RESUMO
To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Cesárea , Feminino , Transmissão Vertical de Doenças Infecciosas , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Gravidez , Suínos , VirulênciaRESUMO
Acquired resistance to lapatinib is a highly problematic clinical barrier that has to be overcome for a successful cancer treatment. Despite efforts to determine the mechanisms underlying acquired lapatinib resistance (ALR), no definitive genetic factors have been reported to be solely responsible for the acquired resistance in breast cancer. Therefore, we performed a cross-platform meta-analysis of three publically available microarray datasets related to breast cancer with ALR, using the R-based RankProd package. From the meta-analysis, we were able to identify a total of 990 differentially expressed genes (DEGs, 406 upregulated, 584 downregulated) that are potentially associated with ALR. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs showed that "response to organic substance" and "p53 signaling pathway" may be largely involved in ALR process. Of these, many of the top 50 upregulated and downregulated DEGs were found in oncogenesis of various tumors and cancers. For the top 50 DEGs, we constructed the gene coexpression and protein-protein interaction networks from a huge database of well-known molecular interactions. By integrative analysis of two systemic networks, we condensed the total number of DEGs to six common genes (LGALS1, PRSS23, PTRF, FHL2, TOB1, and SOCS2). Furthermore, these genes were confirmed in functional module eigens obtained from the weighted gene correlation network analysis of total DEGs in the microarray datasets ("GSE16179" and "GSE52707"). Our integrative meta-analysis could provide a comprehensive perspective into complex mechanisms underlying ALR in breast cancer and a theoretical support for further chemotherapeutic studies.
Assuntos
Antineoplásicos , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Quinazolinas , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Lapatinib , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas , Quinazolinas/uso terapêuticoRESUMO
Human cardiac fibroblasts (HCFs) have various voltage-dependent K(+) channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca(2+)-activated K(+) (KCa) currents but not delayed rectifier K(+) or transient outward K(+) currents, all of which are VDKCs. H2O2-stimulated KCa currents were blocked by iberiotoxin (IbTX, a large conductance KCa blocker). The H2O2-stimulating effect on large-conductance KCa (BKCa) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BKCa currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BKCa currents. Using RT-PCR and western blot analysis, three subtypes of KCa channels were detected in HCFs: BKCa channels, small-conductance KCa (SKCa) channels, and intermediate-conductance KCa (IKCa) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BKCa currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BKCa channels.
RESUMO
BACKGROUND: Achieving euglycaemia by continuous subcutaneous insulin infusion (CSII) therapy alone has been shown to restore ß-cell function in patients with newly diagnosed type 2 diabetes. However, the efficacy has not been evaluated in patients with non-newly diagnosed type 2 diabetes and suboptimal glycaemic control. METHODS: Of the 1220 patients with type 2 diabetes who began CSII therapy from March 2000 to March 2007, we retrospectively selected patients using the following inclusion criteria: glycosylated haemoglobin (HbA1c ) ≥ 7.0%, diabetes duration ≥ 1 year before CSII therapy, and duration of CSII therapy ≥ 6 months. We evaluated sequential changes in HbA1c and serum C-peptide levels measured at a 6- to 12-month intervals during CSII therapy. RESULTS: In the 521 subjects included in this study [median diabetes duration 10 years; interquartile range (IQR) 6.0-17.0; CSII therapy ≤ 30 months], median HbA1c decreased from 8.7% (IQR 7.7-10.0) at baseline to 6.3% (IQR 5.9-6.9) after 6 months of CSII therapy (p < 0.0001). During the subsequent 24 months, median HbA1c levels were maintained between 6.3% and 6.5% (p < 0.0001 for all time points vs baseline). At 12 months after CSII therapy, median C-peptide levels began to increase compared with baseline (fasting level 23% increase, p < 0.0001; 2-h postprandial level 26% increase, p = 0.022), and the increase was maintained at 30 months (fasting level 39%; 2-h postprandial level 53%; p < 0.0001 for all vs baseline). CONCLUSIONS: ß-Cell function was significantly improved in patients with non-newly diagnosed and suboptimally controlled type 2 diabetes after achieving and maintaining optimal glycaemic control with long-term CSII therapy alone.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Sistemas de Infusão de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/administração & dosagem , Idoso , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Infusões Subcutâneas , Resistência à Insulina , Células Secretoras de Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.
