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KEY MESSAGE: Through comprehensive genomic and transcriptomic analyses, we identified a set of 23 genes that act up- or downstream of erucic acid content (EAC) production in rapeseed seeds. We selected example genes to showcase the distribution of single nucleotide polymorphisms, haplotypes associated with EAC phenotypes, and the creation of molecular markers differentiating low EAC and high EAC genotypes. Erucic acid content (EAC) is a crucial trait in rapeseed, with low LEAC oil recognized for its health benefits and high EA oil holding industrial value. Despite its significance, the genomic consequences of intensive LEAC-cultivar selection and the genetic basis underlying EA regulation remain largely unexplored. To address this knowledge gap, we conducted selective signal analyses, genome-wide association studies (GWAS), and transcriptome analyses. Our investigation unveiled the genetic footprints resulting from LEAC selection in germplasm populations, drawing attention to specific loci that contribute to enriching diversity. By integrating GWAS and transcriptome analyses, we identified a set of 23 genes that play a significant role in determining EAC in seeds or are downstream consequences of EA-level alterations. These genes have emerged as promising candidates for elucidating the potential mechanisms governing EAC in rapeseed. To exemplify the findings, we selected specific genes to demonstrate the distribution of single nucleotide polymorphisms and haplotypes associated with different EAC phenotypes. Additionally, we showcased to develop molecular markers distinguishing between LEAC and high EAC genotypes.
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Brassica napus , Ácidos Erúcicos , Polimorfismo de Nucleotídeo Único , Sementes , Sementes/genética , Sementes/crescimento & desenvolvimento , Brassica napus/genética , Ácidos Erúcicos/metabolismo , Fenótipo , Haplótipos , Transcriptoma , Estudo de Associação Genômica Ampla , Genótipo , Perfilação da Expressão Gênica , Genômica/métodos , Regulação da Expressão Gênica de Plantas , Locos de Características QuantitativasRESUMO
Ischemic stroke can induce neurogenesis. However, most stroke-generated newborn neurons cannot survive. It has been shown that MR-409, a potent synthetic agonistic analog of growth hormone-releasing hormone (GHRH), can protect against some life-threatening pathological conditions by promoting cell proliferation and survival. The present study shows that long-term treatment with MR-409 (5 or 10 µg/mouse/d) by subcutaneous (s.c.) injection significantly reduces the mortality, ischemic insult, and hippocampal atrophy, and improves neurological functional recovery in mice operated on for transient middle cerebral artery occlusion (tMCAO). Besides, MR-409 can stimulate endogenous neurogenesis and improve the tMCAO-induced loss of neuroplasticity. MR-409 also enhances the proliferation and inhibits apoptosis of neural stem cells treated with oxygen and glucose deprivation-reperfusion. The neuroprotective effects of MR-409 are closely related to the activation of AKT/CREB and BDNF/TrkB pathways. In conclusion, the present study demonstrates that GHRH agonist MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice.
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Hormônio Liberador de Hormônio do Crescimento/agonistas , Hormônio Liberador de Hormônio do Crescimento/metabolismo , AVC Isquêmico/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Plasticidade Neuronal , Fármacos Neuroprotetores , Proteínas Tirosina Quinases/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Tumor-associated macrophages (TAMs) are a type of functionally plastic immune cell population in tumor microenvironment (TME) and mainly polarized into two phenotypes: M2 and M1-like TAMs. The M2-like TAMs could stimulate tumor growth and metastasis, tissue remodeling and immune-suppression, whereas M1-like TAMs could initiate immune response to dampen tumor progression. TAMs with different polarization phenotypes can produce various kinds of cytokines, chemokines and growth factors to regulate immunity and inflammatory responses. It is an effective method to treat cancer through ameliorating TME and modulating TAMs by converting M2 into M1-like phenotype. However, intracellular signaling mechanisms underlying TAMs polarization are largely undefined. Phosphoinositide 3-kinase (PI3K)/Akt is an important signaling pathway participating in M2-like TAMs polarization, survival, growth, proliferation, differentiation, apoptosis and cytoskeleton rearrangement. In the present review, we analyzed the mechanism of TAMs polarization focusing on PI3K/Akt and its downstream mitogenactivated protein kinase (MAPK) as well as nuclear factor kappa B (NF-κB) signaling pathways, thus provides the first evidence of intracellular targets for cancer immunotherapy.
