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2.
Isr Med Assoc J ; 21(7): 504, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31507133

RESUMO

BACKGROUND: Klotho is a transmembrane protein that can be shed and can act as a circulating hormone in three forms: soluble klotho (KL1 + KL2), KL1, and KL2. Klotho was discovered as a gene implicated in aging through inhibition of the IGF-I pathway. Our laboratory discovered the role of klotho as a tumor suppressor in breast cancer and other malignancies. Furthermore, we showed that the KL1 domain mediates this activity. Altered cancer cell metabolism is a hallmark of cancer and our lab demonstrated various effects of klotho on breast cancer cell metabolism. Thus, klotho inhibited glycolysis and activated adenosine monophosphate activating kinase (AMPK), an energy sensor pathway. Moreover, inhibition of AMPK reduced the tumor suppressor activity of klotho. OBJECTIVES: To assess the effect of KL1 on breast tumor cells metabolism, as KL1 possesses the tumor suppressor activity of klotho. METHODS: We used MCF-7 breast cancer cells treated with soluble or over-expressed KL1 and klotho. Glycolysis was assessed by measuring mRNA levels of key glycolytic enzymes using reverse transcription polymerase chain reaction and by measuring lactate and glucose levels in media. The AMPK pathway was studied by monitoring AMPK phosphorylation as well as its down-stream target, acetyl-CoA carboxylase, using western blotting. Wound healing assay was used to assess cell migration. RESULTS: KL1 treatment reduced glycolytic enzymes mRNA levels and the activity of hexokinase, similar to klotho treatment. Furthermore, KL1 reduced glucose uptake and decreased lactate production. KL1 elevated phosphorylated acetyl-CoA carboxylase and phosphorylated AMPK levels. Inhibition AMPK (using a mutant AMPK activator) stopped KL1 from inhibiting cell migration, suggesting AMPK underlies klotho's tumor suppressor activity. CONCLUSIONS: Our data indicate KL1 as a regulator of metabolic activity in breast cancer and suggest that metabolic alterations underlie KL1 tumor suppressor activities. Furthermore, as KL1 and klotho share a similar effect on cell metabolism, our results further support the central role KL1 domain plays in klotho's tumor suppressor activity.


Assuntos
Neoplasias da Mama/metabolismo , Glucuronidase/metabolismo , Glicólise/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Movimento Celular/fisiologia , Feminino , Humanos , Células MCF-7 , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Acta Virol ; 63(3): 286-291, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507194

RESUMO

Schmallenberg virus (SBV), a neurotropic member of the genus Orthobunyavirus, infects ruminants and causes neurological lesions and fetal malformations including cerebellar hypoplasia, hydranencephaly, and porencephaly. The aim of this study is to establish intracerebral (i.c.) infection of SBV in newborn BALB/c mice and to investigate some of the transcription factors in brain. For this aim, brain samples of newborn BALB/c mice which were infected with SBV i.c. were analyzed by plaque titration and real-time RT-PCR for T-bet, Gata3, RoRγt, Foxp3 and Eomes mRNA levels. Study results showed that SBV can replicate in BALB/c mice brain and cause death of newborn mice with generation of infectious viral particles. Analyses of transcription factor mRNA levels indicated up-regulation of T-bet, Gata3, RoRγt, Foxp3 and down-regulation of Eomes. In this report, we introduce preliminary data of T cell transcription factors affected by SBV infection of BALB/c mice. Keywords: Eomes; Foxp3; Gata3; RoRγt; Schmallenberg virus; T-bet.


