall-disturbing agents that activate the cell wall integrity MAPK pathway. The null mutant missing the octamer-exclusive core septin (ΔaspDcdc10) showed similar sensitivity, but only to a single cell wall-disturbing agent and the null mutant missing the noncore septin (ΔaspE) showed only very mild sensitivity to a different single agent. Core septin mutants showed changes in wall polysaccharide composition and chitin synthase localization. Mutants missing any of the five septins resisted ergosterol-disrupting agents. Hexamer mutants showed increased sensitivity to sphingolipid-disrupting agents. Core septins mislocalized after treatment with sphingolipid-disrupting agents, but not after ergosterol-disrupting agents. Our data suggest that the core septins are involved in cell wall integrity signaling, that all five septins are involved in monitoring ergosterol metabolism, that the hexamer septins are required for sphingolipid metabolism and that septins require sphingolipids to coordinate the cell wall integrity response.


Assuntos
Aspergillus nidulans , Septinas , Aspergillus nidulans/metabolismo , Parede Celular/metabolismo , Metabolismo dos Lipídeos , Septinas/genética , Septinas/metabolismo , Esfingolipídeos/metabolismo
9.
Ann Lab Med ; 42(1): 36-46, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374347

RESUMO

Background: The emergence of carbapenemase-producing Enterobacteriaceae (CPE) represents a major clinical problem. Recently, the occurrence of CPE has increased globally, but epidemiological patterns vary across region. We report the trends in the genotypic distribution and antimicrobial susceptibility of CPE isolated from rectal and clinical samples during a four-year period. Methods: Between January 2016 and December 2019, 1,254 nonduplicated CPE isolates were obtained from four university hospitals in Korea. Carbapenemase genotypes were determined by multiplex real-time PCR. Antimicrobial susceptibility was profiled using the Vitek 2 system (bioMérieux, Hazelwood, MO, USA) or MicroScan Walkaway-96 system (Siemens West Sacramento, CA, USA). The proportions of carbapenemase genotypes and nonsusceptibility were analyzed using Pearson's chi-square test. Results: Among the 1,254 CPE isolates, 486 (38.8%), 371 (29.6%), 357 (28.5%), 8 (0.6%), 8 (0.6%), and 24 (1.9%) were Klebsiella pneumoniae carbapenemase (KPC), oxacillinase (OXA)-48-like, New Delhi metallo-ß-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-ß-lactamase (VIM), and multiple producers, respectively. The predominant species was K. pneumoniae (72.6%), followed by Escherichia coli (6.5%). More than 90% of the isolates harboring KPC, NDM, and OXA-48-like were nonsusceptible to cephalosporins, aztreonam, and carbapenems. Conclusions: The impact of CPE is primarily due to KPC-, NDM-, and OXA-48-like-producing K. pneumoniae isolates. Isolates carrying these carbapenemase are mostly multidrug-resistant. Control strategies based on these genotypic distributions and antimicrobial susceptibilities of CPE isolates are required.


Assuntos
Anti-Infecciosos , Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Infecções por Enterobacteriaceae/epidemiologia , Genótipo , Hospitais Universitários , Humanos , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , República da Coreia , beta-Lactamases/genética
10.
Ann Lab Med ; 42(1): 54-62, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374349

RESUMO

Background: Associations between IgA nephropathy (IgAN) and HLA-DRB1 and -DQB1 alleles have been reported in several ethnic groups. We investigated the association of HLA-DRB1 and -DQB1 alleles with the predisposition for IgAN and disease progression to end-stage kidney disease (ESKD) in Korean patients. Methods: We analyzed HLA-DRB1 and -DQB1 genotypes in 399 IgAN patients between January 2000 and January 2019 using a LIFECODES sequence-specific oligonucleotide (SSO) typing kit (Immucor, Stamford, CT, USA) or a LABType SSO Typing Test (One Lambda, Canoga Park, CA, USA). Alleles with a significant difference in two-digit resolution were further analyzed using in-house sequence-based typing and sequence-specific primer PCR. As controls, 613 healthy hematopoietic stem cell donors were included. Kidney survival was analyzed in 281 IgAN patients with available clinical and laboratory data using Cox regression analysis. Where needed, P-values were adjusted using Bonferroni correction. Results: The allele frequencies of HLA-DRB1*04:05 (corrected P [Pc]<0.001), -DQB1 *04:01 (Pc=0.048), and -DQB1*03:02 (Pc=0.021) were significantly higher in IgAN patients than in controls, whereas those of HLA-DRB1*07:01, -DRB1*15:01, -DQB1*02:02, and -DQB1*06:02 (Pc<0.001 for all) were significantly lower in IgAN patients than in controls. The allele frequency of HLA-DQB1*05:03 (Pc=0.016) was significantly lower in the ESKD group than in the non-ESKD group; however, there was no significant difference for ESKD progression between these groups. Conclusions: We report novel associations of HLA-DRB1*15:01, DQB1*02:02, -DQB1*03:02, and -DQB1*04:01 with IgAN. Further studies of HLA alleles associated with IgAN progression in a larger cohort and in various ethnic groups are needed.


