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1.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
2.
Aging (Albany NY) ; 11(20): 8745-8759, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31631065

RESUMO

Diabetic nephropathy (DN) is one of the leading causes of end-stage renal diseases worldwide. This study is designed to investigate the underlying function and mechanism of a novel lncRNA GAS5 in the progression of DN. We found that lncRNA GAS5 expression level was decreased in type 2 diabetes (T2D) with DN compared with that in patients without DN. Moreover, lncRNA GAS5 expression level was negatively associated with the severity of DN-related complications. lncRNA GAS5 inhibited MCs proliferation and caused G0/1 phase arrest. lncRNA GAS5 overexpression alleviated the expression of fibrosis-related protein in mesangial cells (MCs). The dual-luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay results revealed that lncRNA GAS5 functions as an endogenous sponge for miR-221 via both the directly targeting way and Ago2-dependent manner. Furthermore, SIRT1 was confirmed as a target gene of miR-221. lncRNA GAS5 upregulated SIRT1 expression and inhibited MCs proliferation and fibrosis by acting as an miR-221 sponge. Finally, we found that lncRNA GSA5 suppressed the development of DN in vivo. Thus, lncRNA GAS5 was involved in the progression of DN by sponging miR-221 and contributed to lncRNA-directed diagnostics and therapeutics in DN.

3.
ACS Chem Biol ; 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31613083

RESUMO

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in Saccharomyces cerevisiae, a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1' positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1' position, respectively. Comparably, it exhibited efficient recognition of most residues at the P2' position, except Trp. Further biochemical characterization through site mutagenesis, enzyme structural modeling, and comparison with other 3C proteases indicated that the S1 pocket of HRV 3C-P was constituted by neutral and basic amino acids, in which His160 and Thr141 specifically interacted with Gln or Glu residues at the substrate P1 position. Additionally, the stringent S1' pocket determined its unique property of only accommodating residues without or with short side chains. Based on our characterization, LEVLFQ↓GM was identified as a more favorable substrate than the original LEVLFQ↓GP at high temperature, which might be caused by the conversion of random coils to ß-turns in HRV 3C-P along with the temperature increase. Our studies prompted a further understanding of the substrate specificity and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined with the enzyme modeling strategy in this study provides a general strategy for deciphering the substrate specificities of proteases.

4.
Nucleic Acids Res ; 47(21): 11461-11475, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31647102

RESUMO

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.

5.
J Cell Physiol ; 234(10): 18492-18503, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30927260

RESUMO

Diabetic nephropathy (DN) is an important factor leading to end-stage kidney disease that affects diabetes mellitus patients globally. Our previous transcriptome sequencing has identified a large group of differentially expressed long noncoding RNA (lncRNA) in early development of DN. On basis of this, we aimed to investigate the function of lncRNA NONHSAG053901 in DN pathogenesis. In this study, we revealed that the expression of NONHSAG053901 was drastically elevated in both DN mouse model and mesangial cells (MCs). It was found that overexpression of NONHSAG053901 remarkably promoted inflammation, fibrosis and proliferation in MCs. Consistently, further investigations suggested that the stimulation of NONHSAG053901 on proinflammatory cytokines via direct binding to early growth response protein 1 (Egr-1). Interaction between Egr-1 and transforming growth factor ß (TGF-ß) could augment TGF-ß function in DN inflammation. Furthermore, the effects of NONHSAG053901 on stimulation of proinflammatory cytokines were abolished by knockdown of Egr-1. These results together suggested that NONHSAG053901 promoted proinflammatory cytokines via stimulating Egr-1/TGF-ß mediated renal inflammation.