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NF-kappa B , Fosfatidilinositol 3-Quinases , Linhagem Celular Tumoral , Citocinas/metabolismo , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Plásticos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
Macrophages are a type of functionally plastic cells that can create a pro-/anti-inflammatory microenvironment for organs by producing different kinds of cytokines, chemokines, and growth factors to regulate immunity and inflammatory responses. In addition, they can also be induced to adopt different phenotypes in response to extracellular and intracellular signals, a process defined as M1/M2 polarization. Growing evidence indicates that glycobiology is closely associated with this polarization process. In this research, we review studies of the roles of glycosylation, glucose metabolism, and key lectins in the regulation of macrophages function and polarization to provide a new perspective for immunotherapies for multiple diseases.
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Ativação de Macrófagos , Macrófagos/imunologia , Animais , Citocinas/imunologia , Glucose/imunologia , Glicosilação , Humanos , Imunidade , Lectinas/imunologia , Macrófagos/citologiaRESUMO
Thienopyridines and related heterocycles were prepared in a straightforward manner in moderate to good yields and under mild conditions by palladium-catalysed cross-coupling of ortho-fluorinated iodopyridines and terminal alkynes, followed by a reagent-capsule-assisted thiolation cyclization process. By applying paraffin wax capsules to prevent catalyst poisoning and undesired side reactions, the separation and purification processes were reduced.
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Silkworm (Bombyx mori) pupae lipid profiles were analyzed using high-resolution mass spectrometry-based lipidomics. A total of 241 lipid molecular species were annotated with high confidence in male and female silkworm pupae. Triacylglycerol (TG), phosphoethanolamine (PE) and phosphocholine (PC) were the main lipid subclasses of silkworm pupae, accounting for 63, 41 and 38 lipid molecular species, respectively. No unique lipid molecular species were identified, but there were differences in the abundance of lipid molecular species between male and female silkworm pupae. Therefore, the differences in the lipid abundance of male and female silkworm pupae were analyzed by chemometrics. As a result, 8 lipid molecular species were screened for differential lipids. Hierarchical clustering analysis (HCA) showed that male and female silkworm pupae samples formed two distinct branches, indicating that these differential lipids could potentially be used as biomarkers to distinguish between male and female silkworm pupae.
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Background: Fibroblast activation protein (FAP) is a cell-surface serine protease that has both dipeptidyl peptidase as well as endopeptidase activities and could cleave substrates at post-proline bond. Previous findings showed that FAP was hard to be detected in normal tissues but significantly up-regulated in remodeling sites like fibrosis, atherosclerosis, arthritis and embryonic tissues. Though increasing evidence has demonstrated the importance of FAP in cancer progression, no multifactorial analysis has been developed to investigate its function in gastrointestinal cancers until now. Methods: By comprehensive use of datasets from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), scTIME Portal and Human Protein Atlas (HPA), we evaluated the carcinogenesis potential of FAP in gastrointestinal cancers, analyzing the correlation between FAP and poor outcomes, immunology in liver, colon, pancreas as well as stomach cancers. Then liver cancer was selected as example to experimentally validate the pro-tumor and immune regulative role of FAP in gastrointestinal cancers. Results: FAP was abundantly expressed in gastrointestinal cancers, such as LIHC, COAD, PAAD and STAD. Functional analysis indicated that the highly-expressed FAP in these cancers could affect extracellular matrix organization process and interacted with genes like COL1A1, COL1A2, COL3A1 and POSTN. In addition, it was also observed that FAP was positively correlated to M2 macrophages infiltration across these cancers. To verify these findings in vitro, we used LIHC as example and over-expressed FAP in human hepatic stellate LX2 cells, a main cell type that produce FAP in tumor tissues, and then investigate its role on LIHC cells as well as macrophages. Results showed that the medium from FAP-over-expressed LX2 cells could significantly promote the motility of MHCC97H and SK-Hep1 LIHC cells, increase the invasion of THP-1 macrophages and induce them into pro-tumor M2 phenotype. Conclusion: In summary, we employed bioinformatic tools and experiments to perform a comprehensive analysis about FAP. Up-regulation of FAP in gastrointestinal cancers was primarily expressed in fibroblasts and contributes to tumor cells motility, macrophages infiltration and M2 polarization, revealing the multifactorial role of FAP in gastrointestinal cancers progression.
