RESUMO
Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (VNARs), was considered to have the high antigen binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6 and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized Chiloscyllium plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs.
RESUMO
In pear (Pyrus bretschneideri), pollen tube growth is critical for the double fertilization associated with seed setting, which in turn affects fruit yield. The normal deposition of callose mediates the polar growth of pollen tubes. However, the mechanism regulating callose synthesis in pollen tubes remains relatively uncharacterized. In this study, we revealed that the typical pear pollen tube lifecycle has a semi-growth duration (GD50) of 16.16 h under in vitro culture conditions. Moreover, callose plugs were deposited throughout the pollen tube lifecycle. The formation of callose plugs was inhibited by 2-deoxy-D-glucose, which also accelerated the senescence of pear pollen tubes. Additionally, PbrCalS1B.1, which encodes a plasma membrane-localized callose synthase, was expressed specifically in pollen tubes and restored the fertility of the Arabidopsis (Arabidopsis thaliana) cals5 mutant, in which callose synthesis is inhibited. However, this restoration of fertility was impaired by the transient silencing of PbrCalS1B.1, which restricts callose plug formation and shortens the pear pollen tube lifecycle. More specifically, PbrbZIP52 regulated PbrCalS1B.1 transcription by binding to promoter A-box elements to maintain the periodic formation of callose plugs and normal pollen tube growth, ultimately leading to double fertilization. This study confirmed that PbrbZIP52 positively affects pear pollen tube longevity by promoting callose synthesis. This finding may be useful for breeding high-yielding pear cultivars and stabilizing fruit setting in commercial orchards.
Assuntos
Arabidopsis , Pyrus , Tubo Polínico , Pyrus/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Longevidade , Melhoramento Vegetal , Arabidopsis/metabolismoRESUMO
IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (â¼12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VANR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Tubarões , Tubarões/imunologia , Tubarões/genética , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Antígenos/imunologia , Antígenos/genética , Doenças dos Peixes/imunologiaRESUMO
BACKGROUND: The relationship between Alzheimer's disease biomarkers and postoperative complications, such as postoperative delirium (POD) and postoperative cognitive dysfunction (POCD), remains a subject of ongoing debate. OBJECTIVE: This meta-analysis aimed to determine whether there is an association between perioperative Alzheimer's disease biomarkers and postoperative complications. DESIGN: We conducted a meta-analysis of observational clinical studies that explored the correlation between Alzheimer's disease biomarkers and POD or POCD in patients who have undergone surgery, following PRISMA guidelines. The protocol was previously published (INPLASY: INPLASY202350001). DATA SOURCES: A comprehensive search was conducted across PubMed, Embase, Web of Science, and Cochrane databases until March 2023. ELIGIBILITY CRITERIA: Surgical patients aged at least 18 years, studies focusing on POD or POCD, research involving Alzheimer's disease biomarkers, including Aß or tau in blood or cerebrospinal fluid (CSF), and availability of the full text. RESULTS: Our meta-analysis included 15 studies: six focusing on POD and nine on POCD. The findings revealed a negative correlation between preoperative CSF ß-amyloid 42 (Aß42) levels and the onset of POD [mean difference -86.1, 95% confidence interval (CI), -114.15 to -58.05, I2 : 47%]; this association was strongly supported by trial sequential analysis (TSA). A similar negative correlation was discerned between preoperative CSF Aß42 levels and the incidence of POCD (-165.01, 95% CI, -261.48 to -68.53, I2 : 95%). The TSA also provided robust evidence for this finding; however, the evidence remains insufficient to confirm a relationship between other Alzheimer's disease biomarkers [ß-amyloid 40 (Aß40), total tau (T-tau), phosphorylated tau (P-tau), and Aß42/T-tau ratio] and POD or POCD. CONCLUSION: The study results indicate a negative correlation between preoperative CSF Aß42 levels and the occurrence of both POD and POCD. Future investigations are warranted to identify the predictive cutoff value of preoperative CSF Aß42 for POD and POCD.