RESUMO
Despite the routine use of porcine reproductive and respiratory syndrome (PRRS)-modified live vaccines, serious concerns are currently being raised due to their quick reversion to virulence and limited cross-protection against divergent PRRS virus (PRRSV) strains circulating in the field. Therefore, a PRRS chimeric vaccine (JB1) was produced using a DNA-launched infectious clone by replacing open reading frames (ORFs) 3-6 with those from a mixture of two genetically different PRRSV2 strains (K07-2273 and K08-1054) and ORF1a with that from a mutation-resistant PRRSV strain (RVRp22) exhibiting an attenuated phenotype. To evaluate the safety and cross-protective efficacy of JB1 in a reproductive model, eight PRRS-negative pregnant sows were purchased and divided into four groups. Four sows in two of the groups were vaccinated with JB1, and the other 4 sows were untreated at gestational day 60. At gestational day 93, one vaccinated group and one nonvaccinated group each were challenged with either K07-2273 or K08-1054. All of the sows aborted or delivered until gestation day 115 (24 days post challenge), and the newborn piglets were observed up to the 28th day after birth, which was the end of the experiment. Overall, pregnant sows of the JB1-vaccinated groups showed no meaningful viremia after vaccination and significant reductions in viremia with K07-2273 and K08-1054, exhibiting significantly higher levels of serum virus-neutralizing antibodies than non-vaccinated sows. Moreover, the JB1-vaccinated groups did not exhibit any abortion due to vaccination and showed improved piglet viability and birth weight. The piglets from JB1-vaccinated sows displayed lower viral concentrations in serum and fewer lung lesions compared with those of the piglets from the nonvaccinated sows. Therefore, JB1 is a safe and effective vaccine candidate that confers simultaneous protection against two genetically different PRRSV strains.
RESUMO
Porcine epidemic diarrhea (PED) is a devastating disease that causes considerable economic damage to the global pig industry. Although the causative agent, the porcine epidemic diarrhea virus (PEDV), was identified about a half century ago, there is still much debate on the preventive measures against the disease, especially regarding the PED vaccine. Recent reports on PEDV variants make the vaccination for PEDV more confusing. Therefore, we systematically reviewed published articles on PED and vaccines against the disease and performed a meta-analysis of vaccine efficacy based on the clinical signs, fecal score and survival rates. A total of 299 articles on the efficacy of PED vaccines were found online, and 21 articles were selected that fulfilled all the criteria. A meta-analysis was performed on the 21 articles based on the fecal scores and survival rates. This analysis showed the efficacy of PED vaccines, and no significant differences in the efficacy depending on vaccine type (killed vs. live) or administration route (intramuscular vs. oral) were found. The results from our study suggest that any vaccination against PED is a useful strategy to control the disease regardless of the type of vaccine and administration route.
