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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic and contagious coronavirus that caused a global pandemic with 5.2 million fatalities to date. Questions concerning serologic features of long-term immunity, especially dominant epitopes mediating durable antibody responses after SARS-CoV-2 infection, remain to be elucidated. OBJECTIVE: We aimed to dissect the kinetics and longevity of immune responses in coronavirus disease 2019 (COVID-19) patients, as well as the epitopes responsible for sustained long-term humoral immunity against SARS-CoV-2. METHODS: We assessed SARS-CoV-2 immune dynamics up to 180 to 220 days after disease onset in 31 individuals who predominantly experienced moderate symptoms of COVID-19, then performed a proteome-wide profiling of dominant epitopes responsible for persistent humoral immune responses. RESULTS: Longitudinal analysis revealed sustained SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies in COVID-19 patients, along with activation of cytokine production at early stages after SARS-CoV-2 infection. Highly reactive epitopes that were capable of mediating long-term antibody responses were shown to be located at the spike and ORF1ab proteins. Key epitopes of the SARS-CoV-2 spike protein were mapped to the N-terminal domain of the S1 subunit and the S2 subunit, with varying degrees of sequence homology among endemic human coronaviruses and high sequence identity between the early SARS-CoV-2 (Wuhan-Hu-1) and current circulating variants. CONCLUSION: SARS-CoV-2 infection induces persistent humoral immunity in COVID-19-convalescent individuals by targeting dominant epitopes located at the spike and ORF1ab proteins that mediate long-term immune responses. Our findings provide a path to aid rational vaccine design and diagnostic development.
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COVID-19 , Anticuerpos Antivirales , Epítopos , Humanos , Inmunidad Humoral , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
Hematopoiesis is a complicated process involving a series of biological sub-processes that lead to the formation of various blood components. A widely accepted model of early hematopoiesis proceeds from long-term hematopoietic stem cells (LT-HSCs) to multipotent progenitors (MPPs) and then to lineage-committed progenitors. However, the molecular mechanisms of early hematopoiesis have not been fully characterized. In this study, we applied a computational strategy to identify the gene expression signatures distinguishing three types of closely related hematopoietic cells collected in recent studies: (1) hematopoietic stem cell/multipotent progenitor cells; (2) LT-HSCs; and (3) hematopoietic progenitor cells. Each cell in these cell types was represented by its gene expression profile among a total number of 20,475 genes. The expression features were analyzed by a Monte-Carlo Feature Selection (MCFS) method, resulting in a feature list. Then, the incremental feature selection (IFS) and a support vector machine (SVM) optimized with a sequential minimum optimization (SMO) algorithm were employed to access the optimal classifier with the highest Matthews correlation coefficient (MCC) value of 0.889, in which 6698 features were used to represent cells. In addition, through an updated program of MCFS method, seventeen decision rules can be obtained, which can classify the three cell types with an overall accuracy of 0.812. Using a literature review, both the rules and the top features used for building the optimal classifier were confirmed to be commonly used or potential biological markers for distinguishing the three cell types of HSPCs. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang.
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Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/metabolismo , Máquina de Vectores de Soporte , Transcriptoma/fisiología , HumanosRESUMEN
SLC39A7 (zip7) is a zinc transporter that plays a key role in intestinal epithelial self-renewal. However, little is known about SLC39A7 in colorectal cancer. To assess the biological function of SLC39A7 in colorectal cancer, the expression of SLC39A7 in human colorectal tumors and five colorectal cancer cell lines were evaluated by Oncomine Cancer Microarray Database and western blot analysis. In addition, short hairpin RNAs specifically targeting SLC39A7 were transfected into HCT116 and SW1116 cells to knockdown SLC39A7 expression. Then, the effects of SLC39A7 knockdown on colorectal cancer cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, colony-forming assay and flow cytometry. Our results showed that colorectal tumors have higher expression levels of SLC39A7 than normal colon tissues. Knockdown of SLC39A7 exhibited a significant decrease in cell viability and proliferation of colorectal cancer cells. It was also shown that knockdown of SLC39A7 interfered with cell cycle progression and induced G2/M cell cycle arrest, as well as boosted early and late apoptosis in colorectal cancer cells. Furthermore, downregulation of SLC39A7 promoted the cleavage of PARP and enhanced the expression of Bad, Caspase-9, and cleaved-Caspase-3, as well as suppressed Bcl-2 expression. In conclusion, our results suggest that SLC39A7 plays a crucial role in the proliferation and survival of colorectal cancer cells, which associates with colorectal tumorigenesis.