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Delírio do Despertar , Complicações Cognitivas Pós-Operatórias , Humanos , Adolescente , Adulto , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/líquido cefalorraquidiano , Estudos Prospectivos , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/epidemiologia , Disfunção Cognitiva/etiologia , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Complicações Cognitivas Pós-Operatórias/diagnóstico , Complicações Cognitivas Pós-Operatórias/epidemiologia , Complicações Cognitivas Pós-Operatórias/etiologia , Biomarcadores/líquido cefalorraquidiano , Fragmentos de PeptídeosRESUMO
Hepatocellular carcinoma (HCC) is one of the most common malignant diseases associated with a high rate of mortality. Frequent intrahepatic spread, extrahepatic metastasis, and tumor invasiveness are the main factors responsible for the poor prognosis of patients with HCC. Hypoxia-inducible factor 1 (HIF-1) has been verified to play a critical role in the metastasis of HCC. HIFs are also known to be modulated by small molecular metabolites, thus highlighting the need to understand the complexity of their cellular regulation in tumor metastasis. In this study, lower expression levels of oxoglutarate dehydrogenase-like (OGDHL) were strongly correlated with aggressive clinicopathologic characteristics, such as metastasis and invasion in three independent cohorts featuring a total of 281 postoperative HCC patients. The aberrant expression of OGDHL reduced cell invasiveness and migration in vitro and HCC metastasis in vivo, whereas the silencing of OGDHL promoted these processes in HCC cells. The pro-metastatic role of OGDHL downregulation is most likely attributed to its upregulation of HIF-1α transactivation activity and the protein stabilization by promoting the accumulation of L-2-HG to prevent the activity of HIF-1α prolyl hydroxylases, which subsequently causes an epithelial-mesenchymal transition process in HCC cells. These results demonstrate that OGDHL is a dominant factor that modulates the metastasis of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/patologia , Prognóstico , Estabilidade ProteicaRESUMO
BACKGROUND AND AIMS: NASH, which is a common clinical condition predisposing to advanced liver diseases, has become a worldwide epidemic. A large and growing unmet therapeutic need for this condition reflects incomplete understanding of its pathogenesis. In the current study, we identified a transcription factor, zinc fingers and homeoboxes 2 (ZHX2), in hepatocytes as a protective factor against steatohepatitis. APPROACH AND RESULTS: We found that hepatic ZHX2 was significantly suppressed in NASH models and steatotic hepatic cells. Hepatocyte-specific ablation of ZHX2 exacerbated NASH-related phenotypes in mice, including lipid accumulation, enhanced inflammation, and hepatic fibrosis. Conversely, hepatocyte-specific overexpression of ZHX2 significantly alleviated the progression of NASH in an experimental setting. Integrated analysis of transcriptomic profiling and chromatin immunoprecipitation sequencing data demonstrated that the phosphatase and tensin homolog (PTEN) was a target gene of ZHX2 in hepatocyte. ZHX2 bound to the promoter of PTEN gene and subsequently promoted the transcription of PTEN, which mediated the beneficial role of ZHX2 against NASH. CONCLUSIONS: The current findings demonstrate a protective role of ZHX2 against NASH progression by transcriptionally activating PTEN. These findings shed light on the therapeutic potential of targeting ZHX2 for treating NASH and related metabolic disorders.