6.
J Cell Physiol ; 234(7): 10640-10645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30536533

RESUMO

Asymmetric dimethylarginine (ADMA) plays a vital role in the regulation of insulin sensitivity and has been shown as a potential marker for various disease, including type 2 diabetes mellitus (DM2). However, the correlation between ADMA and impaired glucose tolerance (IGT) and obesity has not been studied. A total of 195 subjects were involved in our study. The characteristics of the subjects in the study cohort were measured and analyzed. We found that the serum ADMA and C-reactive protein levels were significantly increased in IGT and diabetic patients, whereas the levels of lipoprotein A and adiponectin were decreased, especially in diabetic patients with obesity. The serum ADMA level was positively correlated to a homeostatic model assessment for insulin resistance, and multivariate regression analysis further indicated that ADMA was an independent factor for DM patients with obesity. Our study expands the understanding of the complicated relationship between obesity, insulin resistance, IGT, and ADMA. In addition, we demonstrated that the serum ADMA level could serve as a diagnositic biomarker of the early signs for IGT patients with obesity.

7.
J Cell Physiol ; 2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30515796

RESUMO

Accumulating evidence has indicated the significant roles of long noncoding RNAs (lncRNAs) in the pathophysiology of diabetic nephropathy (DN). LncRNA nuclear enriched abundant transcript 1 (NEAT1) has been reported to exert a key role in the progression of several diseases including diabetes. However, the role of NEAT1 in the regulation of DP progression remains barely known. Therefore, our study aimed to investigate the role of NEAT1 in a streptozotocin-induced diabetes model (DM) of rats and glucose-induced mouse mesangial cell models. Currently, we found that NEAT1 was greatly upregulated in DM rats and glucose-induced mice mesangial cells, in which a high activation of Akt/mTOR signaling was also observed. Then, it was shown that knockdown of NETA1 was able to reduce renal injury in DM rats obviously. In addition, cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay were carried out and we observed downregulation of NEAT1 significantly inhibited mesangial cell proliferation. Meanwhile, extracellular matrix proteins and messenger RNA (transforming growth factor ß1, fibronectin, and collagen IV) expression was dramatically restrained by silencing of NEAT1 in the high glucose-induced mesangial cells. Finally, knockdown of NEAT1 greatly reduced the expression of the phosphorylation of Akt and mammalian target of rapamycin (mTOR) in vitro. These findings revealed that the decrease of NEAT1 repressed the proliferation and fibrosis in DN via activating the Akt/mTOR signaling pathway, which might represent a novel pathological mechanism of DN progression.

8.
J Cell Biochem ; 2018 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-30506883

RESUMO

Insulin resistance is associated with impaired glucose uptake and altered protein kinase B (Akt) signaling. Previous studies have suggested asymmetric dimethylarginine (ADMA) and inflammation are two distinguish factors that correlate with insulin resistance (IR). How ADMA and inflammation factors interact and synchronize in the regulation of IR in liver remain to be elucidated. In this study, we systematically investigated whether ADMA is involved in IR using primary hepatocytes, if yes, by via which molecular mechanism. Our results demonstrated that ADMA inhibits insulin sensitivity in a concentration-dependent manner by activating inflammation factors tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 in primary hepatocytes. Further analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway act downstream of ADMA and inflammation factors, and inhibition of MAPK pathway rescued the IR. Furthermore, metformin effects has been found which could reverse ADMA-induced IR by suppressing MAPK signaling pathway. To our knowledge, we, for the first time, unveiled the complicated regulatory network and interactions among ADMA, inflammation, and MAPK signaling pathway, which advanced current research on the development and regulation of IR in liver. This study also certainly provided novel insights on comprehensive diagonistics roles of ADMA as a potential biomarker.