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Neoplasias Gastrointestinais , Proteômica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
The glycosylation levels of proteins in cancer cells are closely related to cancer invasion and migration. CD44 is a transmembrane glycoprotein that is significantly overexpressed in a variety of tumor cells and has been proven to promote the migration and motility of cancer cells, but the effect of its N-glycosylation modification on CD44 protein function in tumors is less studied. Here, we investigated the effect of six N-glycan chains (N25/57/100/110/120/255) on CD44s localization, function and stability in hepatocarcinoma cells. When the six sites were mutated, we found that CD44s lost its membrane localization in Huh7 and MHCC-97H cells. On this basis, we identified three glycosylation sites on CD44s (N57, N100 and N110) that played key roles in intracellular localization. When N57, N100 and N110 were mutated together, CD44 localized to the cytoplasm, while another three-site mutant (N25/N120/N255) was still anchored to the membrane. In addition, the ability of CD44-N57Q/N100Q/N110Q to promote the metastasis and invasion of Huh7 and 97H cells was weakened compared with that of CD44-N25Q/N120Q/N255Q. Furthermore, CD44-N57Q/N100Q/N110Q accumulated abnormally in the ER, and a high level of the ER stress (ERS) marker BiP was detected at the same time compared with wild-type CD44. When the lysosome inhibitor CQ was added, the content of mutant protein that triggered ERS significantly increased, which indicated that the degradation mode of CD44-N57Q/N100Q/N110Q after ERS was mainly through the lysosomal pathway (ERLAD). The results revealed that the N-glycosylation sites N57, N100 and N110 mutated on CD44s affected its function and degraded it by lysosomes after triggering ERS. These findings provide data for new studies on ER-related degradation, further promote the study of the glycan chain function of CD44 and furnish new ideas for the treatment of liver cancer metastasis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Glicosilação , Citoplasma/metabolismo , Polissacarídeos , Receptores de Hialuronatos/metabolismoRESUMO
Drug-loaded liposomes have been shown to be effective in the treatment of hepatocellular carcinoma (HCC). However, the systemic non-specific distribution of drug-loaded liposomes in tumor patients is a critical therapeutic challenge. To address this issue, we developed galactosylated chitosan-modified liposomes (GC@Lipo) that could selectively bind to the asialoglycoprotein receptor (ASGPR), which is highly expressed on the membrane surface of HCC cells. Our study demonstrated that the GC@Lipo significantly enhanced the anti-tumor efficacy of oleanolic acid (OA) by enabling targeted drug delivery to hepatocytes. Remarkably, treatment with OA-loaded GC@Lipo inhibited the migration and proliferation of mouse Hepa1-6 cells by upregulating E-cadherin expression and downregulating N-cadherin, vimentin, and AXL expressions, compared to a free OA solution and OA-loaded liposomes. Furthermore, using an axillary tumor xenograft mouse model, we observed that OA-loaded GC@Lipo led to a significant reduction in tumor progression, accompanied by concentrated enrichment in hepatocytes. These findings strongly support the clinical translation of ASGPR-targeted liposomes for the treatment of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Ácido Oleanólico , Camundongos , Humanos , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Lipossomos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos , Hepatócitos , Modelos Animais de DoençasRESUMO