Assuntos
Proteínas de Homeodomínio , Hepatopatia Gordurosa não Alcoólica , Fatores de Transcrição , Animais , Genes Homeobox , Hepatócitos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tensinas/genética , Tensinas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Dedos de ZincoRESUMO
Single domain antibodies (sdAb) are promising candidates in cancer and anti-virus biotherapies for their unique structure characters. Though VHH and IgNAR have been discovered in camelidae and nurse shark (Ginlymostoma cirratum) respectively serval decades ago, expense of these large animals still limits the studies and applications of sdAb. Recently, IgNAR has been found in whitespotted bamboo shark (Chiloscyllium plagiosum), a small-sized sharks, while how to characterize and achieved the IgNAR of whitespotted bamboo shark is still unclear. In our research, we identified four IgNAR coding gene loci in whitespotted bamboo shark chromosome 44 (NC_057753.1), and primers were designed for single domain variable regions of IgNAR (VNAR) libraries preparation. Following sequencing results revealed that all plasmids constructed with our predicted VNAR libraries contained VNAR coding sequences, which confirmed the specificities of our primers in VNAR amplification. To our surprise, ≥90% VNAR sequences were encoded by IgNAR1, which suggests IgNAR1 is the most active IgNAR transcription locus in whitespotted bamboo shark. Interestingly, we found IgNAR(ΔC2-C3) were encoded by IgNAR3. Our findings gave a new sight of whitespotted bamboo shark IgNAR, which would broad the way of IgNAR studies and applications in biotherapies.
Assuntos
Loci Gênicos , Tubarões , Animais , Tubarões/genéticaRESUMO
Myeloperoxidase (MPO) is a critical proinflammatory enzyme implicated in cardiovascular, neurological, and rheumatological diseases. Emerging therapies targeting inflammation have raised interest in tracking MPO activity in patients. We describe 18F-MAPP, an activatable MPO activity radioprobe for positron emission tomography (PET) imaging. The activated radioprobe binds to proteins and accumulates at sites of MPO activity. The radioprobe 18F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood-brain barrier. The 18F-MAPP imaging detected sites of elevated MPO activity in living mice embedded with human MPO and in mice induced with chemical inflammation or myocardial infarction. The 18F-MAPP PET imaging noninvasively differentiated varying amounts of MPO activity, competitive inhibition, and MPO deficiency in living animals, confirming specificity and showing that the radioprobe can quantify changes in in vivo MPO activity. The radiosynthesis has been optimized and automated, an important step in translation. These data indicate that 18F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients.
Assuntos
Peroxidase/metabolismo , Animais , Feminino , Radioisótopos de Flúor/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Tomografia por Emissão de Pósitrons/métodosRESUMO
An effective chromatography process was developed and validated for simultaneous purification and separation of total lignans and flavonoids from Valeriana amurensis. The total lignans and flavonoids in Valeriana amurensis extract were prepurified with macroporous resin column chromatography, and the conditions were optimized as follows: 40 mg/mL Valeriana amurensis extract (2.0 g) solution was loaded onto an AB-8 resin column with a diameter-to-height ratio of 1:7, followed by adsorption for 6 h; then, the column was eluted successively with 5 BV water and 10% and 50% ethanol at a flow rate 2 BV/h. The obtained 50% ethanol fraction was further repurified and separated by polyamide resin column chromatography to obtain the total lignans and flavonoids, respectively. The chromatography conditions were optimized as follows: a 50% ethanol fraction (1.0 g) was mixed with 1.0 g polyamide resin and loaded onto a polyamide resin (60-100 mesh) column with a diameter-to-height ratio of 1:3; then, the column was eluted successively with 6 BV water and 40% and 80% ethanol at a flow rate of 4 BV/h. The total lignans and flavonoids were obtained from water and 80% ethanol fraction, respectively. The content and recovery of standard compounds in total lignans and flavonoids were analyzed with HPLC-PDA, and the feasibility of the process was confirmed.