Assuntos
Glomerulonefrite por IGA , Alelos , Predisposição Genética para Doença , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/genética , Cadeias HLA-DRB1/genética , Teste de Histocompatibilidade , Humanos , República da Coreia
11.
Ann Lab Med ; 42(1): 79-88, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374352

RESUMO

Background: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are genomic imprinting disorders that are mainly caused by a deletion on 15q11-q13, the uniparental disomy of chromosome 15, or an imprinting defect. We evaluated the utility of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a diagnostic tool and for demonstrating the relationship between molecular mechanisms and clinical presentation. Methods: We performed MS-MLPA using DNA samples from 93 subjects (45 PWS, 24 AS, and 24 non-PWS/AS controls) who had previously undergone MS-PCR for the diagnosis of PWS/AS. We compared the results of both assays, and patients' clinical phenotypes were reviewed retrospectively. Results: MS-MLPA showed a 100% concordance rate with MS-PCR. Among the 45 PWS patients, 26 (57.8%) had a deletion of 15q11-q13, and the others (42.2%) had uniparental disomy 15 or an imprinting defect. Among the 24 AS patients, 16 (66.7%) had a deletion of 15q11-q13, 7 AS patients (29.2%) had uniparental disomy 15 or an imprinting defect, and one AS patient (4.2%) showed an imprinting center deletion. Conclusions: MS-MLPA has clinical utility for the diagnosis of PWS/AS, and it is superior to MS-PCR in that it can identify the molecular mechanism underlying the disease.


Assuntos
Síndrome de Angelman , Síndrome de Prader-Willi , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/genética , Cromossomos Humanos Par 15/genética , Metilação de DNA , Humanos , Metilação , Reação em Cadeia da Polimerase Multiplex , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Estudos Retrospectivos
12.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
13.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771931

RESUMO

The lipid composition of the primary cilia membrane is emerging as a critical regulator of cilia formation, maintenance and function. Here, we show that conditional deletion of the phosphoinositide 5'-phosphatase gene Inpp5e, mutation of which is causative of Joubert syndrome, in terminally developed mouse olfactory sensory neurons (OSNs), leads to a dramatic remodeling of ciliary phospholipids that is accompanied by marked elongation of cilia. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], which is normally restricted to the proximal segment redistributed to the entire length of cilia in Inpp5e knockout mice with a reduction in phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] and elevation of phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] in the dendritic knob. The redistribution of phosphoinositides impaired odor adaptation, resulting in less efficient recovery and altered inactivation kinetics of the odor-evoked electrical response and the odor-induced elevation of cytoplasmic Ca2+. Gene replacement of Inpp5e through adenoviral expression restored the ciliary localization of PI(4,5)P2 and odor response kinetics in OSNs. Our findings support the role of phosphoinositides as a modulator of the odor response and in ciliary biology of native multi-ciliated OSNs.


Assuntos
Neurônios Receptores Olfatórios , Animais , Cílios , Camundongos , Odorantes , Fosfolipídeos , Monoéster Fosfórico Hidrolases/genética
14.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000034

RESUMO

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.


Assuntos
Fosfatidilserinas , Saccharomyces cerevisiae , Membrana Celular , Microdomínios da Membrana , Fosfolipídeos , Saccharomyces cerevisiae/genética , Esfingolipídeos
15.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34080635

RESUMO

Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21 (also known as ZDHHC21), a member of the DHHC family of mammalian protein acyltransferases, mediates T cell receptor-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice, which express a functionally deficient version of DHHC21, we show that DHHC21 is a Ca2+/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We find that disruption of the Ca2+/calmodulin-binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+ T cells into Th1, Th2 and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.


Assuntos
Linfócitos T CD4-Positivos , Cálcio , Acetiltransferases , Aciltransferases/genética , Animais , Diferenciação Celular , Camundongos , Receptores de Antígenos de Linfócitos T/genética
16.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34479991

RESUMO

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.


Assuntos
Coronavirus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/virologia , Macrófagos/metabolismo , Animais , COVID-19/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais
17.
Virol J ; 18(1): 180, 2021 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-34482844