N-glycosylation has been revealed to be tightly associated with cancer metastasis. As a key transferase that catalyzes the formation of ß1,4 N-acetylglucosamine (ß1,4GlcNAc) branches on the mannose core of N-glycans, N-acetylglucosaminyltransferase IVa (GnT-IVa) has been reported to be involved in hepatocellular carcinoma (HCC) metastasis by forming N-glycans; however, the underlying mechanisms are largely unknown. In the current study, we found that GnT-IVa was upregulated in HCC tissues and positively correlated with worse outcomes in HCC patients. We found that GnT-IVa could promote tumor growth in mice; notably, this effect was attenuated after mutating the enzymatic site (D445A) of GnT-IVa, suggesting that GnT-IVa regulated HCC progression by forming ß1,4GlcNAc branches. To mechanistically investigate the role of GnT-IVa in HCC, we conducted GSEA and GO functional analysis as well as in vitro experiments. The results showed that GnT-IVa could enhance HCC cell migration, invasion and adhesion ability and increase ß1,4GlcNAc branch glycans on integrin ß1 (ITGB1), a tumor-associated glycoprotein that is closely involved in cell motility by interacting with vimentin. Interruption of ß1,4GlcNAc branch glycan modification on ITGB1 could suppress the interaction of ITGB1 with vimentin and inhibit cell motility. These results revealed that GnT-IVa could promote HCC cell motility by affecting the biological functions of ITGB1 through N-glycosylation. In summary, our results revealed that GnT-IVa is highly expressed in HCC and can form ß1,4GlcNAc branches on ITGB1, which are essential for interactions with vimentin to promote HCC cell motility. These findings not only proposed a novel mechanism for GnT-IVa in HCC progression but also revealed the significance of N-glycosylation on ITGB1 during the process, which may provide a novel target for future HCC therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , N-Acetilglucosaminiltransferases , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Glicosilação , Integrina beta1/genética , Integrina beta1/metabolismo , Neoplasias Hepáticas/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Vimentina/genética , Vimentina/metabolismo , HumanosRESUMO
Febrifugine, a natural alkaloid, exhibits specific anti-phytophthora activity; however, its mode of action is unclear. In this study, halofuginone, a synthetic derivative of febrifugine, showed significantly higher anti-phytophthora activities than those of febrifugine and the commercial drug metalaxyl against Phytophthora sojae, Phytophthora capsici, and Phytophthora infestans with effective concentration for 50% inhibition (EC50) values of 0.665, 0.673, and 0.178 µg/mL, respectively. Proline could alleviate the growth inhibition of halofuginone on P. capsici, implying that halofuginone might target prolyl-tRNA synthetase (PcPRS). The anti-phytophthora mechanism of halofuginone was then investigated by molecular docking, fluorescence titration, and enzymatic inhibition assays. The results revealed that halofuginone could bind to PcPRS and shared a similar binding site with the substrate proline. Point mutations at Glu316 and Arg345 led to 24.5 and 16.1% decreases in the enzymatic activity of PcPRS but 816.742- and 459.557-fold increases in the resistance to halofuginone, respectively. The results further confirmed that halofuginone was a competitive inhibitor of proline against PcPRS, and Glu316 and Arg345 played important roles in the binding of halofuginone and proline. Taken together, the results indicated that halofuginone is an alternative anti-phytophthora drug candidate and that PcPRS represents a potential target for the development of new pesticides.