Assuntos
Encéfalo , Infecções por Bunyaviridae , Regulação da Expressão Gênica , Orthobunyavirus , Fatores de Transcrição , Animais , Animais Recém-Nascidos , Encéfalo/virologia , Infecções por Bunyaviridae/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ruminantes , Fatores de Transcrição/genética , Replicação Viral
4.
Pestic Biochem Physiol ; 158: 47-53, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378360

RESUMO

Buprofezin is a chitin synthesis inhibitor that is very effective against Homopteran pests, such as the white-backed planthopper (WBPH), S. furcifera (Horvath). In the present study, resistance selection, cross-resistance and mechanisms of buprofezin resistance were investigated in this planthopper species. However, the mechanism associated with resistance to growth regulator insecticides (IGRs) remains largely unknown. A resistant strain (Bup-R) with a resistance level (22-fold) to buprofezin was developed through continuous selection for 47 generations from a laboratory susceptible strain (Bup-S). The results showed that the Bup-R exhibited no cross-resistance to other tested insecticides. Synergism tests showed that piperonyl butoxide (PBO) (SR = 3.9-fold) and diethyl maleate (DEM) (SR = 1.8-fold) had synergistic effects on buprofezin toxicity in the resistant strain (F47). Enzyme activity results revealed an approximate 5.7-fold difference in cytochrome P450 monooxygenase and a 2-fold difference in glutathione S-transferase (GST) between the resistant and susceptible strains, suggesting that the increased activity of these two enzymes is likely the main detoxification mechanism involved in resistance to buprofezin in this species. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) and GST genes by quantitative real-time PCR results indicated that sixteen P450 and one GST gene were significantly overexpressed in the Bup-R strain, among which thirteen P450 genes and one GST gene were >2-fold higher than in the Bup-S strain. The present study increases our knowledge of the buprofezin resistance mechanism in S. furcifera and provides a useful reference for integrated pest management (IPM) strategies.


Assuntos
Hemípteros/efeitos dos fármacos , Inseticidas/farmacologia , Tiadiazinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemípteros/metabolismo , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Maleatos/metabolismo , Butóxido de Piperonila/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
5.
Braz J Med Biol Res ; 52(8): e8341, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365693

RESUMO

MicroRNAs (miRNAs), as post-transcriptional regulators, have been reported to be involved in the initiation and progression of various types of cancer, including gastric cancer (GC). The present study aimed to investigate the role of miR-383-5p in gastric carcinogenesis. Cell viability was analyzed using CCK-8 kit. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was used to evaluate cell apoptosis. The expression levels of miR-383-5p and histone deacetylase 9 (HDAC9) mRNA in GC tissues and cell lines were analyzed using RT-qPCR. The protein expression of HDAC9 was detected by western blotting. We found that HDAC9 was up-regulated and miR-383-5p was down-regulated in GC tissues and cell lines. High HDAC9 expression or low miR-383-5p expression was closely related to poor prognosis and metastasis in GC patients. HDAC9 knockout or miR-383-5p mimics led to growth inhibition and increased apoptosis in AGS and SGC-7901 cells. More importantly, we validated that miR-383-5p as a post-transcriptional regulator inhibited HDAC9 expression and was inversely correlated with HDAC9 expression in GC tissues. miR-383-5p had the opposite effects to HDAC9 in gastric carcinogenesis. miR-383-5p played an important role in gastric carcinogenesis, and it is one of the important mechanisms to regulate oncogenic HDAC9 in GC, which might be helpful in the development of novel therapeutic strategies for the treatment of GC.


Assuntos
Carcinoma/patologia , Histona Desacetilases/metabolismo , MicroRNAs/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias Gástricas/patologia , Apoptose , Carcinogênese/genética , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células/genética , Progressão da Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
6.
Gene ; 716: 144016, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377318

RESUMO

Drug resistance of malaria parasites remains a problem affecting antimalarial treatment and control of the disease. We previously synthesized an antimalarial endoperoxide, N-89, having high antimalarial effects in vitro and in vivo. In this study we seek to understand the resistant mechanism against N-89 by establishing a highly N-89-resistant clone, named NRC10H, of the Plasmodium falciparum FCR-3 strain. We describe gene mutations in the parent FCR-3 strain and the NRC10H clone using whole-genome sequencing and subsequently by expression profiling using quantitative real-time PCR. Seven genes related to drug resistance, proteolysis, glycophosphatidylinositol anchor biosynthesis, and phosphatidylethanolamine biosynthesis exhibited a single amino acid substitution in the NRC10H clone. Among these seven genes, the multidrug resistance protein 2 (mdr2) variant A532S was found only in NRC10H. The genetic status of the P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC), a potential target of N-89, was similar between the NRC10H clone and the parent FCR-3 strain. These findings suggest that the genetic alterations of the identified seven genes, in particular mdr2, in NRC10H could give rise to resistance of the antimalarial endoperoxide N-89.