RESUMO

BACKGROUND: Covid-19 has the respiratory tract as the main target of infection, and patients present mainly dyspnea, pneumonia, dry cough, and fever. Nevertheless, organs outside the respiratory tract had been reported in recent studies, including the gastrointestinal tract and liver. The host innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptor (PRRs). Toll-like receptor 7 (TLR-7) is a pattern recognition receptor recognizing ssRNA (SARS-CoV-2 is an ssRNA). Polymorphisms are characterized by two or more alternative forms of a distinct phenotype in the same population. Polymorphisms in tlrs genes can negatively influence the immune response to infectious diseases. There are several references in the literature to non-synonymous single nucleotide (rs) polymorphisms related to several genes. Some of them are important for the innate immunity, as rs 179008 (tlr-7), rs3775291 (tlr3), rs8177374 (tir domain-containing adaptor protein, tirap), rs1024611 (monocyte chemoattractant protein-1, mcp-1) and rs61942233 (2'-5'-oligoadenylate synthase-3, oas-3). CASE PRESENTATION: We identified a 5-year-old-male child with gastrointestinal symptoms and fever presenting acholic stool and jaundice, who was positive for SARS-CoV-2 IgM, IgA, and IgG and presenting the Gln11Leu rs 179008 in tlr-7. The child presented high levels of aspartate aminotransferase, alanine aminotransferase, bilirubin, C-reactive protein, D-dimer, gamma-glutamyl transferase, alkaline phosphatase, and was negative for serological tests for hepatitis A, B, C, E, HIV 1 and 2, herpes virus, cytomegalovirus, Epstein-Barr virus, and negative for RTqPCR for Influenza A and B, RSV and SARS-CoV-2. We also investigated other SNPs in the tlr-3 (rs3775291), tirap (rs8177374), mcp-1 (rs1024611), and oas-3 (rs61942233) genes, and no mutation was detected. After an interview with the child's caregivers, any possible accidental ingestion of drugs or hepatotoxic substances was ruled out. CONCLUSION: To our knowledge, this is the first report of a SARS-CoV-2 caused hepatitis in a male child that has the tlr-7 Gln11Leu rs 179008, which could impair an efficient initial immune response. The knowledge of the patient's immune deficiency could improve the treatment to correct this deficiency with specific medications.


Assuntos
COVID-19/genética , COVID-19/virologia , Hepatite Viral Humana/genética , Hepatite Viral Humana/virologia , Receptor 7 Toll-Like/genética , Anticorpos Antivirais/sangue , COVID-19/imunologia , Pré-Escolar , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Fezes/virologia , Hepatite Viral Humana/imunologia , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imunidade Inata , Influenza Humana , Masculino , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/isolamento & purificação
18.
Front Immunol ; 12: 693775, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484190

RESUMO

Small number of SARS-CoV-2 epidemic lineages did not efficiently exhibit a neutralization profile, while single amino acid mutation in the spike protein has not been confirmed in altering viral antigenicity resulting in immune escape. To identify crucial mutations in spike protein that escape humoral immune response, we evaluated the cross-neutralization of convalescent plasmas and RBD-specific monoclonal antibodies (mAbs) against various spike protein-based pseudoviruses. Three of 24 SARS-CoV-2 pseudoviruses containing different mutations in spike protein, including D614G, A475V, and E484Q, consistently showed an altered sensitivity to neutralization by convalescent plasmas. A475V and E484Q mutants are highly resistant to neutralization by mAb B38 and 2-4, suggesting that some crucial mutations in spike protein might evolve SARS-CoV-2 variants capable of escaping humoral immune response.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/imunologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Anticorpos Neutralizantes/imunologia , Convalescença , Humanos , Evasão da Resposta Imune , Imunidade Humoral , Testes de Neutralização , Ligação Proteica
19.
Front Immunol ; 12: 728513, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484238

RESUMO

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.


Assuntos
Anticorpos/efeitos adversos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Púrpura Trombocitopênica Idiopática/induzido quimicamente , Púrpura Trombocitopênica Idiopática/fisiopatologia , Adenoviridae/genética , Animais , Plaquetas/imunologia , Plaquetas/patologia , Vacinas contra COVID-19/imunologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Camundongos , Ativação Plaquetária/imunologia , Fator Plaquetário 4 , Coelhos
20.
Front Immunol ; 12: 728936, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484239

RESUMO

The use of minimal peptide sets offers an appealing alternative for design of vaccines and T cell diagnostics compared to conventional whole protein approaches. T cell immunogenicity towards peptides is contingent on binding to human leukocyte antigen (HLA) molecules of the given individual. HLA is highly polymorphic, and each variant typically presents a different repertoire of peptides. This polymorphism combined with pathogen diversity challenges the rational selection of peptide sets with broad immunogenic potential and population coverage. Here we propose PopCover-2.0, a simple yet highly effective method, for resolving this challenge. The method takes as input a set of (predicted) CD8 and/or CD4 T cell epitopes with associated HLA restriction and pathogen strain annotation together with information on HLA allele frequencies, and identifies peptide sets with optimal pathogen and HLA (class I and II) coverage. PopCover-2.0 was benchmarked on historic data in the context of HIV and SARS-CoV-2. Further, the immunogenicity of the selected SARS-CoV-2 peptides was confirmed by experimentally validating the peptide pools for T cell responses in a panel of SARS-CoV-2 infected individuals. In summary, PopCover-2.0 is an effective method for rational selection of peptide subsets with broad HLA and pathogen coverage. The tool is available at https://services.healthtech.dtu.dk/service.php?PopCover-2.0.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Peptídeos/imunologia , Alelos , Alergia e Imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Genótipo , Antígenos HLA/classificação , Humanos , Imunogenicidade da Vacina , Técnicas Imunológicas , Peptídeos/classificação , SARS-CoV-2/imunologia
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