Assuntos
Flavonoides , Lignanas , Flavonoides/química , Cromatografia Líquida de Alta Pressão , Resinas Vegetais/química , Extratos Vegetais/química , Adsorção , Etanol , Água , Resinas Sintéticas/químicaRESUMO
BACKGROUND: Gene transcripts that show invariant abundance during development are ideal as reference genes (RGs) for accurate gene expression analyses, such as RNA blot analysis and reverse transcription-quantitative real time PCR (RT-qPCR) analyses. In a genome-wide analysis, we selected three "Commonly used" housekeeping genes (HKGs), fifteen "Traditional" HKGs, and nine novel genes as candidate RGs based on 80 publicly available transcriptome libraries that include data for receptacle development in eight strawberry cultivars. RESULTS: The results of the multifaceted assessment consistently revealed that expression of the novel RGs showed greater stability compared with that of the "Commonly used" and "Traditional" HKGs in transcriptome and RT-qPCR analyses. Notably, the majority of stably expressed genes were associated with the ubiquitin proteasome system. Among these, two 26 s proteasome subunits, RPT6A and RPN5A, showed superior expression stability and abundance, and are recommended as the optimal RGs combination for normalization of gene expression during strawberry receptacle development. CONCLUSION: These findings provide additional useful and reliable RGs as resources for the accurate study of gene expression during receptacle development in strawberry cultivars.
Assuntos
Fragaria , Fragaria/genética , Perfilação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/genética , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Transcriptoma , Ubiquitina/genéticaRESUMO
S-RNase is the female determinant of self-incompatibility (SI) in pear (Pyrus bretschneideri). After translocation to the pollen tube, S-RNase degrades rRNA and induces pollen tube death in an S-haplotype-specific manner. In this study, we found that the actin cytoskeleton is a target of P. bretschneideri S-RNase (PbrS-RNase) and uncovered a mechanism that involves phosphatidic acid (PA) and protects the pollen tube from PbrS-RNase cytotoxicity. PbrS-RNase interacts directly with PbrActin1 in an S-haplotype-independent manner, causing the actin cytoskeleton to depolymerize and promoting programmed cell death in the self-incompatible pollen tube. Pro-156 of PbrS-RNase is essential for the PbrS-RNase-PbrActin1 interaction, and the actin cytoskeleton-depolymerizing function of PbrS-RNase does not require its RNase activity. PbrS-RNase cytotoxicity enhances the expression of phospholipase D (PbrPLDδ1), resulting in increased PA levels in the incompatible pollen tube. PbrPLDδ1-derived PA initially prevents depolymerization of the actin cytoskeleton elicited by PbrS-RNase and delays the SI signaling that leads to pollen tube death. This work provides insights into the orchestration of the S-RNase-based SI response, in which increased PA levels initially play a protective role in incompatible pollen, until sustained PbrS-RNase activity reaches the point of no return and pollen tube growth ceases.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Ácidos Fosfatídicos/metabolismo , Polinização/fisiologia , Ribonucleases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Pectin methylesterase (PME) is a hydrolytic enzyme that catalyzes the demethylesterification of homogalacturonans and controls pectin reconstruction, being essential in regulation of cell wall modification. During fruit ripening stage, PME-mediated cell wall remodeling is an important process to determine fruit firmness and softening. Strawberry fruit is a soft fruit with a short postharvest life, due to a rapid loss of firm texture. Hence, preharvest improvement of strawberry fruit rigidity is a prerequisite for extension of fruit refreshing time. Although PME has been well characterized in model plants, knowledge regarding the functionality and evolutionary property of PME gene family in strawberry remain limited. RESULTS: A total of 54 PME genes (FvPMEs) were identified in woodland strawberry (Fragaria vesca 'Hawaii 4'). Phylogeny and gene structure analysis divided these FvPME genes into four groups (Group 1-4). Duplicate events analysis suggested that tandem and dispersed duplications effectively contributed to the expansion of the PME family in strawberry. Through transcriptome analysis, we identified FvPME38 and FvPME39 as the most abundant-expressed PMEs at fruit ripening stages, and they were positively regulated by abscisic acid. Genetic manipulation of FvPME38 and FvPME39 by overexpression and RNAi-silencing significantly influences the fruit firmness, pectin content and cell wall structure, indicating a requirement of PME for strawberry fruit softening. CONCLUSION: Our study globally analyzed strawberry pectin methylesterases by the approaches of phylogenetics, evolutionary prediction and genetic analysis. We verified the essential role of FvPME38 and FvPME39 in regulation of strawberry fruit softening process, which provided a guide for improving strawberry fruit firmness by modifying PME level.