Assuntos
Antimaláricos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/farmacologia , Resistência a Medicamentos/genética , Genômica , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Sequenciamento Completo do Genoma
7.
Gene ; 716: 144036, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31381952

RESUMO

Nebulin is a 770 kDa protein that is localized along the thin filaments of skeletal muscles in vertebrates. It is also present in the striated muscles of Amphioxus, an invertebrate cephalochordate that is phylogenetically close to vertebrates. However, the nebulin of urochordate ascidians or its expression in invertebrate hearts has not been investigated. In this study, we investigated the structure and cardiac expression of the nebulin gene in Ciona intestinalis, a urochordate whose phylogeny lies between cephalochordates and vertebrates. As a result of the gene structure analysis, we found that the Ciona nebulin gene predicted to be 62 kb and consists of 143 exons. The nebulin was expected to consist of a unique N-terminal region, followed by 155 nebulin repeats, another unique region, a Ser-rich region and a C-terminal SH3 domain. Whole-mount in situ hybridization experiments showed that the Ciona nebulin gene was expressed in a variety of muscles, including hearts. However, Western blot analysis using antibody to Ciona nebulin did not detect the presence of full-length nebulin. Alternatively, RT-PCR experiments on samples of Ciona heart detected the expression of nebulette-like and nrap-like isoforms from the Ciona nebulin gene. These results indicate that, similarly to vertebrate hearts, Ciona hearts do not express nebulin, but rather nrap- and nebulette-like isoforms. These results also imply that the nebulin, nebulette and nrap genes in vertebrates were separated from an ancestral invertebrate nebulin gene during vertebrate evolution.


Assuntos
Ciona intestinalis/genética , Família Multigênica , Proteínas Musculares/genética , Miocárdio/metabolismo , Animais , Ciona intestinalis/metabolismo , Evolução Molecular , Éxons , Íntrons , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Domínios Proteicos , RNA Mensageiro/metabolismo
8.
BMC Plant Biol ; 19(1): 349, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399044

RESUMO

BACKGROUND: AFP is a negative regulator of ABA signaling that promotes ABI5 protein degradation and weakens regulation of ABA signaling by targeting upstream genes of ABI5, and TaABI5 gene was seed-specific, and accumulated during wheat grain maturation and dormancy acquisition, which played an important role in seed dormancy; TaAFP has a conserved domain with AFP, so TaAFP may also play an important role in seed dormancy in wheat. RESULTS: Two allelic variants of TaAFP were identified on chromosome 2BS in common wheat, and designated as TaAFP-B1a and TaAFP-B1b. Sequence analysis showed a 4-bp deletion in the 5'UTR region of TaAFP-B1b compared with TaAFP-B1a. Based on the 4-bp deletion, a co-dominant functional marker of TaAFP-B was developed and designated as AFPB. The genotype generating a 203-bp fragment (TaAFP-B1b) was more resistant to pre-harvest sprouting than the genotype producing a 207-bp fragment (TaAFP-B1a) in a test of 91 white-grained Chinese wheat cultivars and advanced lines. The average germination index(GI) values of TaAFP-B1a and that of TaAFP-B1b were 45.18 and 30.72%, respectively, indicating a significant difference (P < 0.001). Moreover, the 4-bp deletion located in the 5'UTR not only affected the transcription level of TaAFP-B but also affected the mRNA decay, reduced the translation level of GUS and tdTomatoER and GUS activity in wheat leaves of transient expression. The transcript expression and the mRNA half-life value of TaAFP-B1a in developing seeds and mature seeds were much higher than those of TaAFP-B1b. CONCLUSION: We identified a 4-bp InDel in the 5'UTR of TaAFP-B, which affected the mRNA transcription level, mRNA decay, translation levels of GUS and tdTomatoER, GUS activity, and was significantly associated with seed dormancy in common wheat. A functional marker was developed and validated based on this InDel.