9.
J Exp Clin Cancer Res ; 37(1): 166, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30041673

RESUMO

BACKGROUND: The KRAS mutation is the driving force of pancreatic ductal adenocarcinoma (PDAC). Downstream effectors of KRAS signal pathways are crucial to the development of PDAC. The purpose of this study was to investigate the relationship between KRAS mutation and transgelin-2. Transgelin-2 is highly expressed in PDAC tissues compared with adjacent normal tissues. The underlying mechanism for upregulating transgelin-2 is largely unknown. METHODS: Expression of transgelin-2 was analyzed by microarray data and qRT-PCR. The effect of KRAS signaling on transgelin-2 expression was examined in PDAC cells in the presence or absence of the ERK inhibitor. The interaction of transgelin-2 with ERK was confirmed by immunoprecipitation. ERK-mediated Phosphorylation of transglein-2 was detected by in vivo and in vitro kinase assays. The gain-of-function and loss-of-function approaches were used to examine the role of phosphorylation of transgelin-2 on cell proliferation. Phosphorylation of transgelin-2 was detected by immunohistochemistry in PDAC tissues. RESULTS: Here we found transgelin-2 expression was induced by KRAS mutation. In the case of KRAS mutation, ERK2 interacted with 29-31 amino acids of transgelin-2 and subsequently phosphorylated the S145 residue of transgelin-2. S145 phosphorylation of transgelin-2 played important roles in cell proliferation and tumorigenesis of PDAC. In addition, S145 phosphorylation of transgelin-2 was associated with a poor prognosis in patients with PDAC. CONCLUSIONS: This study indicated that KRAS-ERK-mediated transeglin-2 phosphorylation played an important role in the development of PDAC. Inhibition of transgelin-2 phosphorylation may be a potential therapeutic strategy for targeting PDAC with KRAS mutation.

10.
J Cell Physiol ; 233(11): 8551-8557, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29319171

RESUMO

Although diabetes mellitus (DM) is reported as an independent risk factor for colorectal cancer (CRC) in many researches, the underlying pathophysiology is still unclear. We investigated the differentially expressed genes (DEGs) for the diabetes and CRC to reveal the underlying pathophysiological association between the type 2-diabetic (T2D) and CRC. Gene expression profiles for T2D (GSE55650), CRC (GSE8671), and Metformin treated cell lines (GSE67342) were downloaded from GEO database. The DEGs between T2D samples and their control samples were identified with t-test and variance analysis. After cluster analysis and functional enrichment analysis, protein-protein interaction (PPI) network was constructed to find potential genes for diabetes and CRC in Metformin's treatment. Totally, we identified 583 overlapped genes, 169 common DEGs, and 414 independent DEGs between T2D and CRC samples. The common genes contained 89 up-regulated (DEGs1) and 80 down-regulated genes (DEGs3); and independent DEGs contained 270 down-regulated genes (DEGs4) in diabetes and 144 down-regulated genes (DEGs2) in CRC. In enrichment analysis, the Ribosome pathway was significantly enriched by the independent DEGs. The common genes were mainly enriched in some inflammatory related pathways. Two target genes of Metformin were significantly interacted with six hub genes (HADHB, NDUFS3, TAF1, MYC, HNFF4A, and MAX) with significant changes in expression values (P < 0.05, t-test). To summary, it is suggested that the six hub genes might play important roles in the process of Metformin treatment for diabetes and CRC. However, specific pathology remains to be further studied.

11.
J Biol Chem ; 292(50): 20707-20719, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29038295

RESUMO

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.


Assuntos
Aminoácidos Aromáticos/química , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Retículo Endoplasmático/enzimologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Técnicas do Sistema de Duplo-Híbrido
12.
Nucleic Acids Res ; 45(15): 8978-8992, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911114

RESUMO

CRISPR-Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR-Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements.


Assuntos
Proteínas Arqueais/genética , Sistemas CRISPR-Cas , Reparo do DNA , DNA Arqueal/genética , Regulação da Expressão Gênica em Archaea , Sulfolobus/genética , Ativação Transcricional , Proteínas Arqueais/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/genética , DNA Helicases/imunologia , DNA Polimerase II/genética , DNA Polimerase II/imunologia , DNA Arqueal/imunologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/imunologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Sulfolobus/imunologia
13.
Microbiol Res ; 196: 118-128, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28164786

RESUMO

Yeast surface display (YSD) system has been widely used in protein engineering since it was established 20 years ago. Combined with fluorescence-activated cell sorting (FACS) technology and directed evolution, YSD has been proven of its extraordinary effectiveness for molecular engineering of various target proteins, especially for antibodies. Recently, a few remarkable efforts were exploited to modify the original Aga1-Aga2 YSD for the non-antibody protein engineering with successful outcomes, expanding its application on oxidase, Class II major histocompatibility complex (MHC-II), protease, sortase, lipoic acid ligase etc. Here, the methodologies of these optimized Aga1-Aga2 YSD technologies were introduced, and the recent progress of non-antibody protein engineering using these methods was summarized.