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Apoptosis/genética , Proteínas de Transporte de Catión/genética , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Interferencia de ARN , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HEK293 , HumanosRESUMEN
Numerous eukaryotic genes are alternatively spliced. Recently, deep transcriptome sequencing has skyrocketed proportion of alternatively spliced genes; over 95% human multi-exon genes are alternatively spliced. One fundamental question is: are all these alternative splicing (AS) events functional? To look into this issue, we studied the most common form of alternative 5' splice sites-GYNNGYs (Y = C/T), where both GYs can function as splice sites. Global analyses suggest that splicing noise (due to stochasticity of splicing process) can cause AS at GYNNGYs, evidenced by higher AS frequency in non-coding than in coding regions, in non-conserved than in conserved genes and in lowly expressed than in highly expressed genes. However, â¼20% AS GYNNGYs in humans and â¼3% in mice exhibit tissue-dependent regulation. Consistent with being functional, regulated GYNNGYs are more conserved than unregulated ones. And regulated GYNNGYs have distinctive sequence features which may confer regulation. Particularly, each regulated GYNNGY comprises two splice sites more resembling each other than unregulated GYNNGYs, and has more conserved downstream flanking intron. Intriguingly, most regulated GYNNGYs may tune gene expression through coupling with nonsense-mediated mRNA decay, rather than encode different proteins. In summary, AS at GYNNGY 5' splice sites is primarily splicing noise, and secondarily a way of regulation.
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Empalme Alternativo , Sitios de Empalme de ARN , Animales , Secuencia de Bases , Secuencia Conservada , Humanos , Macaca mulatta , Ratones , Especificidad de ÓrganosRESUMEN
In female mammals most X-linked genes are subject to X-inactivation. However, in humans some X-linked genes escape silencing, these escapees being candidates for the phenotypic aberrations seen in polyX karyotypes. These escape genes have been reported to be under stronger purifying selection than other X-linked genes. Although it is known that escape from X-inactivation is much more common in humans than in mice, systematic assays of escape in humans have to date employed only interspecies somatic cell hybrids. Here we provide the first systematic next-generation sequencing analysis of escape in a human cell line. We analyzed RNA and genotype sequencing data obtained from B lymphocyte cell lines derived from Europeans (CEU) and Yorubans (YRI). By replicated detection of heterozygosis in the transcriptome, we identified 114 escaping genes, including 76 not previously known to be escapees. The newly described escape genes cluster on the X chromosome in the same chromosomal regions as the previously known escapees. There is an excess of escaping genes associated with mental retardation, consistent with this being a common phenotype of polyX phenotypes. We find both differences between populations and between individuals in the propensity to escape. Indeed, we provide the first evidence for there being both hyper- and hypo-escapee females in the human population, consistent with the highly variable phenotypic presentation of polyX karyotypes. Considering also prior data, we reclassify genes as being always, never, and sometimes escape genes. We fail to replicate the prior claim that genes that escape X-inactivation are under stronger purifying selection than others.