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Aminoacil-tRNA Sintetases , Praguicidas , Phytophthora , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Simulação de Acoplamento Molecular , Praguicidas/farmacologia , Piperidinas , Prolina/farmacologia , QuinazolinonasRESUMO
Propensity to relapse, even after long-term abstinence, is a crucial feature of methamphetamine (METH) abuse. We and other laboratories have reported that acute treatment of oxytocin (OXT), a hormone and neuropeptide, could inhibit reinstatement of METH seeking in animal studies. However, the effects of repeated OXT treatment on METH reinstatement as well as underlying mechanisms are still unclear. In the present study, the effects of repeated OXT treatment during abstinence on context- or restraint stress-induced reinstatement were investigated using the mice conditioned place preference (CPP) paradigm. After three intermittent injections of METH (2 mg/kg, i.p.) to induce CPP, mice received a daily bilateral intra-hippocampus injection of OXT (0.625, 1.25 or 2.5 µg) for 8 consecutive days before the context- or restraint stress-induced reinstatement test. Meanwhile, adult hippocampal neurogenesis (AHN) level was detected using immunostaining. To further clarify the role of AHN underlying OXT's effects on METH-CPP reinstatement, temozolomide (TMZ, 25 mg/kg, i.p.) was employed to deplete AHN prior to OXT treatment. The data showed that repeated OXT treatment (1.25 and 2.5 µg, intra-hippocampus) significantly inhibited both context- and restraint stress-induced METH-CPP reinstatement and concomitantly promoted AHN in a dose-dependent manner. Notably, TMZ pre-treatment markedly abolished all the above-mentioned effects of OXT, suggesting that AHN was closely involved in OXT's inhibition on reinstatement induced by both triggers. Taken together, the present study indicated that repeated OXT treatment during abstinence could inhibit both context- and restraint stress-induced METH-CPP reinstatement possibly by promoting AHN in mice, which provided a better understanding for OXT's beneficial effects on METH addiction.
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Condicionamento Operante/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Metanfetamina/administração & dosagem , Neurogênese/efeitos dos fármacos , Ocitocina/administração & dosagem , Restrição Física/psicologia , Animais , Condicionamento Operante/fisiologia , Inibidores da Captação de Dopamina/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Hipocampo/citologia , Hipocampo/fisiologia , Bombas de Infusão Implantáveis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Restrição Física/efeitos adversosRESUMO
Citri reticulatae pericarpium (CRP) shows multiple bioactivities, including antioxidant, anti-tumor, and anti-inflammation. The folk proverb "CRP, the older, the better" means storing for longer time would improve its quality, which attributed to the influence of bioactive compounds. The aim of this work was to study which compounds are the factors that long storage would influence the quality of CRP. 161 compounds, including 65 flavonoids, 51 phenolic acids, 27 fatty acids, and 18 amino acids were identified through derivatization and non-derivatization liquid chromatography mass spectrometry approaches. Their dynamic changes indicated phenolic acids, which were reported to have various activities, were the main increased components. Furthermore, the representative phenolic acids were quantified and correlation analysis between their contents and antioxidant activity implicated they were the possible indicators that long storage would improve CRP quality. The results would provide basis for quality control of CRP during storage.
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Citrus , Medicamentos de Ervas Chinesas , Antioxidantes , FlavonoidesRESUMO
Drug addiction is one of the leading causes of mortality worldwide. Despite great advances were achieved in understanding the neurobiology of drug addiction, the therapeutic options are severely limited, with poor effectiveness and serious side effects. The neuropeptide oxytocin (OXT) is well known for its effects on uterine contraction, sexual/maternal behaviors, social affiliation, stress and learning/memory by interacting with the OXT receptor and other neuromodulators. Emerging evidence suggests that the acute or chronic exposure to drugs can affect the OXT system. Additionally, OXT administration can ameliorate a wide range of abused drug-induced neurobehavioral changes. Overall, OXT not only suppresses drug reward in the binge stage of drug addiction, but also reduces stress responses and social impairments during the withdrawal stage and, finally, prevents drug/cue/stress-induced reinstatement. More importantly, clinical studies have also shown that OXT can exert beneficial effects on reducing substance use disorders of a series of drugs, such as heroin, cocaine, alcohol, cannabis and nicotine. Thus, the present review focuses on the role of OXT in treating drug addiction, including the preclinical and clinical therapeutic potential of OXT and its analogs on the neurobiological perspectives of drugs, to provide a better insight of the efficacy of OXT as a clinical addiction therapeutic agent.