RESUMO
Alzheimer's disease (AD) is a chronic neurodegenerative disease, and typical pathologic findings include abnormally hyperphosphorylated tau aggregation and neurofibrillary tangles. Insulin resistance and hyperglycaemia have been proposed as risk factors for AD development. As the maintenance of optimal blood glucose level is an important indicator of diabetes mellitus (DM) treatment, diet control is essential. AMPK is a crucial sensor of cellular bioenergetics for controlling anabolic and catabolic metabolism. Since AMPK is a direct regulator of tau phosphorylation, we hypothesized that strict diet control to achieve euglycaemia affects tau protein phosphorylation through increased AMPK activity in the hippocampus of DM rats. To test this hypothesis, we generated insulin-deficient DM rats by subtotal pancreatectomy and the animals were categorized into the diet-restriction (R) group and ad libitum (AL) feeding group. We found that tau phosphorylation was significantly higher in the R group than that in the sham-control (C) or AL group. AMPK activity in the R group was significantly higher than that in the C or AL group, as expected. Furthermore, the R group showed more critical tau pathology in the hippocampus than the other groups. These results suggest that diet control to achieve euglycaemia in an insulin-deficient DM condition may be harmful because of the greater possibility of AD development through increased tau phosphorylation by AMPK activation in the hippocampus.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/dietoterapia , Hipocampo/efeitos dos fármacos , Proteínas tau/metabolismo , Doença de Alzheimer , Animais , Dieta , Modelos Animais de Doenças , Insulina/sangue , Insulina/deficiência , Masculino , Pancreatectomia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
While liver histopathology is heterogeneous in diabetes, the underlying mechanisms remain unclear. We investigated whether glycemic variation resulting from differential diets can induce heterogeneity in diabetic liver and the underlying molecular mechanisms. We generated end-stage non-obese diabetic model rats by subtotal-pancreatectomy in male Sprague- Dawley rats and ad libitum diet for 7 weeks (n = 33). The rats were then divided into three groups, and fed a standard- or a low-protein diet (18 or 6 kcal%, respectively), for another 7 weeks: to maintain hyperglycemia, 11 rats were fed ad libitum (18AL group); to achieve euglycemia, 11 were calorierestricted (18R group), and 11 were both calorie- and proteinrestricted with the low-protein diet (6R group). Overnightfasted liver samples were collected after the differential diets together with sham-control (18S group), and histology and molecular changes were compared. Hyperglycemic-18AL showed glycogenic hepatopathy (GH) without steatosis, with the highest GSK-3ß inactivation because of Akt activation during hyperglycemia; mitochondrial function was not impaired, compared to the 18S group. Euglycemic-18R showed neither GH nor steatosis, with intermediate GSK-3ß activation and mitochondrial dysfunction. However, euglycemic-6R showed both GH and steatosis despite the highest GSK-3ß activity and no molecular evidence of increased lipogenesis or decreased ApoB expression, where mitochondrial dysfunction was highest among the groups. In conclusion, heterogeneous liver histopathology developed in end-stage non-obese diabetic rats as the glycemic levels varied with differential diets, in which protein content in the diets as well as glycemic levels differentially influenced GSK-3ß activity and mitochondrial function in insulin-deficient state. [BMB Reports 2020; 53(2): 100-105].
Assuntos
Diabetes Mellitus Experimental/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Hiperglicemia/patologia , Fígado/patologia , Mitocôndrias/metabolismo , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Glicemia/metabolismo , Restrição Calórica , Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Experimental/metabolismo , Dieta da Carga de Carboidratos , Fígado Gorduroso/dietoterapia , Fígado Gorduroso/enzimologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Índice Glicêmico/fisiologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Hiperglicemia/dietoterapia , Hiperglicemia/enzimologia , Hiperglicemia/metabolismo , Insulina/metabolismo , Lipogênese , Fígado/enzimologia , Fígado/metabolismo , Masculino , Mitocôndrias/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/farmacologia , Animais , Animais Recém-Nascidos , Chlorocebus aethiops , Temperatura Baixa , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Feminino , Genótipo , Vírus da Diarreia Epidêmica Suína/genética , Gravidez , Distribuição Aleatória , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Vacinas Atenuadas/farmacologia , Células VeroRESUMO
We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2-3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.
Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Doenças dos Suínos/prevenção & controle , Potência de Vacina , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Colostro/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Diarreia/prevenção & controle , Feminino , Genótipo , Imunização Secundária , Injeções Intramusculares , Vírus da Diarreia Epidêmica Suína/genética , Gravidez , Taxa de Sobrevida , Suínos , Doenças dos Suínos/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Virulência , Eliminação de Partículas ViraisRESUMO
Since the 2013-2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate-scale regional outbreaks have recurred. In particular, areas of Jeju Island with extensive swine production have faced repeated epidemics since the re-emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full-length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non-structural protein (nsp)- or spike (S) protein-coding regions. The 2018 Jeju isolates shared 96.7%-98.7% and 98.5%-99.4% identity at the S gene and whole-genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re-emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU-1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV-induced syncytia formation. Genomic sequencing identified a unique 8-nt DEL in the extreme C-terminal region of the S gene at the 4th passage (KNU-1807-P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU-1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.