Assuntos
Engenharia de Proteínas/métodos , Leveduras/genética , Leveduras/metabolismo , Anticorpos Monoclonais/biossíntese , Citometria de Fluxo/métodos , Leveduras/enzimologia
14.
Nucleic Acids Res ; 45(4): 1983-1993, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-27986854

RESUMO

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Complexos Multiproteicos/metabolismo , Plasmídeos/genética , Ligação Proteica , RNA Arqueal/química , RNA Arqueal/genética , RNA Arqueal/metabolismo , Ribonucleoproteínas/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo
15.
Nucleic Acids Res ; 45(4): 1902-1913, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-27980065

RESUMO

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Regulação da Expressão Gênica em Archaea , Sulfolobus/genética , Sulfolobus/virologia , Transcrição Genética , Archaea/genética , Proteínas Arqueais/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Evolução Molecular , Técnicas de Inativação de Genes , Filogenia , Sulfolobus/metabolismo
16.
Ann Endocrinol (Paris) ; 77(6): 633-640, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27641081

RESUMO

PURPOSE: To investigate the effects of asymmetric dimethyl-arginine (ADMA), adiponectin (APN) and apelin in predicting macroangiopathy in impaired glucose regulation (IGR) patients. METHODS: A total of 210 patients undergoing oral glucose tolerance test were included in this study. They were classified to normal glucose tolerance (NGT, n=42), impaired fasting glucose (IFG, n=36), impaired glucose tolerance (IGT, n=92, including 44 IGT1 and 48 IGT2 patients) and IFG+IGT (n=40) groups. APN, apelin and ADMA levels, blood pressure, blood lipid, insulin, body mass index (BMI), and homeostasis model assessment of insulin resistance (HOMA-IR) were detected. The severity and extent of coronary atherosclerosis were determined by the Gensini score. RESULTS: The prevalence of coronary heart disease and Gensini scores in IGT and IFG+IGT groups were similar but both were higher than NGT and IFG groups (all P<0.05). Lower APN, higher ADMA and apelin levels were witnessed in IGT and IGT+IFG groups compared with NGT and IFG groups (all P<0.05). IGT2 group had higher 2-h PG and apelin levels and Gensini scores but lower APN levels than IGT1 group (all P<0.05). Gensini score was positively correlated with apelin (r=0.669) and ADMA (r=0.764), but were negatively correlated with APN (r=-0.555, all P<0.001). ADMA and APN were the independent factors affecting Gensini score. CONCLUSION: ADMA and APN levels could be predictive factors for macroangiopathy in IGR patients, especially in IGT cases.


Assuntos
Adiponectina/sangue , Arginina/análogos & derivados , Doença das Coronárias/sangue , Intolerância à Glucose/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Idoso , Apelina , Arginina/sangue , Aterosclerose/sangue , Aterosclerose/complicações , Glicemia/metabolismo , Doença das Coronárias/complicações , Doença das Coronárias/diagnóstico , Feminino , Intolerância à Glucose/complicações , Intolerância à Glucose/diagnóstico , Teste de Tolerância a Glucose , Humanos , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/sangue , Estado Pré-Diabético/complicações , Estado Pré-Diabético/diagnóstico , Prognóstico
17.
Aging Ment Health ; 20(6): 647-54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25880710