Assuntos
Hidrolases de Éster Carboxílico/genética , Fragaria , Frutas/metabolismo , Pectinas/metabolismo , Ácido Abscísico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/metabolismo , Fragaria/genética , Fragaria/metabolismo , Frutas/genética , Perfilação da Expressão Gênica , Genes de Plantas , Filogenia , Interferência de RNARESUMO
BACKGROUND: The B-BOX (BBX) proteins are the class of zinc-finger transcription factors and can regulate plant growth, development, and endure stress response. In plants, the BBX gene family has been identified in Arabidopsis, rice, and tomato. However, no systematic analysis of BBX genes has been undertaken in grapevine. RESULTS: In this study, 24 grapevine BBX (VvBBX) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Phylogenetic analysis divided VvBBX genes into five subgroups. Numerous cis-acting elements related to plant development, hormone and/or stress responses were identified in the promoter of the VvBBX genes. The tissue-specific expressional dynamics of VvBBX genes demonstrated that VvBBXs might play important role in plant growth and development. The transcript analysis from transcriptome data and qRT-PCR inferred that 11 VvBBX genes were down-regulated in different fruit developmental stages, while three VvBBX genes were up-regulated. It is also speculated that VvBBX genes might be involved in multiple hormone signaling (ABA, ethylene, GA3, and CPPU) as transcriptional regulators to modulate berry development and ripening. VvBBX22 seems to be responsive to multiple hormone signaling, including ABA, ethylene GA3, and CPPU. Some VvBBX genes were strongly induced by Cu, salt, waterlogging, and drought stress treatment. Furthermore, the expression of VvBBX22 proposed its involvement in multiple functions, including leaf senescence, abiotic stress responses, fruit development, and hormone response. CONCLUSIONS: Our results will provide the reference for functional studies of BBX gene family, and highlight its functions in grapevine berry development and ripening. The results will help us to better understand the complexity of the BBX gene family in abiotic stress tolerance and provide valuable information for future functional characterization of specific genes in grapevine.
Assuntos
Frutas/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Vitis/genética , Frutas/crescimento & desenvolvimento , Família Multigênica , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Vitis/crescimento & desenvolvimentoRESUMO
Ammonia (NH3) is considered as environmental pollutant and toxic agent for animals and humans including poultry. Previous reports demonstrated that NH3 suppressed broilers immunity. However, the harmful effects of NH3 on broilers bursa of fabricius (BF) is still unknown. Functionally, apoptosis is very important for many physiological processes including homeostasis of lymphocyte population. Therefore, the present study was aimed to investigate the underlying mechanisms of NH3 toxicity in the broilers BF. Histological observation showed lymphocyte accumulation, cavities and increased interstitial cells in BF. Ultrastructural observation indicated mitochondrial vacuoles, deformation and disappearance of mitochondrial membranes. Oxidative stress markers (CAT, MDA, H2O2, GGT, GSH-Px and GSH) showed that NH3-induced oxidative stress in BF. Meanwhile, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed increased apoptotic cells. In addition, the mRNA and protein expression of dynamin-related protein 1 (Drp1), mitochondrial fission factor (Mff), mitofusin 1 and 2 (Mfn1 and Mfn2), optic atrophy 1 (Opa1) indicated imbalance between mitochondrial inner and outer membrane and results in mitochondrial dysfunction in broilers BF. The mRNA and protein expression of apoptosis-related genes including Caspase-3, Caspase-9, Caspase-8, Cytochrome-C (Cyt-C), p53, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) were significantly altered in broilers BF. Conclusively, these results displayed that excessive NH3 causes BF damage and mitochondrial dysfunction through oxidative stress and apoptosis in BF and could affect immune function of BF. These findings provide possible therapeutic targets to prevent NH3 induced toxicity in the BF of broilers.