Assuntos
Infecções por Coronavirus/veterinária , Genoma Viral/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Filogenia , República da Coreia , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Sus scrofa , Suínos , Células Vero , VirulênciaRESUMO
The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.
Assuntos
Genótipo , Macrófagos Alveolares/virologia , Fenótipo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Linhagem Celular , Imunogenicidade da Vacina , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Inoculações Seriadas/veterinária , Suínos , VirulênciaRESUMO
OBJECTIVES: In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, altered reactivity of blood vessels to vasoactive agonists is frequently associated with an elevation in blood pressure. Canonical transient receptor potential (TRPC) channels are believed to encode receptor-operated cation channels (ROC), the activation of which is involved in smooth muscle depolarization and vasoconstriction. The aims of the present study were to investigate whether the ROC current is increased in DOCA-hypertensive rats and determine whether aldosterone directly enhances the expression of TRPC. METHODS: The nystatin-perforated patch-clamp technique was used for the recording of receptor-stimulated ion currents in mesenteric arterial smooth muscle cells, which were enzymatically dispersed from sham-operated and DOCA-salt hypertensive rats. Expressions of TRPCs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analysis. RESULTS: Receptor-stimulated currents activated by 5-hydroxytryptamine (serotonin) and norepinephrine were increased significantly in the mesenteric arterial smooth muscle cells of DOCA-salt hypertensive rats compared to sham-operated rats. Ion-substitution experiments revealed that the enhanced currents were cation currents (ROC currents). Enhanced expression of TRPC6 in mesenteric arteries from DOCA-salt hypertensive rats was demonstrated by real-time RT-PCR. Up-regulation of TRPC6 by aldosterone treatment in vitro was also observed in A7r5 cells by RT-PCR and in western blots. CONCLUSION: These results suggest that aldosterone enhances TRPC6 expression and ROC currents in vascular smooth muscle cells, and that this may in turn contribute to altered vascular reactivity and to hypertension.
Assuntos
Artérias/citologia , Canais de Cálcio/biossíntese , Canais de Cálcio/efeitos dos fármacos , Hipertensão/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPC/biossíntese , Canais de Cátion TRPC/efeitos dos fármacos , Aldosterona/farmacologia , Animais , Aorta/citologia , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Desoxicorticosterona , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Artéria Mesentérica Superior/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Norepinefrina/farmacologia , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/farmacologia , Serotoninérgicos/farmacologia , Regulação para Cima/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Porcine epidemic diarrhea virus (PEDV) has emerged or re-emerged worldwide, posing a significant financial threat to major pig-producing countries. In the present study, a virulent Korean pandemic PEDV strain, KNU-141112, was serially propagated in Vero cells for up to 100 passages. Through cell culture adaptation, we obtained four distinct deletion (DEL) mutants by plaque purification followed by nucleotide sequencing of the spike (S)/ORF3 gene-coding region, which were designated KNU-141112-S DEL2, -S DEL5, -S DEL2/ORF3, and -S DEL5/ORF3. Further whole genome sequencing identified 12 or 14 amino acid changes in the cell-adapted DEL strains. Animal inoculation studies revealed that the virulence of both S DEL2/ORF3 and S DEL5/ORF3 viruses with a large 46-nt deletion in the intergenic portion of S and ORF3 was remarkably diminished, indicating viral attenuation in the natural host. Furthermore, these cell-adapted strains elicited potent neutralizing antibody responses in immunized pigs. Taken together, our data indicate that the cell-attenuated S DEL2/ORF3 and S DEL5/ORF3 strains are promising candidates for the development of a safe and effective live PEDV vaccine.
