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Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 µM, in contrast to an IC50 of 28.3 µM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 µM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 µM). Notably, (-)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.
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COVID-19 , Estilbenos , Humanos , Pandemias , Fenóis , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: Chronic hepatitis B virus (HBV) infection (CHB) is a significant public health problem that could benefit from treatment with immunomodulators. Here we describe a set of therapeutic HBV vaccines that target the internal viral proteins. METHODS: Vaccines are delivered by chimpanzee adenovirus vectors (AdC) of serotype 6 (AdC6) and 7 (AdC7) used in prime only or prime-boost regimens. The HBV antigens are fused into an early T cell checkpoint inhibitor, herpes simplex virus (HSV) glycoprotein D (gD), which enhances and broadens vaccine-induced cluster of differentiation (CD8)+ T cell responses. RESULTS: Our results show that the vaccines are immunogenic in mice. They induce potent CD8+ T cell responses that recognize multiple epitopes. CD8+ T cell responses increase after a boost, although the breadth remains similar. In mice, which carry high sustained loads of HBV particles due to a hepatic infection with an adeno-associated virus (AAV)8 vector expressing the 1.3HBV genome, CD8+ T cell responses to the vaccines are attenuated with a marked shift in the CD8+ T cells' epitope recognition profile. CONCLUSIONS: Our data show that in different stains of mice including those that carry a human major histocompatibility complex (MHC) class I antigen HBV vaccines adjuvanted with a checkpoint inhibitor induce potent and broad HBV-specific CD8+ T cell responses and lower but still detectable CD4+ T cell responses. CD8+ T cell responses are reduced and their epitope specificity changes in mice that are chronically exposed to HBV antigens. Implications for the design of therapeutic HBV vaccines are discussed.
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Hepatite B Crônica , Hepatite B , Animais , Linfócitos T CD8-Positivos , Epitopos de Linfócito T/genética , Vacinas contra Hepatite B , Vírus da Hepatite B/genética , Camundongos , Infecção PersistenteRESUMO
BACKGROUND: Ischemic stroke (IS) is a major public health issue causing mortality and disability and is more difficult to treat than other cerebral diseases. Previous study reported that miR-376a was upregulated in the serum of stroke patients, indicating that miR-376a played potential role in occurrence and development of stroke. METHODS: IS cell model was induced by oxygen-glucose deprivation (OGD) exposed HCMEC/D3 cells. The mRNA level of SNHG1, miR-376a and inflammatory cytokines were detected by q-PCR. Protein level of CBS, apoptotic proteins were examined by Western blot. Apoptosis was analyzed by flow cytometry, and H2S level was measured by kit. Interaction among lncRNA, miRNA and target gene was validated by luciferase assay. RESULTS: Our research revealed that mRNA level of SNHG1 and CBS in HCMEC/D3 cells was downregulated while miR-376a was upregulated under OGD conditions. Further results demonstrated that miR-376a overexpression promoted apoptosis and inflammation while SNHG1 overexpressing alleviated such processes. Mechanistically, SNHG1 directly targeted miR-376a, and CBS was a target of miR-376a. Moreover, SNHG1 exert its function via inhibiting miR-376a to regulate CBS expression. CONCLUSION: LncRNA SNHG1 depressed apoptosis and inflammation of IS cell model via inhibiting miR-376a and upregulating CBS/H2S signal. These results show light on underlying mechanisms of IS and provide potential targets for IS therapy.