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Expresión Génica , Genes Ligados a X , Discapacidad Intelectual/genética , Inactivación del Cromosoma X , Animales , Pueblo Asiatico/genética , Línea Celular , Evolución Molecular , Femenino , Variación Genética , Humanos , Masculino , Ratones , Tasa de Mutación , Fenotipo , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ARN , Población Blanca/genética , Cromosoma XRESUMEN
We report on a patient with a 47,XXY karyotype who presents a normal female phenotype, which is an extremely rare observation worldwide. The patient is infertile. Type B ultrasound scans and other tests suggested that her ovaries had completely failed. Microsatellite DNA marker analysis revealed that the 2 X chromosomes were derived from her mother and that this abnormality was caused by non-disjunction of the maternal X chromosomes during meiosis II. Copy number variation analysis identified 2 large de novo deletions in her Y chromosome. Remarkably, one of the deleted regions includes the SRY gene locus, which might explain her female phenotype. However, the genetic mechanism of her ovarian failure remains unclear. This paper is the first report of a 47,XXY female with ovarian failure.
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Síndrome de Klinefelter/diagnóstico , Insuficiencia Ovárica Primaria/diagnóstico , Adulto , Cromosomas Humanos Y/genética , Femenino , Eliminación de Gen , Genes sry , Humanos , Síndrome de Klinefelter/genética , Técnicas de Diagnóstico Molecular , Linaje , Polimorfismo de Nucleótido Simple , Insuficiencia Ovárica Primaria/genéticaRESUMEN
Genome-wide interaction-based association (GWIBA) analysis has the potential to identify novel susceptibility loci. These interaction effects could be missed with the prevailing approaches in genome-wide association studies (GWAS). However, no convincing loci have been discovered exclusively from GWIBA methods, and the intensive computation involved is a major barrier for application. Here, we developed a fast, multi-thread/parallel program named "pair-wise interaction-based association mapping" (PIAM) for exhaustive two-locus searches. With this program, we performed a complete GWIBA analysis on seven diseases with stringent control for false positives, and we validated the results for three of these diseases. We identified one pair-wise interaction between a previously identified locus, C1orf106, and one new locus, TEC, that was specific for Crohn's disease, with a Bonferroni corrected P < 0.05 (P = 0.039). This interaction was replicated with a pair of proxy linked loci (P = 0.013) on an independent dataset. Five other interactions had corrected P < 0.5. We identified the allelic effect of a locus close to SLC7A13 for coronary artery disease. This was replicated with a linked locus on an independent dataset (P = 1.09 × 10â»7). Through a local validation analysis that evaluated association signals, rather than locus-based associations, we found that several other regions showed association/interaction signals with nominal P < 0.05. In conclusion, this study demonstrated that the GWIBA approach was successful for identifying novel loci, and the results provide new insights into the genetic architecture of common diseases. In addition, our PIAM program was capable of handling very large GWAS datasets that are likely to be produced in the future.
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Enfermedad/genética , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de Crohn/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Femenino , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: T cells are key players in the tumor immune microenvironment (TIME), as they can recognize and eliminate cancer cells that express neoantigens derived from somatic mutations. However, the diversity and specificity of T-cell receptors (TCRs) that recognize neoantigens are largely unknown, due to the high variability of TCR sequences among individuals. METHODS: To address this challenge, we applied GLIPH2, a novel algorithm that groups TCRs based on their predicted antigen specificity and HLA restriction, to cluster the TCR repertoire of 1,702 patients with digestive tract cancer. The patients were divided into five groups based on whether they carried tumor-infiltrating or clonal-expanded TCRs and calculated their TCR diversity. The prognosis, tumor subtype, gene mutation, gene expression, and immune microenvironment of these groups were compared. Viral specificity inference and immunotherapy relevance analysis performed for the TCR groups. RESULTS: This approach reduced the complexity of TCR sequences to 249 clonally expanded and 150 tumor-infiltrating TCR groups, which revealed distinct patterns of TRBV usage, HLA association, and TCR diversity. In gastric adenocarcinoma (STAD), patients with tumor-infiltrating TCRs (Patients-TI) had significantly worse prognosis than other patients (Patients-nonTI). Patients-TI had richer CD8+ T cells in the immune microenvironment, and their gene expression features were positively correlated with immunotherapy response. We also found that tumor-infiltrating TCR groups were associated with four distinct tumor subtypes, 26 common gene mutations, and 39 gene expression signatures. We discovered that tumor-infiltrating TCRs had cross-reactivity with viral antigens, indicating a possible link between viral infections and tumor immunity. CONCLUSION: By applying GLIPH2 to TCR sequences from digestive tract tumors, we uncovered novel insights into the tumor immune landscape and identified potential candidates for shared TCRs and neoantigens.