Assuntos
Dormência de Plantas/genética , Proteínas de Plantas/genética , Triticum/genética , Regiões 5' não Traduzidas/genética , Ácido Abscísico/metabolismo , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal/genética , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Deleção de Sequência , Transdução de Sinais/genética , Triticum/crescimento & desenvolvimento
9.
Mol Biol (Mosk) ; 53(4): 692-704, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397443

RESUMO

miRNAs regulate the expression of many genes and are involved in the development of diseases. We studied miRNAs that interact partly or fully complementarily with the 5'UTR, CDS and 3'UTR of mRNAs of target genes. The MirTarget program used in this study allows for the discovery of miRNA binding sites (BS) in the entire nucleotide sequence of the mRNA and for determining the characteristics of the interactions of miRNAs with mRNAs. We identified five pairs of fully complementary BS for miR-127-5p and miR-127-3p, miR-136-5p and miR-136-3p, miR-431-5p and miR-431-3p, miR-432-5p and miR-432-3p, and miR-433-5p and miR-433-3p in the CDS of the human and animal mRNA of RTL1 gene. The fully complementary BS for miR-6720-5p, miR-6720-3p were identified in the CDS of the FOXF2 gene; BS for miR-3187-5p, miR-3187-3p were found in the CDS of the PLPPR3 gene; BS for miR-4665-5p, miR-4665-3p were found in the 5'UTR of the KIAA2026 gene; BS for miR-135a-5p, miR-135a-3p were found in the 3'UTR of the GLYCTK gene; BS for miR-7106-5p, miR-7106-3p were found in the 3'UTR of the CCDC42B gene. The miRNA-5p and miRNA-3p associated with the RTL1 gene have BS in the mRNAs of 32 target human genes. The miRNA-5p and miRNA-3p associated with the FOXF2, PLPPR3, KIAA2026, GLYCTK and CCDC42B genes have BS in the mRNAs of 27 target genes, involved in development of several diseases. Nucleotide sequences of miRNA-5p and miRNA-3p and BS are conserved over tens of millions of years of divergence of the studied animal species. Binding characteristics of miR-3120-3p and miR-3120-5p, miR-196b-3p and miR-196b-5p, miR-125a-3p and miR-125a-3p, let-7e-3p and let-7e-5p, miR-99b-3p in fully complementary BS of non-coding DMN3OS, HOXA10-AS, SPACA6P-AS genes have been established.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação/genética , Humanos
10.
Exp Parasitol ; 204: 107732, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31374184

RESUMO

In the present study, the cytotoxic effects of amitraz, an octopamine receptor agonist on the reproductive system of engorged adult females of Rhipicephalus (Boophilus) annulatus were assessed using histology, electron microscopy and octopamine beta (OCTß) receptor transcriptional expression analysis. Adult immersion test (AIT) was performed by immersing the fully engorged female ticks for 2 min in different concentrations of amitraz (200, 250, 300, 350 ppm). Amitraz at the dose of 300 ppm, caused an adult tick mortality of 16.66 ±â€¯6.80 per cent, inhibition of fecundity of 75.80 per cent and hatching of 50 per cent of ova laid by treated ticks. Histological changes in the ovaries of ticks collected after 24 h of treatment with amitraz (300 ppm), in comparison with controls (distilled water/methanol) were identified by microscopical examination of sections (4  µm) stained using haematoxylin and eosin. These changes included reduction in size and basophilia of stage I oocytes, presence of cytoplasmic vacuoles of various sizes around germinal vesicle of stage II oocytes, wavy basement membrane of stage III oocytes and reduction in size and number of mature stage IV and V oocytes. Electron microscopy was employed for understanding the structural changes in the ultrathin sections (60 nm) of ovaries. Ticks treated with amitraz showed major ultrastructural changes such as irregular nuclear membrane, crystolysis of mitochondria and detachment of external and internal layers of basal lamina of oocytes. The cDNA synthesized from the total RNA of whole ticks and ovaries of ticks treated with amitraz along with controls were used for relative quantification of Octopamine ß receptor (OCTß-R) expression based on the 2-ΔΔCT method by quantitative real time PCR (qRT PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Down regulation of expression of OCTß-R mRNA in the ovaries of amitraz treated ticks was observed compared to controls. Thus, the inhibition of fecundity observed in the ticks treated with amitraz can be attributed to the major structural changes and decreased expression of OCT ß receptor mRNA induced by it in the ovary.


