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SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.
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Proteínas SNARE/química , Proteínas SNARE/metabolismo , Enfermedad/genética , Humanos , Fusión de Membrana , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Mutación , Pinzas Ópticas , Fosforilación , Dominios Proteicos , Pliegue de Proteína , Proteínas SNARE/genéticaRESUMEN
The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.
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COVID-19 , SARS-CoV-2 , Animales , Ratones , Conejos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Macaca mulatta , Macrófagos , Nanovacunas , Fagocitosis , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.
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Sistemas CRISPR-Cas , Chlamydomonas reinhardtii , Codón , Edición Génica , Edición Génica/métodos , Chlamydomonas reinhardtii/genética , Codón/genética , Mutación Puntual/genéticaRESUMEN
Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαßs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αß paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/ß clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/ß clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.
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Linfocitos T CD8-positivos , Privilegio Inmunológico , Infección por el Virus Zika , Animales , Masculino , Ratones , Encéfalo/inmunología , Encéfalo/virología , Linfocitos T CD8-positivos/inmunología , Receptor de Interferón alfa y beta/genética , Virus Zika , Infección por el Virus Zika/inmunología , Ratones Noqueados , Testículo/inmunología , Testículo/virologíaRESUMEN
Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, glucose transporter (GLUT4) translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggests that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes - Munc18-1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate the binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of 0.054 s-1 for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite the similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.
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Fusión de Membrana , Proteína 2 de Membrana Asociada a Vesículas , Sintaxina 1 , Chaperonas Moleculares , Proteínas Qa-SNARE/genética , InsulinaRESUMEN
Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.
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Fusión de Membrana , Proteínas SNARE , Cinética , Fusión de Membrana/fisiología , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.
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Trasplante de Células Madre Hematopoyéticas , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Linfocitos T , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunoterapia Adoptiva/métodosRESUMEN
Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.
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Tiburones , Anticuerpos de Dominio Único , Animales , Humanos , alfa-Fetoproteínas , Tiburones/metabolismo , Anticuerpos , Suero/metabolismo , Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , AntígenosRESUMEN
Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.
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Glicerolfosfato Deshidrogenasa , Hepatitis B , Proteína 28 que Contiene Motivos Tripartito , Proteínas Reguladoras y Accesorias Virales , Animales , Ratones , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Mitocondrias/enzimología , Fosfatos/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.
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Calcio , Pinzas Ópticas , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Lípidos/análisisRESUMEN
The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving "coupling"-the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote "coupling," and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.
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Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/enzimología , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Hidrólisis , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Tiempo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.
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Dominios C2 , Calcio , Neurotransmisores , Sinaptotagmina I , Animales , Calcio/metabolismo , Lípidos , Ratones , Neurotransmisores/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismoRESUMEN
Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers worldwide. The molecular mechanisms of serous ovarian cancer (SOC) remain unclear and not well understood. SOC cases are primarily diagnosed at the late stage, resulting in a poor prognosis. Advances in molecular biology techniques allow us to obtain a better understanding of precise molecular mechanisms and to identify the chromosome instability region and key driver genes in the carcinogenesis and progression of SOC. Whole-exome sequencing was performed on the normal ovarian cell line IOSE80 and the EOC cell lines SKOV3 and A2780. The single-nucleotide variation burden, distribution, frequency and signature followed the known ovarian mutation profiles, without chromosomal bias. Recurrently mutated ovarian cancer driver genes, including LRP1B, KMT2A, ARID1A, KMT2C and ATRX were also found in two cell lines. The genome distribution of copy number alterations was found by copy number variation (CNV) analysis, including amplification of 17q12 and 4p16.1 and deletion of 10q23.33. The CNVs of MED1, GRB7 and MIEN1 located at 17q12 were found to be correlated with the overall survival of SOC patients (MED1: p = 0.028, GRB7: p = 0.0048, MIEN1: p = 0.0051), and the expression of the three driver genes in the ovarian cell line IOSE80 and EOC cell lines SKOV3 and A2780 was confirmed by western blot and cell immunohistochemistry.
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Neoplasias Ováricas , Humanos , Femenino , Carcinoma Epitelial de Ovario/genética , Neoplasias Ováricas/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , Inestabilidad Cromosómica/genética , Proteínas de Neoplasias/genética , Péptidos y Proteínas de Señalización Intracelular/genéticaRESUMEN
Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.