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Neoplasias Gastrointestinales , Receptores de Antígenos de Linfocitos T , Humanos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T CD8-positivos , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismo , Inmunoterapia , Antígenos de Neoplasias , Microambiente TumoralRESUMEN
The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines.
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Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Serina Proteasas/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Fluorescentes Verdes , Humanos , Lentivirus , Ratones , Microscopía Fluorescente , Fenilalanina/análogos & derivados , Serina Endopeptidasas , Transducción de Señal/genética , Especificidad por Sustrato , Tiofenos , Transducción GenéticaRESUMEN
Congenital cataracts cause 10-30% of all blindness in children, with one-third of cases estimated to have a genetic cause. Lamellar cataract is the most common type of infantile cataract. We carried out whole-genome linkage analysis of Chinese individuals with lamellar cataract, and found that the disorder is associated with inheritance of a 5.11-cM locus on chromosome 16. This locus coincides with one previously described for Marner cataract. We screened individuals of three Chinese families for mutations in HSF4 (a gene at this locus that encodes heat-shock transcription factor 4) and discovered that in each family, a distinct missense mutation, predicted to affect the DNA-binding domain of the protein, segregates with the disorder. We also discovered an association between a missense mutation and Marner cataract in an extensive Danish family. We suggest that HSF4 is critical to lens development.
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Catarata/genética , Proteínas de Unión al ADN/genética , Mutación Missense , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Catarata/congénito , Preescolar , Cromosomas Humanos Par 16 , Secuencia Conservada , Femenino , Ligamiento Genético , Genoma Humano , Factores de Transcripción del Choque Térmico , Humanos , Lactante , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
X chromosome dosage compensation (XDC) refers to the process by which X-linked genes acquire expression equivalence between two sexes. Ohno proposed that XDC is achieved by two-fold upregulations of X-linked genes in both sexes and by silencing one X chromosome (X chromosome inactivation, XCI) in females. However, genes subject to two-fold upregulations as well as the underlying mechanism remain unclear. It's reported that gene dosage changes may only affect X-linked dosage-sensitive genes, such as protein complex coding genes (PCGs). Our results showed that in human PCGs are more likely to escape XCI and escaping PCGs (EsP) show two-fold higher expression than inactivated PCGs (InP) or other X-linked genes at RNA and protein levels in both sexes, which suggest that EsP may achieve upregulations and XDC. The higher expressions of EsP possibly result from the upregulations of the single active X chromosome (Xa), rather than escaping expressions from the inactive X chromosome (Xi). EsP genes have relatively high expression levels in humans and lower dN/dS ratios, suggesting that they are likely under stronger selection pressure over evolutionary time. Our study also suggests that SP1 transcription factor is significantly enriched in EsP and may be involved in the up-regulations of EsP on the active X. Finally, human EsP genes in this study are enriched in the toll-like receptor pathway, NF-kB pathway, apoptotic pathway, and abnormal mental, developmental and reproductive phenotypes. These findings suggest misregulations of EsP may be involved in autoimmune, reproductive, and neurological diseases, providing insight for the diagnosis and treatment of these diseases.
