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1.
J Clin Microbiol ; 53(10): 3165-75, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202119

RESUMO

Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to <20%; and minor, 1% to <5%. The rates of MHR variation that were present in the major and intermediate viral population were significantly greater in patients with advanced liver disease than those in chronic patients. The most frequent MHR variants related to immune evasion in the major and intermediate populations were P120Q/T, T123A, P127T, Q129H/R, M133L/T, and G145R. The major population of MHR variants causing impaired of HBsAg secretion (e.g., G119R, Q129R, T140I, and G145R) was detected only in advanced liver disease patients. This is the first study to use ultradeep sequencing for the detection of MHR variants of HBV quasispecies in Indonesian patients. We found that a greater number of MHR variations was related to disease severity and reduced likelihood of HBsAg titer.


Assuntos
Variação Genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Adulto , Idoso , Substituição de Aminoácidos , Feminino , Frequência do Gene , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Humanos , Indonésia , Masculino , Pessoa de Meia-Idade
2.
Intervirology ; 57(6): 384-92, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25382636

RESUMO

OBJECTIVE: The long-term administration of a nucleos(t)ide analogue (NA) for the treatment of chronic hepatitis B may encourage the emergence of viral mutations associated with drug resistance. Minor populations of viruses may exist before treatment, but are difficult to detect because of technological limitations. Identifying minor viral quasispecies should be useful in the clinical management of hepatitis B virus (HBV) infection. METHODS: Six treatment-naïve Indonesian patients with chronic HBV infection participated in this study. The polymerase region of the HBV genome, including regions with known drug-resistant mutations, was subjected to capillary sequencing and MiSeq sequencing (Illumina). Mutations were analyzed with Genomics Workbench software version 6.0.1 (CLC bio). RESULTS: The mean mapping reads for the six samples was 745,654, and the mean number of amplified fragments ranged from 17,926 to 25,336 DNA reads. Several known drug-resistant mutations in the reverse transcriptase region were identified in all patients, although the frequencies were low (0.12-1.06%). The proportions of the total number of reads containing mutations I169L/M, S202R, M204I/L or N236S were >1.0%. CONCLUSION: Several known NA-resistant mutations were detected in treatment-naïve patients in Indonesia using deep sequencing. Careful management of such patients is essential to prevent drug-resistant mutations from spreading to other patients.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , DNA Polimerase Dirigida por RNA/genética , Análise de Sequência de DNA/métodos , Adulto , Idoso , Sequência de Aminoácidos , Genótipo , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Humanos , Indonésia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , DNA Polimerase Dirigida por RNA/química , Adulto Jovem
3.
Sci Adv ; 10(35): eadn7167, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39213345

RESUMO

Dietary protein intake modulates growth, reproduction, and longevity by stimulating amino acid (AA)-sensing pathways. Essential AAs are often considered as limiting nutrients during protein scarcity, and the role of dietary non-essential AAs (NEAAs) is less explored. Although tyrosine has been reported to be crucial for sensing protein restriction in Drosophila larvae, its effect on adult physiology and longevity remains unclear. Here, using a synthetic diet, we perform a systematic investigation of the effect of single NEAA deprivation on nutrient-sensing pathways, reproductive ability, starvation resistance, feeding behavior, and life span in adult female flies. Specifically, dietary tyrosine deprivation decreases internal tyrosine levels and fecundity, influences AA-sensing machineries, and extends life span. These nutritional responses are not observed under higher total AA intake or in infertile female flies, suggesting a context-dependent influence of dietary tyrosine. Our findings highlight the unique role of tyrosine as a potentially limiting nutrient, underscoring its value for dietary interventions aimed at enhancing health span.


Assuntos
Longevidade , Tirosina , Animais , Tirosina/metabolismo , Feminino , Fertilidade , Drosophila melanogaster/fisiologia , Dieta , Comportamento Alimentar/fisiologia , Proteínas Alimentares/metabolismo , Reprodução , Drosophila/fisiologia , Aminoácidos/metabolismo
4.
Kidney Int ; 84(3): 545-54, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23615507

RESUMO

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/prevenção & controle , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Fibrose , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Córtex Renal/metabolismo , Córtex Renal/patologia , Masculino , Camundongos , Camundongos Mutantes , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
5.
Biochem Biophys Res Commun ; 430(3): 999-1004, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23261456