Assuntos
Amônia/toxicidade , Bolsa de Fabricius/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Bolsa de Fabricius/imunologia , Bolsa de Fabricius/metabolismo , Galinhas , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.
Assuntos
Sequência Conservada , Evolução Molecular , Proteínas Ativadoras de GTPase/química , Tubarões/metabolismo , Proteínas rab de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/genética , Cristalografia por Raios X , Glicina/química , Glicina/genética , Humanos , Lisina/química , Lisina/genética , Camundongos , Mutação , Domínios Proteicos , Alinhamento de Sequência , Tubarões/genética , Especificidade por Substrato , Suínos , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The leading cause of mortality due to colorectal cancer (CRC) is highly associated with the development of liver metastases. Recently, we described cGAMP that is closely related to the metastatic state wherein the progress of metastatic tumors is associated with favorable outcomes in a zebrafish xenograft model. cGAMP was administered and the expression levels of type-I interferons were induced amongst tumor tissues to illuminate the overall measure of the induced STING/STAT3 axis in colorectal liver metastases. Furthermore, cGAMP-STING dependent STAT3 activation resulted in the inhibition of tumor cell proliferation, viability, and invasion in vitro. The subtotal reduction in tumor growth attributed to a large number of infiltrating inflammatory cells in vivo. We showed that cGAMP inhibited migration through angiogenesis by up-regulating IL-2, TNF-α, and IFN-γ, whereas STAT3 down-regulation inhibited CXCL8, BCL-2, and VEGFA expression. The importance of cGAMP in inhibiting the invasion front of CRC confirmed that the cGAMP dependent activation of STING/STAT3 axis played a key role in the inhibition of tumor progression.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/veterinária , Xenoenxertos/patologia , Neoplasias Hepáticas/veterinária , Nucleotídeos Cíclicos/farmacologia , Transdução de Sinais , Animais , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Neoplasias Hepáticas/secundário , Proteínas de Membrana/genética , Metástase Neoplásica , Fator de Transcrição STAT3/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Chlorpyrifos (CPF) is an environmental pollutant due to its high toxicity to aquatic animals. Because CPF was detected in aquatic environments in many countries, it has been widely concerned by researchers. Although the immunotoxicity of CPF to fish had been reported, the immunotoxicity mechanism is still not clear. Recently, transcriptome analysis has become a major method to study the toxic mechanism of pollutants in environmental toxicology. However, the immunotoxicity identification of CPF on fish had not been reported by transcriptome analysis. In the present study, we examined the effects of CPF on organismal system in the spleen of common carp by transcriptome analysis. We have successfully constructed a database of transcriptome analysis of carp spleens under exposure to CPF and found 773 differentially expressed genes (DEGs) (including 498 up-regulated DEGs and 275 down-regulated DEGs) and 4 branches (containing 33 known KEGG pathways). Some genes associated with the 4 pathways (Complement and coagulation cascades, PPAR signaling pathway, Fat digestion and absorption, and Collecting duct acid secretion) contained in organismal system were validated by quantitative real-time PCR and showed significant improvement compared with the control group. Our results indicated that exposure to CPF caused a change in the signal pathways of organismal system in carp spleens. The present study provides new insights into the immunotoxicity mechanism and risk assessment of CPF, as well as references for comparative medicine.