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Apoptose/fisiologia , Cistationina beta-Sintase/metabolismo , Citocinas/metabolismo , Sulfeto de Hidrogênio/metabolismo , Inflamação/metabolismo , AVC Isquêmico/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Células Cultivadas , Regulação para Baixo , Humanos , Hipoglicemia/metabolismo , Hipóxia/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Glioma is one of the most common and aggressive malignant primary brain tumor with invariably poor 5-year survival rates. Because of the high recurrence rate and mortality rate, effective therapies for glioma are still weak. Recently, several studies has been proved that long non-coding RNAs (lncRNAs) have been identified to play regulatory mediators in the tumorigenesis of glioma. Nevertheless, the role of lncRNAs and their downstream transcripts are still elusive in the progression of glioma. Small nucleolar RNA host gene 16 (SNHG16), a newly identified lncRNA, has been verified to be up-regulated in human malignant carcinomas. In the present study, we confirmed that lncRNA SNHG16 was highly expressed in glioma and may exert oncogenic function as a competing endogenous RNA (ceRNA) to regulate EGFR by sponging of miR-373-3p through activating PI3K/AKT pathway, which providing a new insight of the regulatory network of lncRNA SNHG16 in the development of glioma.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Humanos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/fisiologia , Transdução de SinaisRESUMO
The erythropoietin-producing hepatocellular carcinoma A3 (EphA3) receptor tyrosine kinase (RTK) regulates morphogenesis during development and is overexpressed and mutated in a variety of cancers. EphA3 activation is believed to follow a 'seeding mechanism' model, in which ligand binding to the monomeric receptor acts as a trigger for signal-productive receptor clustering. We study EphA3 lateral interactions on the surface of live cells and we demonstrate that EphA3 forms dimers in the absence of ligand binding. We further show that these dimers are stabilized by interactions involving the EphA3 sterile α-motif (SAM) domain. The discovery of unliganded EphA3 dimers challenges the current understanding of the chain of EphA3 activation events and suggests that EphA3 may follow the 'pre-formed dimer' model of activation known to be relevant for other receptor tyrosine kinases. The present work also establishes a new role for the SAM domain in promoting Eph receptor lateral interactions and signalling on the cell surface.
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Multimerização Proteica/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/genética , Receptor EphA3RESUMO
Human adenovirus (AdHu)-based candidate AIDS vaccine can provide protection from simian immunodeficiency virus (SIV) transmission and disease progression. However, their potential use may be limited by widespread preexisting immunity to the vector. In contrast, preexisting immunity to chimpanzee adenoviruses (AdC) is relatively rare. In this study, we utilized two regimens of prime-boost immunizations with AdC serotype SAd-V23 (also called AdC6) and SAd-V24 (also called AdC7) expressing SIV Gag/Tat to test their immunogenicity and ability to protect rhesus macaques (RMs) from a repeated low-dose SIVmac239 challenge. Both AdC6 followed by AdC7 (AdC6/7) and AdC7 followed by AdC6 (AdC7/6) induced robust SIV Gag/Tat-specific T cell responses as measured by tetramer staining and functional assays. However, no significant protection from SIV transmission was observed in either AdC7/6- or AdC7/6-vaccinated RMs. Interestingly, in the RMs showing breakthrough infections, AdC7/6-SIV immunization was associated with a transient but significant (P = 0.035 at day 90 and P = 0.033 at day 120 postinfection) reduction in the setpoint viral load compared to unvaccinated controls. None of the measured immunological markers (i.e., number or functionality of SIV-specific CD8(+) and CD4(+) T cell responses and level of activated and/or CCR5(+) CD4(+) target cells) at the time of challenge correlated with protection from SIV transmission in the AdC-SIV-vaccinated RMs. The robust immunogenicity observed in all AdC-immunized RMs and the transient signal of protection from SIV replication exhibited by AdC7/6-vaccinated RMs even in the absence of any envelope immunogen suggest that AdC-based vectors may represent a promising platform for candidate AIDS vaccines.
Assuntos
Adenovirus dos Símios/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene tat/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adenovirus dos Símios/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene gag/genética , Produtos do Gene tat/genética , Vetores Genéticos , Humanos , Imunização Secundária , Pan troglodytes/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Doenças Retais , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Replicação ViralRESUMO
Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.