Assuntos
Genótipo , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Cultura de Vírus/veterinária , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Filogenia , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Doenças dos Suínos/virologia , Vacinas Atenuadas , Células Vero , Proteínas da Cauda Viral/genética , Proteínas da Cauda Viral/metabolismo , Virulência , Cultura de Vírus/métodosRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common and world-widespread viral pathogen of swine. We previously reported genomic sequences and pathogenicity of type 2 Korean PRRSV strains belonging to the virulent lineage 1 family, which contain remarkable amino acid deletions in nonstructural protein 2 (nsp2 DEL) compared to VR-2332. Here, a virulent type 2 Korean PRRSV nsp2 DEL strain, CA-2, was serially propagated in MARC-145 cells for up to 100 passages (CA-2-P100). As the passage number increased, the phenotypic characteristics of cell-adapted CA-2 strains were altered, in terms of higher viral titers and larger plaque sizes compared to the parental virus. Pro-inflammatory cytokine genes, including TNF-α, IL-8, MCP-1, and MCP-2, were found to be significantly down-regulated in PAM cells with the CA-2-P100 strain compared to its parental nsp2 DEL virus. Animal inoculation studies demonstrated that the virulence of CA-2-P100 was reduced significantly, with showing normal weight gain, body temperatures, and lung lesions comparable to the control group. Furthermore, high-passage CA-2-P100 showed declined and transient viremia kinetics, as well as delayed and low PRRSV-specific antibody responses in infected pigs. In addition, we determined whole genome sequences of low to high-passage derivatives of CA-2. The nsp2 DEL pattern was conserved for 100 passages, whereas no other deletions or insertions arose during the cell adaptation process. However, CA-2-P100 possessed 54 random nucleotide substitutions that resulted in 27 amino acid changes distributed throughout the genome, suggesting that these genetic drifts provide a possible molecular basis correlated with the cell-adapted features in vitro and the attenuated phenotype in vivo. Taken together, our data indicate that the cell-attenuated CA-2-P100 strain is a promising candidate for developing a safe and effective live PRRSV vaccine.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/patologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Virulência/genética , Substituição de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Regulação para Baixo , Genoma Viral/genética , Interleucina-8/genética , Pulmão/patologia , Mutação , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Distribuição Aleatória , Suínos , Fator de Necrose Tumoral alfa/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Euglycemia is the ultimate goal in diabetes care to prevent complications. However, the benefits of euglycemia in type 2 diabetes are controversial because near-euglycemic subjects show higher mortality than moderately hyperglycemic subjects. We previously reported that euglycemic-diabetic rats on calorie-control lose a critical liver weight (LW) compared with hyperglycemic rats. Here, we elucidated the molecular mechanisms underlying the loss of LW in euglycemic-diabetic rats and identified a potential risk in achieving euglycemia by calorie-control. Sprague-Dawley diabetic rats generated by subtotal-pancreatectomy were fed a calorie-controlled diet for 7 weeks to achieve euglycemia using 19 kcal% (19R) or 6 kcal% (6R) protein-containing chow or fed ad libitum (19AL). The diet in both R groups was isocaloric/kg body weight to the sham-operated group (19S). Compared with 19S and hyperglycemic 19AL, both euglycemic R groups showed lower LWs, increased autophagy, and increased AMPK and caspase-3 and decreased mTOR activities. Though degree of insulin deficiency was similar among the diabetic rats, Akt activity was lower, and PTEN activity was higher in both R groups than in 19AL whose signaling patterns were similar to 19S. In conclusion, euglycemia achieved by calorie-control is deleterious in insulin deficiency due to increased autophagy and apoptosis in the liver via AMPK and caspase-3 activation.
Assuntos
Adenilato Quinase/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Glicemia/metabolismo , Caspase 3/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fígado/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Fígado/patologia , Masculino , Tamanho do Órgão , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Anthracyclines are among the most effective and commonly used chemotherapeutic agents. However, the development of acquired anthracycline resistance is a major limitation to their clinical application. The aim of the present study was to identify differentially expressed genes (DEGs) and biological processes associated with the acquisition of anthracycline resistance in human breast cancer cells. We performed a meta-analysis of publically available microarray datasets containing data on stepwise-selected, anthracyclineresistant breast cancer cell lines using the RankProd package in R. Additionally, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze GO term enrichment and pathways, respectively. A protein-protein interaction (PPI) network was also generated using Cytoscape software. The meta-analysis yielded 413 DEGs related to anthracycline resistance in human breast cancer cells, and 374 of these were not involved in individual DEGs. GO analyses showed the 413 genes were enriched with terms such as 'response to steroid metabolic process', 'chemical stimulus', 'external stimulus', 'hormone stimulus', 'multicellular organismal process', and 'system development'. Pathway analysis revealed significant pathways including steroid hormone biosynthesis, cytokine-cytokine receptor interaction, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, and arachidonic acid metabolism. The PPI network indicated that proteins encoded by TRIM29, VTN, CCNA1, and karyopherin α 5 (KPNA5) participated in a significant number of interactions. In conclusion, our meta-analysis provides a comprehensive view of gene expression patterns associated with acquired resistance to anthracycline in breast cancer cells, and constitutes the basis for additional functional studies.