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Phosphatidylinositol 3-kinase alpha (PI3Kα) inhibitors are currently evaluated for the therapy of esophageal squamous cell carcinoma (ESCC). It is of great importance to identify potential biomarkers to predict or monitor the efficacy of PI3Kα inhibitors in an aim to improve the clinical responsive rate in ESCC. Here, ESCC PDXs with CCND1 amplification were found to be more sensitive to CYH33, a novel PI3Kα-selective inhibitor currently in clinical trials for the treatment of advanced solid tumors including ESCC. Elevated level of cyclin D1, p21 and Rb was found in CYH33-sensitive ESCC cells compared to those in resistant cells. CYH33 significantly arrested sensitive cells but not resistant cells at G1 phase, which was associated with accumulation of p21 and suppression of Rb phosphorylation by CDK4/6 and CDK2. Hypo-phosphorylation of Rb attenuated the transcriptional activation of SKP2 by E2F1, which in turn hindered SKP2-mediated degradation of p21 and reinforced accumulation of p21. Moreover, CDK4/6 inhibitors sensitized resistant ESCC cells and PDXs to CYH33. These findings provided mechanistic rationale to evaluate PI3Kα inhibitors in ESCC patients harboring amplified CCND1 and the combined regimen with CDK4/6 inhibitors in ESCC with proficient Rb.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Carcinoma de Células Escamosas de Esófago/metabolismo , Neoplasias Esofágicas/metabolismo , Proliferación Celular , Fosforilación , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Here, we present a protocol for exploring the effects of PPP1R15A inhibitor, Sephin1, on antitumor immunity of B16F1 subcutaneous tumor in mice. We describe steps for constructing single-cell transcriptome and TCR libraries, sequencing, and using sequencing data for the integration of expression and TCR data. We then detail procedures for gene differentiation, regulon and cell-cell communication analysis, and validation of single-cell analysis results. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
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Comunicación Celular , Neoplasias , Animales , Ratones , Modelos Animales de Enfermedad , Análisis de la Célula Individual , Receptores de Antígenos de Linfocitos TRESUMEN
Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, T cell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1. A special TCR + macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.
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Familial progressive hyperpigmentation (FPH) is an autosomal-dominantly inherited disorder characterized by hyperpigmented patches in the skin, present in early infancy and increasing in size and number with age. The genetic basis for FPH remains unknown. In this study, a six-generation Chinese family with FPH was subjected to a genome-wide scan for linkage analysis. Two-point linkage analysis mapped the locus for FPH at chromosome 12q21.31-q23.1, with a maximum two-point LOD score of 4.35 (Ø = 0.00) at D12S81. Haplotype analysis confined the locus within an interval of 9.09 cM, flanked by the markers D12S1667 and D12S2081. Mutation profiling of positional candidate genes detected a heterozygous transversion (c. 107A-->G) in exon 2 of the KIT ligand (KITLG) gene, predicted to result in the substitution of a serine residue for an asparagine residue at codon 36 (p.N-->S). This mutant "G" allele cosegregated perfectly with affected, but not with unaffected, members of the FPH family. Function analysis of the soluble form of sKITLG revealed that mutant sKITLGN36S increased the content of the melanin by 109% compared with the wild-type sKITLG in human A375 melanoma cells. Consistent with this result, the tyrosinase activity was significantly increased by mutant sKITLGN36S compared to wild-type control. To our knowledge, these data provided the first genetic evidence that the FPH disease is caused by the KITLGN36S mutation, which has a gain-of-function effect on the melanin synthesis and opens a new avenue for exploration of the genetic mechanism of FPH.
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Predisposición Genética a la Enfermedad , Hiperpigmentación/genética , Melaninas/metabolismo , Factor de Células Madre/genética , Cromosomas Humanos Par 12/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Hiperpigmentación/metabolismo , Masculino , Persona de Mediana Edad , Linaje , Factor de Células Madre/metabolismoRESUMEN
In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.