Assuntos
COVID-19 , Animais , Cricetinae , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Vacinas contra COVID-19/genética , Pan troglodytes , Adenoviridae/genética , Nucleocapsídeo , Imunização , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
SARS-CoV-2 vaccines aim to protect against COVID-19 through neutralizing antibodies against the viral spike protein. Mutations within the spike's receptor-binding domain may eventually reduce vaccine efficacy, necessitating periodic updates. Vaccine-induced immunity could be broadened by adding T cell-inducing antigens such as SARS-CoV-2's nucleoprotein (N). Here we describe two replication-defective chimpanzee adenovirus (AdC) vectors from different serotypes expressing SARS-CoV-2 N either in its wild-type form or fused into herpes simplex virus glycoprotein D (gD), an inhibitor of an early T cell checkpoint. The vaccines induce potent and sustained CD8+ T cell responses that are broadened upon inclusion of gD. Depending on the vaccine regimen booster immunizations increase magnitude and breadth of T cell responses. Epitopes that are recognized by the vaccine-induced T cells are highly conserved among global SARS-CoV-2 isolates indicating that addition of N to COVID-19 vaccines may lessen the risk of loss of vaccine-induced protection due to variants.
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Glioma is a malignant brain tumor that accounts for 30% of all brain tumors and 80% of malignant brain tumors. This poor clinical outcome makes the study of molecular mechanisms in glioma as an urgent subject. However, the certain mechanism remains unclear. Long non-coding RNAs (lncRNAs) plays a key role in glioma development and progression. In the present study, we aimed to explore the potential mechanisms of lncRNA SNHG16 in glioma. The levels of lncRNA SNHG16 were qualified in both glioma tissues and cell lines using qRT-PCR assay. The ability of cell proliferation was tested via CCK-8 and colony formation assays. Transfections were performed to knockdown SNHG16 and its target gene p21. The cell cycles and cell apoptosis were evaluated using flow cytometry, and the expression of SNHG16, p21 and apoptosis biomarkers were qualified with qRT-PCR and western blot assays. The expression of SNHG16 were up-regulated in both glioma tissues and cell lines. Knockdown of SNHG16 was associated with poor proliferation, decreased monoclonal formation rates, but increased apoptosis rates, which also caused the high expression of p21. Moreover, p21 could mediate cell proliferation and monoclonal formation, promote cell apoptosis in glioma, which was negatively correlated with lncRNA SNHG16. The molecule mechanism experiments revealed that SNHG16 could not only inhibit the expression of p21 but also suppressed the level of caspase 3 and 9, while promoted cyclinD1 and cyclinB1 expression. lncRNA SNHG16 could promote the cell proliferation and inhibit the apoptosis of glioma through suppressing p21, indicating that lncRNA SNHG16 might be quite vital for the diagnosis and progression of glioma and could even be a novel therapeutic target for glioma.
Assuntos
Neoplasias Encefálicas/patologia , Carcinogênese/genética , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Glioma/patologia , RNA Longo não Codificante/genética , Apoptose/genética , Neoplasias Encefálicas/genética , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , HumanosRESUMO
OBJECTIVE: To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC). METHODS: DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 5 LMP2-DCs by intradermal injection at week 0 and after the second and fourth weeks. Specific responses to LMP2 were detected by enzyme-linked immunospot (ELISPOT) assay at week 0 and at the fifth and eighth weeks. Local clinicians performed the follow-up and tracking of patients. RESULTS: We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders. CONCLUSION: In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.
Assuntos
Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Imunoterapia/métodos , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/uso terapêutico , Adulto , Idoso , China , Feminino , Humanos , Injeções Intradérmicas , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
In this study, Pb-Co powder-pressed alloy was fabricated and used as a suitable anode material to replace Pb-Ca-Sn alloy in electrowinning. The Pb-Co anodes and the traditional Pb-Ca-Sn alloy on the electrochemical properties are investigated in a 160 g L-1 H2SO4 solution at 35 °C using galvanostatic polarization, electrochemical impedance spectroscopy and Tafel test. Thereafter, the anodic oxide layer is observed by energy dispersive X-ray spectroscopy along with scanning electron microscopy and X-ray diffractometry. The results show that the potential and oxygen evolution over-potential of the anodes exhibit a declining trend with increasing the fraction of Co. The anode potential of the Pb-2 wt% Co is approximately 170 mV lower than that of Pb-Ca-Sn alloy and reaches a stable value of 1.291 V at 35 °C, which shows good electrocatalytic performance and commercial application prospect.