Assuntos
Clorpirifos/toxicidade , Baço/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Atrazina/toxicidade , Carpas , Sistema Imunitário/efeitos dos fármacos , Inseticidas/toxicidade , Transdução de Sinais , Baço/fisiologiaRESUMO
BACKGROUND: Flowers open at sunrise and close at sunset, establishing a circadian floral movement rhythm to facilitate pollination as part of reproduction. By the coordination of endogenous factors and environmental stimuli, such as circadian clock, photoperiod, light and temperature, an appropriate floral movement rhythm has been established; however, the underlying mechanisms remain unclear. RESULTS: In our study, we use waterlily as a model which represents an early-diverging grade of flowering plants, and we aim to reveal the general mechanism of flower actions. We found that the intermediate segment of petal cells of waterlily are highly flexible, followed by a circadian cell expansion upon photoperiod stimuli. Auxin causes constitutively flower opening while auxin inhibitor suppresses opening event. Subsequent transcriptome profiles generated from waterlily's intermediate segment of petals at different day-time points showed that auxin is a crucial phytohormone required for floral movement rhythm via the coordination of YUCCA-controlled auxin synthesis, GH3-mediated auxin homeostasis, PIN and ABCB-dependent auxin efflux as well as TIR/AFB-AUX/IAA- and SAUR-triggered auxin signaling. Genes involved in cell wall organization were downstream of auxin events, resulting in the output phenotypes of rapid cell expansion during flower opening and cell shrinkage at flower closure stage. CONCLUSIONS: Collectively, our data demonstrate a central regulatory role of auxin in floral movement rhythm and provide a global understanding of flower action in waterlily, which could be a conserved feature of angiosperms.
Assuntos
Ritmo Circadiano/fisiologia , Flores/fisiologia , Ácidos Indolacéticos/metabolismo , Nymphaea/fisiologia , Reguladores de Crescimento de Plantas/fisiologia , Perfilação da Expressão Gênica , Reguladores de Crescimento de Plantas/metabolismoRESUMO
Ta2N is an effective diffusion barrier material to prevent undesired Cu diffusion in ultra-large scale integration circuits. Previous theoretical work has reported the interesting result that at the Cu/Ta2N interface the Cu layer preferentially bonded with the Ta layer but not the N layer of Ta2N. However, this result was calculated from largely lattice mismatched interface models. To confirm this theoretical result and unravel the cause of strong Cu-Ta bonding at the Cu/Ta2N interface, in this study density functional theory calculations, on the basis of super-cell models, were performed to investigate the Cu(111)/Ta2N(001) interface. We firstly calculated interface cohesive energies and confirmed that the Cu layer preferentially bonded with the Ta layer of Ta2N. Then, electronic structure calculations revealed that the chemical bonding of the Cu-Ta bond at the Cu(111)/Ta2N(001) interface was primarily covalent in character, providing a proper explanation for the close integration of the Cu layer and Ta layer. Lastly, Cu diffusion investigations revealed that Ta2N was able to effectively prevent Cu diffusion. Furthermore, we found that the N layer of Ta2N played the critical role in preventing Cu diffusion.
RESUMO
Previous studies have focused on the effects of propylene glycol alginate sodium sulfate (PSS) against thrombosis, but the anti-inflammatory potential is unknown. Therefore, we specifically focused on the protective effects of PSS on cerulein-induced acute pancreatitis (AP) using a mouse model, and investigated the mechanism of PSS on autophagy and apoptosis via the Mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Cerulein (100 ug/kg) was used to induce AP by ten intraperitoneal injections at hourly intervals in Balb/C mice. Pretreatment with vehicle or PSS was carried out 1 h before the first cerulein injection and two doses (25 mg/kg and 50 mg/kg) of PSS were injected intraperitoneally. The severity of AP was assessed by pathological score, biochemistry, pro-inflammatory cytokine levels, myeloperoxidase (MPO) activity and MEK/ERK activity. Furthermore, pancreatic histological scores, serum amylase and lipase activities, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß interleukin (IL)-6 levels, and MPO activity were significantly reduced by PSS via up-regulated MEK/ERK activity. The representative molecules of apoptosis and autophagy, such as Bcl-2, Bax, Lc-3, Beclin-1, P62, were remarkably reduced. Taken together, these results indicate that PSS attenuates pancreas injury by inhibiting autophagy and apoptosis through a mechanism involving the MEK/ERK signaling pathway.