RESUMO

Progalanin is released from the small cell lung carcinoma line SBC-3A and converted to its active form by plasmin. To elucidate the role of progalanin activation in the extracellular compartment, matrix metalloproteinase (MMP) activity was studied in SBC-3A cells treated with progalanin siRNA, and angiogenesis was measured in tumor tissue originating from SBC-3A cell transplantation into mice. Progalanin siRNA caused downregulation of progalanin expression for approximately 8 days. MMP activity and angiogenesis were reduced in tumors induced by transplantation of progalanin siRNA-treated SBC-3A cells. In contrast, MMP-9 and MMP-2 activity and angiogenesis increased in tumors originating from progalanin siRNA-treated SBC-3A cells in the presence of galanin and progalanin. Furthermore, injection of tranexamic acid, a plasmin inhibitor, more markedly reduced MMP-9 and MMP-2 activity and angiogenesis in tumors originating from progalanin siRNA-treated SBC-3A cells and in tumor tissue originating from progalanin siRNA-treated SBC-3A cells in the presence of progalanin. The reduction of MMP-9 and MMP-2 activity with tranexamic acid was restored by galanin, but not by progalanin. Moreover, tranexamic acid reduced angiogenesis in control siRNA-treated SBC-3A cells. These results suggest that the activation of progalanin by plasmin in the extracellular compartment was involved in MMP-9 and MMP-2 activation and in angiogenesis in tumor tissue.


Assuntos
Galanina/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Patológica/metabolismo , Carcinoma de Pequenas Células do Pulmão/irrigação sanguínea , Animais , Linhagem Celular Tumoral , Fibrinolisina/metabolismo , Galanina/genética , Humanos , Camundongos , Transplante de Neoplasias , Neovascularização Patológica/genética , RNA Interferente Pequeno/genética , Ratos
6.
Biochem Biophys Res Commun ; 435(2): 171-5, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23665024

RESUMO

The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Estresse do Retículo Endoplasmático/fisiologia , Células Endoteliais/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Dobramento de Proteína/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Resposta a Proteínas não Dobradas/efeitos dos fármacos
7.
Molecules ; 18(7): 7448-59, 2013 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-23803716

RESUMO

Three novel multiflorane-type triterpenoids, 3α-p-nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate (1), 3α-acetoxymultiflora-7:9(11)-diene-29-benzoate (2), and 3α-acetoxymultiflora-5(6):7:9(11)-triene-29-benzoate (3), along with two known related compounds 4 and 5 were isolated from the seeds of zucchini (Cucurbita pepo L). Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HREIMS. Triterpenoids possessing a nitro group were not isolated previously.


Assuntos
Cucurbita/metabolismo , Neoplasias/tratamento farmacológico , Sementes/química , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cucurbita/citologia , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Leucemia P388 , Melanoma Experimental/tratamento farmacológico , Camundongos , Extratos Vegetais , Sementes/citologia , Triterpenos/química , Triterpenos/isolamento & purificação
8.
Front Chem ; 11: 1141920, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37065821

RESUMO

Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging. Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo. Results: Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level. Discussion: These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue.

9.
Nat Commun ; 14(1): 7832, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38052797

RESUMO

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension.


Assuntos
Drosophila , Longevidade , Animais , Longevidade/fisiologia , Drosophila/metabolismo , Metionina/metabolismo , Aminoácidos/metabolismo , Racemetionina , Metionina Sulfóxido Redutases/genética
10.
Sci Rep ; 13(1): 8146, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37231024

RESUMO

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Musculares , Humanos , Células Cultivadas , Diferenciação Celular/genética , Músculo Esquelético , Doenças Musculares/metabolismo
11.
Neurosci Res ; 163: 34-42, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32014574

RESUMO

Human induced pluripotent stem cell (hiPSC)-derived neural cells provide valuable disease models for pathophysiological analysis and drug discovery for intractable neurodegenerative diseases. However, neural differentiation of hiPSCs requires a complex and long culture procedure, which has been a bottleneck for analysis. We previously demonstrated rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells (hPSCs). Although optimization of the microenvironment for the differentiation of hPSCs has been considered to achieve more efficient differentiation, it has never been investigated in detail. Here, we demonstrated that three microenvironmental modifiers, oxygen (O2) tension, pH, and cell density, critically affect neural differentiation of hiPSCs. Hypoxia is known to be involved in neural development in vivo and to promote neural differentiation of PSCs. However, in this study, it caused significant cell death in aggregation culture of human embryoid bodies (hEBs) and negatively affected neural differentiation. Modulation of pH by optimized carbon dioxide (CO2) tension improved neural differentiation of hiPSCs, but mild acidosis caused by increased CO2 tension suppressed neural differentiation without cell death. Moreover, high-cell density culture resulted in prominent acidosis and cell death under hypoxic conditions, which synergistically suppressed neural differentiation of hiPSCs. These results suggest that optimization of the microenvironment via O2 tension, pH, and cell density enables more efficient neural differentiation of hiPSCs for the analysis of neurological diseases.