Assuntos
Inseticidas/farmacologia , Rhipicephalus/efeitos dos fármacos , Toluidinas/farmacologia , Análise de Variância , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/ultraestrutura , Regulação para Baixo , Feminino , Fertilidade/efeitos dos fármacos , Expressão Gênica , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Ovário/anatomia & histologia , Ovário/efeitos dos fármacos , Ovário/ultraestrutura , Oviposição/efeitos dos fármacos , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Amina Biogênica/agonistas , Receptores de Amina Biogênica/efeitos dos fármacos , Rhipicephalus/anatomia & histologia , Rhipicephalus/genética , Rhipicephalus/ultraestrutura , Espectrofotometria , Controle de Ácaros e Carrapatos/métodos , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura
11.
Medicine (Baltimore) ; 98(26): e15872, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261495

RESUMO

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer accounts for ∼20% of invasive breast cancers and is associated with poor prognostics. The recent outcome of HER2+ breast cancer treatment has been vastly improved owing to the application of antibody-targeted therapies. Trastuzumab (Herceptin) is a monoclonal antibody designed to target HER2+ breast cancer cells. In addition to improved survival in the adjuvant treatment of HER2+ breast cancer, trastuzumab treatment has also been associated with cardiotoxicity side effect. However, the molecular mechanisms of trastuzumab action and trastuzumab-mediated cardiotoxicity are still not fully understood. Previous research utilized bulk transcriptomics analysis to study the underlining mechanisms, which relied on averaging molecular signals from bulk tumor samples and might have overlooked key expression features within breast cancer tumor. In contrast to previous research, we compared the single cancer cell level transcriptome profile between trastuzumab-treated and nontreated patients to reveal a more in-depth transcriptome profile. A total of 461 significantly differential expressed genes were identified, including previously defined and novel gene expression signatures. In addition, we found that trastuzumab-enhanced MGP gene expression could be used as prognostics marker for longer patient survival in breast invasive carcinoma patients, and validated our finding using TCGA (The Cancer Genome Atlas) breast cancer dataset. Moreover, our study revealed a 48-gene expression signature that is associated with cell death of cardiomyocytes, which could be used as early biomarkers for trastuzumab-mediated cardiotoxicity. This work is the first study to look at single cell level transcriptome profile of trastuzumab-treated patients, providing a new understanding of the molecular mechanism(s) of trastuzumab action and trastuzumab-induced cardiotoxicity side effects.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Transcriptoma/efeitos dos fármacos , Trastuzumab/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/epidemiologia , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/epidemiologia , Carcinoma Ductal de Mama/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Projetos Piloto , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Análise de Sobrevida
12.
Anal Bioanal Chem ; 411(21): 5351-5361, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31267193

RESUMO

Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized. Graphical abstract.