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Ocitocina , Transdução de Sinais , Transtornos Relacionados ao Uso de Substâncias , Humanos , Ocitocina/efeitos dos fármacos , Ocitocina/fisiologia , Transdução de Sinais/fisiologia , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológicoRESUMO
Pathological grading of meningioma is insufficient to predict recurrence after resection and to guide individualized treatment strategies. One hundred and thirty-three patients with meningiomas who underwent total resection were enrolled in this retrospective study. Univariate analyses were conducted to evaluate the association between factors and recurrence. Least absolute shrinkage and selection operator (Lasso) was used to further select variables to build a logistic model. The predictive efficiency of the model and WHO grade was compared by using receiver operating characteristic curve (ROC), decision curve analysis (DCA), and net reclassification improvement (NRI). Patients were given a new risk layer based on a nomogram. The recurrence of meningioma in different groups was observed through the Kaplan-Meier curve. Univariate analysis demonstrated that 11 risk factors were associated with prognosis (P < 0.05). The result of ROC proved that the quantified risk-scoring system (AUC = 0.853) had a higher benefit than pathological grade (AUC = 0.689, P = 0.011). The incidence of recurrence of the high risk cohort (69%) was significantly higher than that of the low risk cohort (9%) by Kaplan-Meier analysis (P < 0.001). And all patients who did not relapse in the high risk group received adjuvant radiotherapy. The novel risk stratification algorithm has a significant value for the recurrence of meningioma and can help in optimizing the individualized design of clinical therapy.
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Drugs of abuse, including morphine and cocaine, can reduce hippocampal neurogenesis (HN). Whereas promotion of HN is being increasingly recognized as a promising strategy for treating morphine and cocaine addiction. The present study is focused on exploring the changes of HN during methamphetamine (METH) administration and further clarify if HN is involved in METH-associated reward memory. After successfully establishing the conditioned place preference (CPP) paradigm to simulate the METH-associated reward memory in C57BL/6 mice, we observed that HN was significantly inhibited during METH (2 mg/kg, i. p.) administration and returned to normal after the extinction of METH CPP, as indicated by the immunostaining of bromodeoxyuridine (BrdU) and doublecortin (DCX) in the hippocampus. To promote/inhibit HN levels, 7,8-dihydroxyflavone (DHF), a small tyrosine kinase receptor B (TrkB) agonist and temozolomide (TMZ), an alkylating agent, were administered intraperitoneally (i.p.), respectively. The data showed that either DHF (5 mg/kg, i. p.) or TMZ (25 mg/kg, i. p.) pre-treatment before METH administration could significantly prolong extinction and enhance reinstatement of the reward memory. Notably, DHF treatment after METH administration significantly facilitated extinction and inhibited METH reinstatement, while TMZ treatment resulted in opposite effects. The present study indicated that METH administration could induce a temporal inhibitory effect on HN. More importantly, promotion of HN after the acquisition of METH-associated reward memory, but not inhibition of HN or promotion of HN before the acquisition of reward memory, could facilitate METH extinction and inhibit METH reinstatement, indicating the beneficial effect of HN on METH addiction by erasing the according reward memory.
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Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Metanfetamina/farmacologia , Neurogênese/efeitos dos fármacos , Recompensa , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Extinção Psicológica/efeitos dos fármacos , Flavonas/farmacologia , Hipocampo/metabolismo , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Temozolomida/farmacologiaRESUMO
B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein expressed on malignant plasma cells of patients with multiple myeloma (MM), and it is a defined therapeutic target. Major histocompatibility complex class I-related chain A (MICA) is frequently expressed in lymphoproliferative malignancies including MM. MICA activates natural killer (NK) cells and costimulates T cells by interaction with its immunoreceptor NK cell receptor G2D (NKG2D). Nonetheless, during full-blown MM, tumor cells promote efficient MICA shedding, which evokes NKG2D internalization and immune suppression. To enhance the directional killing efficacy of immune cells against myeloma cells, we constructed a novel bispecific antibody 2A9-MICA and explored its potential antimyeloma activity against MM. 2A9-MICA consists of human MICA extracellular region and a single-chain antibody fragment (scFv) that targets BCMA generated by phage display technology. In vitro, 2A9-MICA activated NK cell-mediated cytotoxicity and induced NK cells to kill BCMA-positive human myeloma cells. Moreover, in BCMA-positive, MM-bearing nude mice, 2A9-MICA specifically targeted tumor tissue, where it effectively recruited immune cells and inhibited tumor tissue growth showed superior antitumor activity. Taken together, bispecific antibody 2A9-MICA provides a new approach for MM-targeting immunotherapy and has attractive potential for clinical applications.