Assuntos
Acidose , Células-Tronco Pluripotentes Induzidas , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Oxigênio
12.
Biology (Basel) ; 10(11)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34827105

RESUMO

P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology.

13.
Mol Brain ; 13(1): 18, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32070397

RESUMO

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.


Assuntos
Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Atrofia Muscular Espinal/patologia , Sinapses/patologia , Adulto , Animais , Células Cultivadas , Técnicas de Reprogramação Celular , Fibroblastos , Ontologia Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores , Atrofia Muscular Espinal/genética , Neurogênese , Fatores de Transcrição/fisiologia , Expansão das Repetições de Trinucleotídeos , Adulto Jovem
14.
Mol Med Rep ; 17(4): 5528-5537, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29393485

RESUMO

Hepatitis B virus (HBV) polymerase gene is targeted by nucleos(t)ide analogues (NUC), but it is unclear how HBV quasispecies of whole genome changes during early period of NUC treatment. To understand the unknown region of drug sensitivity and treatment resistance, HBV quasispecies of whole genome during early period of NUC treatment was examined using ultra­deep sequencing. Eleven patients with chronic HBV infection who received NUC treatment were enrolled in the current study. Viral DNA was extracted from serum samples before and early period of NUC treatment. Polymerase chain reaction analysis was subsequently performed on the DNA products. The viral quasispecies of the entire genome was analyzed by ultra­deep sequencing. The regions and positions corresponding to the changes in the quasispecies were investigated before and early period of NUC treatment. The secondary structure changes were predicted by mutations/substitutions detected using Lasergene Protean v14.1 software. The frequency of quasispecies variants increased significantly in the polymerase domain from before to early period of NUC treatment (3.08±1.28 vs. 3.51±1.47%, P<0.008), particularly the reverse transcription (RT) domain (3.76±1.25 vs. 4.52±1.37%, P<0.012). In addition, increased variation detected from HBsAg domain showed statistically significant during NUC treatment (6.81±3.26 vs. 7.81±3.26%, P<0.040). The amino acid (aa) mutations/substitutions were detected and compared from before to early period of treatment. Interestingly, most of them were located in the RT region (RT1 motif: aa21­aa51) and small S region in the early duration of NUC treatment. Furthermore, several mutation patterns, such as cI97L and cP130T showed alterations in the secondary structure and predicted antigenicity of HBV protein. Although the HBV whole genome can be affected by NUC treatment, RT 1 motif region and small S region are more sensitive to the early period of NUC treatment. This study suggested the initial changes of HBV quasispecies might affect the long­term drug sensitivity and resistance to NUC treatment.


Assuntos
Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Quase-Espécies/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , DNA Viral , Feminino , Variação Genética , Genoma Viral , Genótipo , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Mutação , Quase-Espécies/imunologia , Carga Viral , Replicação Viral
15.
Infect Agent Cancer ; 13: 7, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29434654

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC. METHODS: Nineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing. RESULTS: There were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC. CONCLUSIONS: Ultra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC. TRIAL REGISTRATION: Ref: R000034382/UMIN000030113; Retrospectively registered 25 November 2017.