Assuntos
Exossomos , Proteômica/métodos , Cromatografia de Afinidade/métodos , Humanos , MicroRNAs/metabolismo , Polietilenoglicóis/química , Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo
13.
BMC Bioinformatics ; 20(1): 379, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286861

RESUMO

BACKGROUND: Unsupervised machine learning methods (deep learning) have shown their usefulness with noisy single cell mRNA-sequencing data (scRNA-seq), where the models generalize well, despite the zero-inflation of the data. A class of neural networks, namely autoencoders, has been useful for denoising of single cell data, imputation of missing values and dimensionality reduction. RESULTS: Here, we present a striking feature with the potential to greatly increase the usability of autoencoders: With specialized training, the autoencoder is not only able to generalize over the data, but also to tease apart biologically meaningful modules, which we found encoded in the representation layer of the network. Our model can, from scRNA-seq data, delineate biological meaningful modules that govern a dataset, as well as give information as to which modules are active in each single cell. Importantly, most of these modules can be explained by known biological functions, as provided by the Hallmark gene sets. CONCLUSIONS: We discover that tailored training of an autoencoder makes it possible to deconvolute biological modules inherent in the data, without any assumptions. By comparisons with gene signatures of canonical pathways we see that the modules are directly interpretable. The scope of this discovery has important implications, as it makes it possible to outline the drivers behind a given effect of a cell. In comparison with other dimensionality reduction methods, or supervised models for classification, our approach has the benefit of both handling well the zero-inflated nature of scRNA-seq, and validating that the model captures relevant information, by establishing a link between input and decoded data. In perspective, our model in combination with clustering methods is able to provide information about which subtype a given single cell belongs to, as well as which biological functions determine that membership.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Neurais (Computação) , RNA Mensageiro/química , Análise de Sequência de RNA/métodos , Aprendizado de Máquina não Supervisionado , Análise por Conglomerados , RNA Mensageiro/metabolismo , Análise de Célula Única
14.
Anticancer Res ; 39(7): 3493-3498, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262873

RESUMO

BACKGROUND/AIM: Pancreatic cancer is the most lethal cancer of the digestive system. IL-29 is a new member of the IFNλ family and well-known for its strong antiviral activity. However, its direct effect on pancreatic cancer is still unclear. This study was performed to investigate if IL-29 has any direct effect on Pan-48 pancreatic cancer cells. MATERIALS AND METHODS: Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the effects of IL-29 on cell survival, proliferation, and apoptosis of Pan-48 pancreatic cancer cells. RT-PCR and IHC were subsequently performed to explore IL-29's potential molecular mechanisms. RESULTS: The percentage of colonies of Pan-48 cells was decreased following the addition of IL-29. This was consistent with a decreased optical density (OD) value of cancer cells. Furthermore, the relative caspase-3 activity in cancer cells was increased after the addition of IL-29, indicating increased apoptosis of cancer cells. The anti-proliferative effect of IL-29 on cancer cells correlated with increased expression of the anti-proliferative molecule p21. The pro-apoptotic effect of IL-29 on cancer cells correlated with an increased expression of the pro-apoptotic molecule Bax. CONCLUSION: IL-29 constrains Pan-48 pancreatic cell growth via up-regulation of p21 and Bax. Our study suggests a potential use of IL-29 in immunotherapy for pancreatic cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Interleucinas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
15.
Gene ; 712: 143945, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279712

RESUMO

Reactive oxygen species, generated in all the aerobic organisms, can cause oxidative stress. Excessive ROS may become a source of carcinogen due to DNA damage, lipid peroxidation, cell injury, and cell death. In order to prevent these adverse effects of ROS, antioxidant enzymes have evolved in aerobic organisms. Catalase is a major antioxidant enzyme that breaks down excessive H2O2 and inhibits apoptotic cell death. Here we molecularly characterized catalase from red-lip mullet. The cDNA sequence of LhCAT consists of an ORF of 1545 bp, which encodes a 527 amino acid peptide (~60 kDa). Based on bioinformatics analysis, LhCAT possesses a domain architecture characteristic of catalases, including a catalase proximal active site signature and a catalase proximal heme-ligand signature. It also has heme and NADPH binding sites homologous to previously described catalases. Pairwise alignment with its homologs revealed that LhCAT shares 95.1% identity with Oplegnathus fasciatus catalase and 97.4% similarity with Sparus aurata catalase. An uprooted phylogenetic tree demonstrated that LhCAT resides in a clade with catalases from other teleosts and exhibits a close relationship with Oplegnathus fasciatus catalase. Among twelve tissue types, we observed the highest LhCAT mRNA expression in the liver, followed by blood. Immune challenge by Lactococcus garvieae, or Poly I:C in the blood or spleen resulted in up-regulation at 24 h post injection. We also tested the antioxidant activity of recombinant LhCAT against hydrogen peroxide and found its optimal concentration to be 12.5 µg/mL. Collectively, these data suggested that LhCAT play an important role in antioxidant defense and immune response of red-lip mullet.