Assuntos
Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Neoplasias da Mama/tratamento farmacológico , Bases de Dados Genéticas , Conjuntos de Dados como Assunto/estatística & dados numéricos , Feminino , Ontologia Genética , Humanos , Redes e Vias Metabólicas/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Mapas de Interação de Proteínas , Análise Serial de TecidosRESUMO
In molecular-targeted cancer therapy, acquired resistance to gemcitabine is a major clinical problem that reduces its effectiveness, resulting in recurrence and metastasis of cancers. In spite of great efforts to reveal the overall mechanism of acquired gemcitabine resistance, no definitive genetic factors have been identified that are absolutely responsible for the resistance process. Therefore, we performed a cross-platform meta-analysis of three publically available microarray datasets for cancer cell lines with acquired gemcitabine resistance, using the R-based RankProd algorithm, and were able to identify a total of 158 differentially expressed genes (DEGs; 76 up- and 82 down-regulated) that are potentially involved in acquired resistance to gemcitabine. Indeed, the top 20 up- and down-regulated DEGs are largely associated with a common process of carcinogenesis in many cells. For the top 50 up- and down-regulated DEGs, we conducted integrated analyses of a gene regulatory network, a gene co-expression network, and a protein-protein interaction network. The identified DEGs were functionally enriched via Gene Ontology hierarchy and Kyoto Encyclopedia of Genes and Genomes pathway analyses. By systemic combinational analysis of the three molecular networks, we could condense the total number of DEGs to final seven genes. Notably, GJA1, LEF1, and CCND2 were contained within the lists of the top 20 up- or down-regulated DEGs. Our study represents a comprehensive overview of the gene expression patterns associated with acquired gemcitabine resistance and theoretical support for further clinical therapeutic studies.
Assuntos
Biomarcadores Tumorais/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Mapas de Interação de Proteínas , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/farmacologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , GencitabinaRESUMO
BACKGROUND/AIM: Despite great effort to elucidate the process of acquired gefitinib resistance (AGR) in order to develop successful chemotherapy, the precise mechanisms and genetic factors of such resistance have yet to be elucidated. MATERIALS AND METHODS: We performed a cross-platform meta-analysis of three publically available microarray datasets related to cancer with AGR. For the top 100 differentially expressed genes (DEGs), we clustered functional modules of hub genes in a gene co-expression network and a protein-protein interaction network. We conducted a weighted correlation network analysis of total DEGs in microarray dataset GSE 34228. The identified DEGs were functionally enriched by Gene Ontology (GO) function and KEGG pathway. RESULTS: We identified a total of 1,033 DEGs (510 up-regulated, 523 down-regulated, and 109 novel genes). Among the top 100 up- or down-regulated DEGs, many genes were found in different types of cancers and tumors. Through integrative analysis of two systemic networks, we selected six hub DEGs (Pre-B-cell leukemia homeobox1, Transient receptor potential cation channel subfamily C member 1, AXL receptor tyrosine kinase, S100 calcium binding protein A9, S100 calcium binding protein A8, and Nucleotide-binding oligomerization domain containing 2) associated with calcium homeostasis and signaling, apoptosis, transcriptional regulation, or chemoresistance. We confirmed a correlation of expression of these genes in the microarray dataset. CONCLUSION: Our study may lead to comprehensive insights into the complex mechanism of AGR and to novel gene expression signatures useful for further clinical studies.