16.
FEBS Open Bio ; 7(11): 1784-1792, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29123986

RESUMO

Small-cell lung carcinoma releases progalanin. The released progalanin is activated via a nonclassical processing pathway, being processed into an active form of galanin (1-20) by plasmin in extracellular components. Plasmin is produced from plasminogen activators. To clarify the regulation of progalanin via plasminogen activation by urokinase and tissue-plasminogen activator (t-PA), we investigated the regulation mechanism for urokinase and t-PA expression and their effect on galanin activation. Additionally, we studied the effect of activated galanin on angiogenesis. To determine the effect of cell density, we measured the expression levels of urokinase and t-PA using real-time PCR and plasminogen/gelatin zymography in a cell culture. The urokinase expression increased under both high cell density and presence of cell membrane fractions. However, urokinase increments induced by conditioned medium were low. These results indicate that expression of plasminogen activators is regulated by cell membrane factors. We used tumor-bearing mice to clarify the expression of plasminogen activators and galanin activation. Real-time PCR showed that urokinase was substantially higher in the central parts of tumors compared to the periphery, and this was confirmed by plasminogen/gelatin zymography. To evaluate the biological effect of plasminogen activators on tumor growth, we used tranexamic acid as a plasminogen inhibitor. Tranexamic acid decreased galanin (1-20) and the hemoglobin content of tumors and suppressed tumor growth. Additionally, galanin had no effect on the hemoglobin content of tumors derived from cells lacking GALR2. These results demonstrate the regulation of urokinase expression in tumors through progalanin activation in extracellular compartments, and confirm that galanin plays a role in angiogenesis.

17.
Gut Pathog ; 9: 21, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28439300

RESUMO

BACKGROUND: Helicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients. RESULTS: We isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p < 0.001 and p < 0.05, respectively). When 5 Vietnamese isolates from GC patients were excluded, CagL-Glu59 still remains significant (p < 0.05), but not Ile234. CagL-Tyr58 was seen in only one isolate. The CagI C-terminal motif was completely conserved across all 40 isolates, and there were no significant AACs in CagI. CONCLUSIONS: Using WGS, we analyzed genetic variants in clinical H. pylori isolates and identified putative novel and candidate variants in uncharacterized CagL and CagI sequences that are related to gastric carcinogenesis. In particular, CagL-Glu59 has the possible association with GC.

18.
Kobe J Med Sci ; 62(1): E1-8, 2016 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-27492206

RESUMO

A nucleos(t)ide analog (NA) is the common antiviral drug available for directly treating hepatitis B virus (HBV) infection. However, its application has led to the emergence of NA-resistant mutations mostly in a conserved region of the reverse transcriptase domain of HBV polymerase. Harboring NA-resistant mutations decreases drug effectiveness and increases the frequency of end-stage liver disease. The invention of next-generation sequencing that can generate thousands of sequences from viral complex mixtures provides opportunities to detect minor changes and early viral evolution under drug stress. The present study used ultra-deep sequencing to evaluate discrepant quasispecies in the reverse transcriptase domain of HBV including NA-resistant hotspots between seven treatment-naïve Indonesian patients infected with HBV and five at the early phase of treatment. The most common sub-genotype was HBV B3 (83.34%). The substitution rate of variants determined among amino acids with a ratio of ≥ 1% changes was higher among the population in conserved regions (23.19% vs. 4.59%, P = 0.001) and in the inter-reverse transcriptase domain (23.95% vs. 2.94%, P = 0.002) in treatment naïve, than in treated patients. Nine hotspots of antiviral resistance were identified in both groups, and the mean frequency of changes in all patients was < 1%. The known rtM204I mutation was the most frequent in both groups. The lower rate of variants in HBV quasispecies in patients undergoing treatment could be associated with virus elimination and the extinction of sensitive species by NA therapy. The present findings imply that HBV quasispecies dynamically change during treatment.


Assuntos
Produtos do Gene pol/genética , Vírus da Hepatite B/enzimologia , Vírus da Hepatite B/genética , DNA Polimerase Dirigida por RNA/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Antivirais/farmacologia , Farmacorresistência Viral/genética , Feminino , Produtos do Gene pol/química , Variação Genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Indonésia , Masculino , Pessoa de Meia-Idade , Domínios Proteicos , DNA Polimerase Dirigida por RNA/química , Análise de Sequência de DNA , Adulto Jovem
19.
Mol Brain ; 8(1): 79, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26626025

RESUMO

BACKGROUND: Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. RESULTS: In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3ß inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis. CONCLUSIONS: Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.


Assuntos
Diferenciação Celular , Neurônios Motores/citologia , Células-Tronco Pluripotentes/citologia , Adulto , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Genes Reporter , Células-Tronco Embrionárias Humanas/citologia , Humanos , Lentivirus/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Junção Neuromuscular/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Adulto Jovem
20.
PLoS One ; 9(9): e106314, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25215888

RESUMO

MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10(-4), and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.


Assuntos
Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Hepáticas/genética , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Idoso , Sequência de Bases , Carcinoma Hepatocelular/diagnóstico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
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