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How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8+ TILs enhance peroxisome proliferator-activated receptor (PPAR)-α signaling and catabolism of fatty acids (FAs) when simultaneously subjected to hypoglycemia and hypoxia. This metabolic switch partially preserves CD8+ TILs' effector functions, although co-inhibitor expression increases during tumor progression regardless of CD8+ TILs' antigen specificity. Further promoting FA catabolism improves the CD8+ TILs' ability to slow tumor progression. PD-1 blockade delays tumor growth without changing TIL metabolism or functions. It synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and even complete cures.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ácidos Graxos/metabolismo , Imunoterapia , Melanoma/imunologia , Melanoma/terapia , Microambiente Tumoral/imunologia , Animais , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/ultraestrutura , Hipóxia Celular , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/patologia , Melanoma/ultraestrutura , Camundongos Endogâmicos C57BL , Oxigênio/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Estresse Fisiológico , Resultado do Tratamento , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Camptothecin (CPT) that targets DNA topoisomerase I is one of the most promising broad-spectrum anticancer drugs in development today. The cytotoxicity of CPT is S phase (S)-specific because the collision of advancing replication forks with CPT-topoisomerase I-DNA complexes results in DNA damage. After DNA damage, proliferating cells could actively slow down the DNA replication through an S checkpoint to provide time for repair. We report now that there is an activated S checkpoint response in CPT-treated mammalian cells. This response is regulated by Ataxia and Rad3-related (ATR)/CHK1 pathway. Compared with their wild-type counterparts, CPT-treated Ku80-/- cells showed stronger inhibition of DNA replication. This stronger inhibition had no relationship with DNA-dependent protein kinase (DNA-PK) activity but correlated with the higher activities of ATR and the higher activities of CHK1 in such cells. Not only caffeine, the nonspecific inhibitor of ATR, or UCN-01, the nonspecific inhibitor of CHK1, but also the specific CHK1 antisense oligonucleotide abolished the stronger inhibition of DNA replication in CPT-treated Ku80-/- cells. These results in aggregate indicated that the stronger S checkpoint in CPT-treated Ku80-/- cells is regulated through the highly activated ATR/CHK1 pathway.
Assuntos
Antígenos Nucleares , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , DNA Helicases , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Fase S/efeitos dos fármacos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Quinase 1 do Ponto de Checagem , DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Fase S/fisiologiaRESUMO
After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas Serina-Treonina Quinases/fisiologia , Fase S/fisiologia , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Quinase 1 do Ponto de Checagem , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Proteínas Supressoras de TumorRESUMO
OBJECTIVE: To explore the methods of isolation, culture and identification of brain tumor stem cells (BTSCs) in neuroepithelial tumor tissues in vitro, and to study the correlation between BTSCs and the patholorical grades of neuroepithelial tumors. METHODS: Tumor cells from patients undergoing neuroepithelial tumors excision were acutely dissociated, triturated into single cells, and then seeded into serum-free medium. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passaged in fresh medium. The expression of Nestin and CD133 of BTSs was detected by immunocytochemistry staining, and the expression of CD133 of tumor specimen sections was detected by immunohistochemistry staining . The expression of CD133 of 46 brain tumors and 5 normal brain tissues were analysed by SABC immunohistochemical staining, and the correlation between the expression and pathological grade of the tumors was analysed. RESULTS: BTSCs from neuroepithelial tumors could be isolated and cultured, and could be generated and passaged in vitro. The expression of Nestin and CD133 could be detected in BTSCs. CD133 could be detected in neuroepithelial tumor tissues, but not in normal brain tissues. There was significant difference between the expression of CD133 and the different grades of tumors (P < 0.01), and there was a positive correlation between the expression of CD133 and the histologic grading of tumors (P < 0.01). CONCLUSION: A small proportion of stem cells have the ability to self-renew in human neuroepithelial tumors, and there is a positive correlation between the expression of CD133 and histologic grading of tumors.