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Celulasa , Sordariales , Celulasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sordariales/metabolismo , Fermentación , Etanol/metabolismo , Celulosa/genética , Celulosa/metabolismoRESUMEN
BACKGROUND: Ovarian cancer is one of the most lethal cancers in women because it is often diagnosed at an advanced stage. The molecular markers investigated thus far have been unsatisfactory. METHODS: We performed whole-exome sequencing on the human ovarian cancer cell lines 3AO and ES2 and the normal ovarian epithelial cell line IOSE-80. Molecular markers of ovarian cancer were screened from shared mutation genes and copy number variation genes in the 6q21-qter region. RESULTS: We found that missense mutations were the most common mutations in the gene (93%). The MUC12, FLG and MUC16 genes were highly mutated in 3AO and ES2 cells. Copy number amplification occurred mainly in 4p16.1 and 11q14.3, and copy number deletions occurred in 4q34.3 and 18p11.21. A total of 23 hub genes were screened, of which 16 were closely related to the survival of ovarian cancer patients. The three genes CCDC170, THBS2 and COL14A1 are most significantly correlated with the survival and prognosis of ovarian cancer. In particular, the overall survival of ovarian cancer patients with high CCDC170 gene expression was significantly prolonged (P < 0.001). The expression of CCDC170 in normal tissues was significantly higher than that in ovarian cancer tissues (P < 0.05), and its expression was significantly decreased in advanced ovarian cancer. Western blotting and immunofluorescence assays also showed that the expression of CCDC170 in ovarian cancer cells was significantly lower than that in normal cells (P < 0.001, P < 0.01). CONCLUSIONS: CCDC170 is expected to become a new diagnostic molecular target and prognostic indicator for ovarian cancer patients, which can provide new ideas for the design of antitumor drugs.
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Neoplasias Ováricas , Femenino , Humanos , Neoplasias Ováricas/patología , Exoma/genética , Variaciones en el Número de Copia de ADN , Mutación , Línea Celular Tumoral , BiomarcadoresRESUMEN
Methyl gallate (MG) is a polyphenolic compound widely found in natural plants. MG has been shown to have a variety of biological functions, including anti-tumor, anti-inflammatory, anti-oxidant, neuroprotective, hepatoprotective and anti-microbial activities, and has broad research and development prospects. A total of 88 articles related to MG were searched using the PubMed, Science Direct, and Google Scholar databases, systematically investigating the pharmacological activity and molecular mechanisms of MG. There were no restrictions on the publication years, and the last search was conducted on June 5, 2023. MG can exert pharmacological effects through multiple pathways and targets, such as PI3K/Akt, ERK1/2, Caspase, AMPK/NF-κB, Wnt/ß-catenin, TLR4/NF-κB, MAPK, p53, NLRP3, ROS, EMT. According to the literature, MG has the potential to be a prospective adjuvant for anticancer therapy and deserves further study.
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FN-kappa B , Fosfatidilinositol 3-Quinasas , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ácido GálicoRESUMEN
Understanding soil organic carbon (SOC), the largest carbon (C) pool of a terrestrial ecosystem, is essential for mitigating climate change. Currently, the spatial patterns and drivers of SOC in the plantations of tea, a perennial leaf crop, remain unclear. Therefore, the present study surveyed SOC across the main tea-producing areas of China, which is the largest tea producer in the world. We analyzed the soil samples from tea plantations under different scenarios, such as provinces, regions [southwest China (SW), south China (SC), south Yangtze (SY), and north Yangtze (NY)], climatic zones (temperate, subtropical, and tropical), and cultivars [large-leaf (LL) and middle or small-leaf (ML) cultivars]. Preliminary analysis revealed that most tea-producing areas (45%) had SOC content ranging from 10 to 20 g kg-1. The highest SOC was recorded for Yunnan among the various provinces, the SW tea-producing area among the four regions, the tropical region among the different climatic zones, and the areas with LL cultivars compared to those with ML cultivars. Further Pearson correlation analysis demonstrated significant associations between SOC and soil variables and random forest modeling (RF) identified that total nitrogen (TN) and available aluminum [Ava(Al)] of soil explained the maximum differences in SOC. Besides, a large indirect effect of geography (latitude and altitude) on SOC was detected through partial least squares path modeling (PLS-PM) analysis. Thus, the study revealed a high spatial heterogeneity in SOC across the major tea-producing areas of China. The findings also serve as a basis for planning fertilization strategies and C sequestration policies for tea plantations.