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Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Mieloma Múltiplo/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/uso terapêutico , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/uso terapêutico , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/etiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
PURPOSE: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). METHODS: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. RESULTS: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. CONCLUSION: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CD24/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Receptores Fc/metabolismo , Transdução de Sinais , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is growing evidence that uridine may act as an endogenous neuromodulator with a potential signaling role in the central nervous system in addition to its function in pyrimidine metabolism. We previously found that acute morphine treatment significantly increased uridine release in the dorsal striatum of mice, indicating that uridine may contribute to morphine-induced neurobehavioral changes. In the present study, we analyzed the mechanism involved in morphine-induced uridine release and the role of uridine in morphine-induced neurobehavioral changes. Uridine release in the dorsal striatum of mice was assessed by in vivo microdialysis coupled with high performance liquid chromatography (HPLC) after morphine treatment. Western blotting and immunofluorescence were used to evaluate the expression of uridine-related proteins. Morphine-induced neurobehavioral changes were assessed by locomotor activity, behavioral sensitization and conditioned place preference (CPP) test. The expression of NT5E, an extracellular enzyme involved in formation of nucleosides, including uridine, was specifically knocked down in the dorsal striatum of mice using adeno-associated virus (AAV)-mediated short hairpin RNA (shRNA). The results indicated that both acute and chronic morphine administration significantly increased uridine release in the dorsal striatum, and this was associated with upregulation of NT5E but not other uridine-related proteins. Inhibition of NT5E with APCP or shRNA markedly inhibited morphine-induced uridine release in the dorsal striatum and related neurobehavioral changes, including hyperlocomotor activity, behavioral sensitization and CPP. Our data give a better understanding of the contribution of NT5E to morphine-induced uridine release and neurobehavioral changes, and identify NT5E as a potential target for treating morphine abuse.
Assuntos
5'-Nucleotidase/metabolismo , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Corpo Estriado/metabolismo , Locomoção/efeitos dos fármacos , Morfina/farmacologia , Uridina/metabolismo , 5'-Nucleotidase/antagonistas & inibidores , Animais , Fosfatos de Dinucleosídeos/farmacologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Masculino , Camundongos , Microdiálise , RNA Interferente Pequeno/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Cluster of differentiation 24 (CD24) is a specific surface marker involved in the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, all reported anti-CD24 antibodies are murine ones with inevitable immunogenicity. To address this, a method using both molecular structure and docking-based complementarity determining region (CDR) grafting was employed for humanization. After xenogeneic CDR grafting into a human antibody, three types of canonical residues (in the VL/VH interface core, in the loop foundation, and interaction with loop residues) that support loop conformation and residues involved in the antigen-binding interface were back-mutated. Four engineered antibodies were produced, among which hG7-BM3 has virtually identical 3-D structure and affinity parameters with the parental chimeric antibody cG7. In vitro, hG7-BM3 demonstrated superior immunogenicity and serum stability to cG7. Antibody-dependent cellular cytotoxicity (ADCC), tumor cell internalization and in vivo targeting assays indicate that hG7-BM3 has the potential for development as an antibody-drug conjugate (ADC). We therefore generated the hG7-BM3-VcMMAE conjugate, which was shown to induce tumor cell apoptosis and effectively suppress nude mice bearing HCC xenografts. In conclusion, our study provides new inspiration for antibody humanization and an ADC candidate for laboratory study and clinical applications.