Assuntos
Catalase/metabolismo , Proteínas de Peixes/metabolismo , Smegmamorpha , Adjuvantes Imunológicos , Animais , Antioxidantes/metabolismo , Catalase/genética , DNA Complementar/genética , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Heme/química , Peróxido de Hidrogênio/química , Sistema Imunitário , Ligantes , Fígado/enzimologia , Estresse Oxidativo , Filogenia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima
16.
Medicine (Baltimore) ; 98(28): e16384, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31305441

RESUMO

Pyrroline-5-carboxylate reductase 1 (PYCR1) is an enzyme involved in cell metabolism and is upregulated in cancer. However, the correlations of PYCR1 expression with the clinicopathological features and prognosis of renal cell carcinoma (RCC) remain unclear. The purpose of this study was to identify the expression of PYCR1 and its clinical relevance in RCC patients.PYCR1 mRNA expression differences between RCC and the adjacent normal renal tissues were assessed using the Cancer Genome Atlas database (TCGA). Subsequently, the expression of PYCR1 mRNA and protein were evaluated by quantitative real-time polymerase chain reaction, Western blot, and immunochemistry using 30 paired frozen samples of RCC and the adjacent normal renal tissues. The protein expression of PYCR1 was evaluated by immunostaining formalin-fixed, paraffin-embedded sections of RCC samples from 96 patients who underwent radical nephrectomy, and its relationship with clinical features were analyzed. Nonpaired t tests were used to statistically analyze the differences between the 2 groups. Cox univariable and multivariable analyses of overall survival (OS) among RCC patients were performed.The expression of PYCR1 mRNA was significantly upregulated in RCC tissues compared to adjacent normal renal tissues in the TCGA database (P < .01). The area under the receiver operating characteristic curve value was 0.748. The expression of PYCR1 mRNA and protein was significantly upregulated in RCC compared with that in paired normal renal tissues (P < .01). Higher PYCR1 levels were associated with metastasis (P < .01). Kaplan-Meier survival curves indicated that higher PYCR1 expression was correlated with poorer OS. Therefore, PYCR1 may act as a novel prognostic marker and therapeutic target in the diagnosis and treatment of RCC.


Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Pirrolina Carboxilato Redutases/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/enzimologia , Rim/patologia , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sobrevida
17.
Nat Commun ; 10(1): 2907, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266958

RESUMO

Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.


Assuntos
Anticorpos/análise , Núcleo Celular/genética , Genômica/métodos , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Córtex Pré-Frontal/química , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
J Microbiol Biotechnol ; 29(7): 1155-1164, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31280524