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Craniopharyngioma (CP) is a mostly benign tumor that is nonetheless one of most formidable skull base lesions. CP tends to recur, and scarce clinical results are available regarding its long-term outcomes. From February 1996 to April 2002, craniopharyngiomas primarily resected by open surgery in a single-center and single-surgeon practice were screened. Medical information regarding patients' baseline, tumor parameters, endocrinological results, complications, and quality of life in those patients with a follow-up longer than 20 years were reviewed. Nineteen out of 187 patients who met the inclusion criteria were eligible, and 78.9% of the patients were admitted due to visual deficits. The transcranial approach was mostly applied for the first attempt of opening resection, with 94.7% gross total resection. The size of the tumor ranged from 25 to 45 mm with a mean maximal diameter of 34.7 mm. Although 7 out of 19 patients received an extra procedure, 6 patients (31.5%) regained fertility, with 3 women bearing a total of 5 children and 3 men fathering a total of 4 children, during the 21.4-year follow-up (range: 20.0-23.3 years). The mean Karnofsky Performance Status (KPS) score was 97.9 (range: 80-100) at the last follow-up, and the physical and mental 36-Item Short Form Health Survey (SF-36) scores were 88.0 and 80.5, respectively. The tumor sizes of the patients who regained fertility were not significantly different from those of the patients who remained infertile (t = 1.29, P > 0.2). The time interval from prior surgery to tumor resection for recurrent cases ranged from 0.3 to 17.4 years (mean, 7.3 years). There was no significant difference in the time until tumor recurrence in the patients who underwent a second surgery, a third surgery, or a fourth surgery. The satisfactory results in the present study revealed favorable long-term outcomes following the transcranial management of CPs, with acceptable endocrinological function and tumor-free survival. A decisive policy of open surgery with the objective of radical tumor removal remains a valid method for the primary treatment of CPs, aiming to avoid retreatment after tumor recurrence involving vital hypothalamic structures or hydrocephalus.
Asunto(s)
Craneofaringioma , Neoplasias Hipofisarias , Masculino , Niño , Humanos , Femenino , Estudios de Seguimiento , Craneofaringioma/cirugía , Craneofaringioma/patología , Resultado del Tratamiento , Recurrencia Local de Neoplasia/cirugía , Recurrencia Local de Neoplasia/patología , Calidad de Vida , Neoplasias Hipofisarias/cirugía , Neoplasias Hipofisarias/patología , Estudios RetrospectivosRESUMEN
Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by â¼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.
Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas SNARE/metabolismo , Exocitosis/fisiología , Cinética , Fusión de Membrana/fisiología , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas del Tejido Nervioso/química , Neuronas/metabolismo , Pinzas Ópticas , Unión Proteica , Dominios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
BACKGROUND: The left primary motor area (M1) stimulation has recently been revealed to promote post-stroke aphasia (PSA) recovery, of which a plausible mechanism might be the semantic and/or the mirror neuron system reorganization, but the direct evidence is still scarce. The aim of this study was to explore the functional connectivity (FC) alterations induced by the left M1 intermittent theta burst stimulation (iTBS), a new transcranial magnetic stimulation paradigm, in the semantic and mirror neuron systems of PSA patients. METHODS: Sixteen PSA patients accepted the left M1 iTBS and underwent a resting-state functional magnetic resonance image (fMRI) scanning before and immediately after the first session of iTBS, of which six underwent another fMRI scanning after twenty sessions of iTBS. Three brain networks covering the semantic and the mirror neuron systems were constructed using the fMRI data, and the FC alterations following one-session iTBS were investigated in the networks. Additional seed-based FC analyses were conducted to explore the longitudinal FC patterns changes during the course of multi-session iTBS. The Aphasia quotient of the Chinese version of the western aphasia battery (WAB-AQ) was used to assess the severity of the language impairments of the participants. The relationship between the longitudinal WAB-AQ and network FC changes was analyzed by Spearman's correlation coefficients in the multi-session iTBS sub-group. RESULTS: Decreased FCs were noted in the bilateral semantic rather than in the mirror neuron networks following one-session of iTBS (p < 0.05, network based statistical corrected). Longitudinal seed-based FC analyses revealed changing FC ranges along the multi-session iTBS course, extending beyond the semantic networks. No significant relationship was found between the longitudinal WAB-AQ and network FC changes in the multi-session iTBS sub-group. CONCLUSIONS: The left M1 iTBS might induce FC changes in the semantic system of PSA patients. CLINICAL TRIAL REGISTRATION: This research was registered on the Chinese Clinical Trial Registry website (http://www.chictr.org.cn/index.aspx), and the registration number is ChiCTR2100041936.