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Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Extensiones de la Superficie Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cristalino/citología , Cristalino/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Regulación hacia Abajo/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Seudópodos/enzimología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance system that eliminates transcripts with premature termination codons. In this study, we show that mRNAs targeted by NMD are also suppressed at the translational level. The low translational efficiency (TE) is a consequence of multiple features acting in concert, including low translation initiation rate, mediated by 5' secondary structure and by use of weak initiation sites, and low translation elongation speed, mediated by low codon usage bias. Despite low elongation rates, NMD transcripts show low ribosome density in the coding sequence, probably owing to low initiation rates, high abortion rates or rapid transit of the ribosome following initiation failure. The low TE is observed in the absence of NMD and is not explained by low transcript abundance. Translational inefficiency is flexible, such that NMD targets have increased TE upon starvation. We propose that the low TE predisposes to NMD and/or that it is part of a mechanism for regulation of NMD transcripts.
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Codón sin Sentido/metabolismo , Regulación Fúngica de la Expresión Génica , Biosíntesis de Proteínas , Estabilidad del ARN/genética , Saccharomyces cerevisiae/genética , Codón sin Sentido/antagonistas & inhibidores , Modelos Genéticos , Sistemas de Lectura Abierta , Estructura Secundaria de Proteína , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Bacterial meningitis shows a higher incidence in children than adults, but signs may be scarce. Although some pathogenic microorganisms of meningitis from cerebrospinal fluid (CSF) have been reported, the signature of the representative microbiota in CSF and blood samples from patients remains incompletely revealed. To extend the understanding of the microbiome in patients, we recruited 32 children with bacterial meningitis, 30 undiagnosed infectious children, and 10 matched healthy individuals, which was followed by untargeted metagenomic next-generation sequencing (mNGS) and bioinformatic analysis. Our results showed that children with bacterial meningitis exhibited different microbiome signatures in their CSF and blood compared with undiagnosed and healthy children, and patients could be divided into varied subsets according to these signatures, including Escherichia coli, Klebsiella pneumoniae, Thermothelomyces thermophila, Lactobacillus acidophilus, and Staphylococcus haemolyticus. To further explore their potential role in patients' conditions, we examined their correlation with clinical parameters. Importantly, microbiome signatures with compositional changes were correlated with the C-reactive protein (CRP) level in blood and granulocyte percentage in CSF. Moreover, the blood in subsets of patients with a predominance of Klebsiella pneumoniae could replace CSF as the main specimen for clinical monitoring. IMPORTANCE This study revealed the microbial compositions in children with bacterial meningitis who were treated with antibiotics and made a comprehensive comparison between blood and CSF specimens for the risk and prognosis assessment. We found that microbiome signatures could distinguish patient subsets in the children and were correlated with the CRP level in blood and granulocyte percentage in CSF. The compositional changes in representative microbiota constituents could provide guidance for clinical monitoring and antibiotic intervention.
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BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer and plays important roles in both malignant and immune cells. The effect of PI3Kα inhibitors on the tumor microenvironment (TME) remains largely unknown. Here, we investigated the modulation of the TME by a clinical PI3Kα-specific inhibitor CYH33. METHODS: The activity of CYH33 against a panel of murine tumors in the immune-competent context or athymic mice was detected. Single-cell RNA sequencing and multi-parameter flow cytometry were performed to determine the immune profiling of TME. The effect of CYH33 on immune cells was conducted with primary murine cells. RESULTS: CYH33 exhibited more potent antitumor activity in immune-competent context. CYH33 enhanced the infiltration and activation of CD8+T and CD4+T cells, while attenuating M2-like macrophages and regulatory CD4+T cells. Increase in memory T cells was confirmed by the induction of long-term immune memory on CYH33 treatment. Mechanistically, CYH33 relieved the suppressed expansion of CD8+T cells via preferential polarization of the macrophages to the M1 phenotype. CYH33 promoted fatty acid (FA) metabolism in the TME, while FA enhanced the activity of CD8+T cells in vitro. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 synergistically inhibited tumor growth with enhanced host immunity. CONCLUSIONS: CYH33 induces immune activation and synergizes with FASN inhibitor to further promote the antitumor immunity, which gains novel insights into how PI3K inhibitors exert their activity by modulating TME and provides a rationale for the concurrent targeting of PI3K and FASN in breast cancer treatment.