RESUMO

OBJECTIVES: To investigate changes of cognitive performances in female elderly patients with osteoporosis and to determine whether any impairments can be attributed to dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. METHODS: This cross-sectional study included 277 postmenopausal women, who were divided into an osteoporosis patients group (n = 170) and an age, gender and educational history matching control group (n = 107). All the subjects completed a set of neuropsychological tests for the elderly for cognitive assessment, which included measures of executive function, episodic memory, attention and processing speed, semantic memory, and visuospatial construction. Blood biomarkers for osteoporosis, as well as diurnal rhythms of cortisol levels were used as cognitive performance correlation parameters in linear multivariate regression analyses. RESULTS: Individuals with osteoporosis had poorer cognitive scores (P < 0.001). When dividing the osteoporosis patients according to their Mini-Mental State Examination scores into mild cognitive impairment (MCI) and normal cognitive (NC) performance groups, Auditory Verbal Learning trial 1-5 scores were lower (P = 0.006) and Trail Making Test-A scores were higher (P = 0.05) in the MCI compared to the NC group. Further comparison of the MCI and NC groups revealed that declarative memory was inversely associated with cortisol levels (P < 0.001), but this association became marginal when 25-hydroxy vitamin D was included in the linear multivariate regression analyses (P = 0.06). CONCLUSIONS: Patients with osteoporosis are prone to cognitive impairments especially declarative memory deficits. The cognitive impairment may be the result of HPA axis dysregulation but 25-hydroxy vitamin D serum concentrations might be compensatory or even a potent contributing factor.


Assuntos
Calcifediol/sangue , Disfunção Cognitiva/fisiopatologia , Hidrocortisona/sangue , Sistema Hipotálamo-Hipofisário/fisiopatologia , Transtornos da Memória/fisiopatologia , Osteoporose Pós-Menopausa/fisiopatologia , Sistema Hipófise-Suprarrenal/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , China/epidemiologia , Disfunção Cognitiva/epidemiologia , Comorbidade , Estudos Transversais , Feminino , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Transtornos da Memória/epidemiologia , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/epidemiologia , Sistema Hipófise-Suprarrenal/metabolismo
18.
Life (Basel) ; 5(1): 783-817, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25764276

RESUMO

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed.

19.
Nucleic Acids Res ; 43(1): 406-17, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25505143

RESUMO

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-ß) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3'-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-α or Cmr-ß system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-ß complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-α complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-ß exhibited much stronger RNA cleavage activity than Cmr-α. Since we previously showed that S. islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA.


Assuntos
Sistemas CRISPR-Cas , Interferência de RNA , RNA Arqueal/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Arqueal/química , Motivos de Nucleotídeos , Plasmídeos/genética , Clivagem do RNA , RNA Arqueal/química , Sulfolobus/genética
20.
J Diabetes Res ; 2014: 717219, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25054160

RESUMO

PURPOSE: The aim was to explore the effect of the chromium picolinate (CrPic) administration on the pancreas and macroangiopathy of type II diabetes mellitus rats. METHODS: The type II diabetes mellitus (T2DM) rat model was induced by low-dose streptozotocin (STZ). The rats were randomly divided into 5 groups (ten rats in each group). After supplementing CrPic for 15 weeks, the histopathological examination was performed by hematoxylin-eosin (HE) staining. Serum insulin and NO level were determined by radioimmunoassay and colorimetry, respectively. Serum glycosylated hemoglobin (HbA1C), adiponectin (APN), advanced glycation end products (AGES), and apelin were measured by ELISA. Real-time reverse transcription polymerase chain reaction (RT-PCR) was applied for detecting the mRNA expression of APN and apelin. RESULTS: After CrPic treatment, compared with the T2DM control group (group 2), pancreas sections stained with HE showed the completed pancreatic cells structure and no inflammatory infiltration in groups 4 and 5. In addition, the levels of serum NO and insulin were significantly increased and the serum levels of HbA1C, AGES, APN, and apelin were significantly decreased in groups 4 and 5 compared with group 2. The mRNA expression of APN and apelin in groups 4 and 5 was also recovered to the normal level. CONCLUSION: CrPic can recover the function of Β-cells and alleviate macroangiopathy in STZ-induced T2DM rats.


Assuntos
Transtornos Cerebrovasculares/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Pâncreas/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Adiponectina/sangue , Animais , Apelina , Transtornos Cerebrovasculares/complicações , Colorimetria , Primers do DNA/química , Complicações do Diabetes/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Hemoglobina A Glicada/metabolismo , Produtos Finais de Glicação Avançada/sangue , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Quelantes de Ferro/farmacologia , Masculino , Óxido Nítrico/sangue , Radioimunoensaio , Ratos , Ratos Wistar
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