RESUMO

Lichens contain diverse bioactive secondary metabolites with various chemical and biological properties, which have been widely studied. However, details of the inhibitory mechanisms of their secondary metabolites against influenza A virus (IAV) have not been documented. Here, we investigated the antiviral effect of lichen extracts, obtained from South Korea, against IAV in MDCK cells. Of the lichens tested, Nipponoparmelia laevior (LC24) exhibited the most potent inhibitory effect against IAV infection. LC24 extract significantly increased cell viability, and reduced apoptosis in IAV-infected cells. The LC24 extract also markedly reduced (~ 3.2 logfold) IAV mRNA expression after 48 h of infection. To understand the antiviral mechanism of LC24 against IAV, proteomic (UPLC-HDMSE) analysis was performed to compare proteome modulation in IAV-infected (V) vs. mock (M) and LC24+IAV (LCV) vs. V cells. Based on Ingenuity Pathway Analysis (IPA), LC24 inhibited IAV infection by modulating several antiviral-related genes and proteins (HSPA4, HSPA5, HSPA8, ANXA1, ANXA2, HIF-1α, AKT1, MX1, HNRNPH1, HNRNPDL, PDIA3, and VCP) via different signaling pathways, including HIF-1α signaling, unfolded protein response, and interferon signaling. These molecules were identified as the specific biomarkers for controlling IAV in vitro and further confirmation of their potential against IAV in vivo is required. Our findings provide a platform for further studies on the application of lichen extracts against IAV.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Líquens/química , Infecções por Orthomyxoviridae/metabolismo , Animais , Antivirais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cães , Líquens/metabolismo , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia , Proteômica , RNA Mensageiro/metabolismo , República da Coreia , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/genética
19.
Chem Biol Interact ; 310: 108750, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319076

RESUMO

Osteoporosis is a major health concern occurring to the aging adult population across the globe. Currently, there is an increasing demand for treatment of osteoporosis with plant-based medicines. In the present study, we report that heraclenin was extracted and purified from unripe fruit portion of Bael (Aegle marmelos Corr.) using silica gel column chromatography. The identification and characterization of heraclenin were carried out by UV-Vis, HPLC, LC-MS, NMR, FT-IR, and XRD analyses. The standardized purification method recorded a yield efficiency of 42% heraclenin microcrystals with 99% purity from bael fruit. SEM image revealed the shape of the purified compound to be an orthorhombic-sphenoid prism. Cytotoxicity studies indicated that heraclenin-treatment did not alter cell viability in mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The mRNA expression of Runx2, a bone transcription factor was found to be stimulated by heraclenin in these cells. At the cellular level, heraclenin-treatment enhanced osteoblast differentiation and mineralization in mMSCs. Thus, these results suggested that heraclenin purified from bael fruit has an osteogenic effect, indicating its potential towards bone regeneration.


Assuntos
Aegle/química , Furocumarinas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Furocumarinas/isolamento & purificação , Camundongos , Osteoblastos/citologia , RNA Mensageiro/metabolismo , Espectrofotometria
20.
Adv Exp Med Biol ; 1143: 75-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338816

RESUMO

As the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), N 6-methyladenosine (m6A) modification has been shown recently to posttranscriptionally regulate expression of thousands of messenger RNA (mRNA) transcripts in each mammalian cell type in a dynamic and reversible manner. This epigenetic mark is deposited by the m6A methyltransferase complex (i.e., the METTL3/METTL14/WTAP complex and other cofactor proteins) and erased by m6A demethylases such as FTO and ALKBH5. Specific recognition of these m6A-modified mRNAs by m6A-binding proteins (i.e., m6A readers) determines the fate of target mRNAs through affecting splicing, nuclear export, RNA stability, and/or translation. During the past few years, m6A modification has been demonstrated to play a critical role in many major normal bioprocesses including self-renewal and differentiation of embryonic stem cells and hematopoietic stem cells, tissue development, circadian rhythm, heat shock or DNA damage response, and sex determination. Thus, it is not surprising that dysregulation of the m6A machinery is also closely associated with pathogenesis and drug response of both solid tumors and hematologic malignancies. In this chapter, we summarize and discuss recent findings regarding the biological functions and underlying mechanisms of m6A modification and the associated machinery in normal hematopoiesis and the initiation, progression, and drug response of acute myeloid leukemia (AML), a major subtype of leukemia usually associated with unfavorable prognosis.


Assuntos
Adenosina , Hematopoese , Leucemia Mieloide Aguda , Metiltransferases , RNA Mensageiro , Adenosina/metabolismo , Animais , Diferenciação Celular , Resistencia a Medicamentos Antineoplásicos/genética , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo
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