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Antígenos CD/análise , Neoplasias Encefálicas/patologia , Glicoproteínas/análise , Neoplasias Neuroepiteliomatosas/patologia , Células-Tronco Neoplásicas/patologia , Peptídeos/análise , Antígeno AC133 , Humanos , Proteínas de Filamentos Intermediários/análise , Proteínas do Tecido Nervoso/análise , Nestina , Células Tumorais CultivadasRESUMO
Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.
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Adenoviridae/genética , Terapia Genética , Vetores Genéticos/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/biossíntese , Animais , Vetores Genéticos/uso terapêutico , Células HEK293 , HIV-1/genética , Humanos , Camundongos , Sorogrupo , Transgenes/genética , Replicação Viral/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/uso terapêuticoRESUMO
Following exposure to genotoxic stress, proliferating cells actively slow down DNA replication through an S phase checkpoint to provide time for repair. The ATM-dependent pathway plays an important role in the S phase checkpoint response following ionizing irradiation. We report that there is a stronger S phase checkpoint response in irradiated Ku80(-/-) cells as compared with their wild-type counterparts, which has no relationship to DNA-dependent protein kinase (DNA-PK) activity but correlates with a higher ATM activity and with more ATM bound to chromatin DNA in such cells. Wortmannin, a nonspecific inhibitor of ATM, not only reduces the higher activity of ATM kinase, but also abolishes the stronger S phase checkpoint response in Ku80(-/-) cells. Furthermore, a specific ATM antisense oligonucleotide abolishes the stronger S checkpoint response in Ku80(-/-) cells and renders these cells practically indistinguishable from Ku80(+/+) cells for this endpoint. These results in aggregate indicate that the stronger S checkpoint in irradiated Ku80(-/-) cells is due to the higher ATM kinase activity.
Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fase S , Fase S/fisiologia , Androstadienos/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células CHO , Proteínas de Ciclo Celular , Cricetinae , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Autoantígeno Ku , Proteínas Nucleares/genética , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Proteínas Supressoras de Tumor , Wortmanina , Raios XRESUMO
Near-Infrared Spectroscopy (NIRS) is the most rapidly developing and the most noticeable spectrographic technique in the 90's (the last century). Its principle and characteristics were explained in this paper, and the development of NIRS instrumentation, the methodology of spectrum pre-processing, as well as the chemical metrology were also introduced. The anthors mainly summarized the applications to agriculture and food, especially in-line analysis methods, which have been used in production procedure by fiber optics. The authors analyzed the NIRS application status in China, and made the first proposal to establish information sharing mode between central database and end-user by using network technology and concentrating valuable resources.
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Agricultura , Análise de Alimentos/métodos , Processamento de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , China , SoftwareRESUMO
Hybridization-based and sequencing-based technologies have found a widespread application in gene expression profiling analysis but much ambiguity exists regarding their reliability. This study developed a framework based on three parameters: detection ability, repeatability, and accuracy to evaluate the reliability of gene expression profiling technologies. The fraction of coverage of detected transcript category, the degree of variance for the number of differentially expressed genes (DEGs), the consistency of DEG category, and suspected false discovery rate (sFDR) were first introduced as statistical indices. In order to validate the availability of these indices, based on the same RNA extract, the analysis was performed by comparing gene expression differences between wild-type and transgenic rice using deep sequencing and microarray. The results suggested that the parameters were available and showed advances in the determination of gene expression differences. Based on relative self-comparison design, suspected false positive genes were easily identified from all DEGs detected, which was difficult for quantitative real-time polymerase chain reaction (qRT-PCR) validation when the count of DEGs was enormous. In addition, sFDRs had advantages in the accuracy evaluation for previous datasets.