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1.
Anal Chem ; 96(22): 9104-9112, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38775358

RESUMO

We developed an optofluidic surface-enhanced Raman scattering chip capable of online fabrication, online molecular detection, and online self-cleaning. In this chip, we harnessed UV light to successfully reduce an AgNO3 solution, resulting in the formation of Ag nanoparticles on carbon fiber cloth coated with titanium dioxide (TiO2). This innovative approach enabled the online fabrication of AgNPs@TiO2-CFC SERS structures. By introducing target molecules into our optofluidic SERS chip, we achieved online molecular Raman detection. Furthermore, by leveraging the UV light-induced self-cleaning properties of TiO2, we achieved continuous online self-cleaning of the molecules. To verify the feasibility and stability of our method, we conducted multiple experiments for online detection and self-cleaning. Experimental results demonstrated impressively low detection limits of 10-8 mol/L for crystal violet and 10-9 mol/L for rhodamine 6G, with an enhancement factor as high as 1.4 × 106. Additionally, we successfully applied our method to polycyclic aromatic hydrocarbons like pyrene.

2.
Plant Biotechnol J ; 22(6): 1610-1621, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38243882

RESUMO

Muscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single-point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. 'Scarlet Royal'. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.


Assuntos
Edição de Genes , Vitis , Vitis/genética , Vitis/metabolismo , Edição de Genes/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Aromatizantes/metabolismo , Monoterpenos/metabolismo , Frutas/genética , Frutas/metabolismo , Mutação Puntual
3.
Plant Biotechnol J ; 22(11): 3121-3134, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39021281

RESUMO

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.


Assuntos
Metilação de DNA , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , RNA não Traduzido , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Plantas Geneticamente Modificadas/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Inativação Gênica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Flores/genética , Arabidopsis/genética
4.
Microb Pathog ; 187: 106513, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38147968

RESUMO

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.


Assuntos
Begoniaceae , Herpesvirus Suídeo 1 , Doenças dos Suínos , Vacinas Virais , Suínos , Animais , Herpesvirus Suídeo 1/genética , Vacinas contra Pseudorraiva , Anticorpos Antivirais , Vacinação/veterinária , Vacinas Virais/genética
5.
Pediatr Res ; 2024 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-39341944

RESUMO

BACKGROUND: Whether portal venous gas (PVG) is a sign of severe neonatal necrotizing enterocolitis (NEC) and predicts poor prognosis remains uncertain. METHODS: Patients from two centres were randomly assigned to a training set or a validation set. A nomogram model for predicting severe NEC was developed on the basis of the independent risk factors selected by least absolute shrinkage and selection operator (LASSO) regression analysis and multivariate logistic regression analysis. The model was evaluated based on the area under the curve (AUC), calibration curve, and decision curve analysis (DCA). RESULTS: A total of 585 patients met the study criteria, and propensity score matching resulted in 141 matched pairs for further analysis. Patients with PVG had a greater risk of surgical intervention or death compared with patients without PVG. A prediction model for severe NEC was established based on PVG, invasive mechanical ventilation (IMV), serum platelet count (PLT) and pH <7.35 at the onset of NEC. The model had a moderate predictive value with an AUC > 0.8. The calibration curve and DCA suggested that the nomogram model had good performance for clinical application. CONCLUSION: A prediction nomogram model based on PVG and other risk factors can help physicians identify severe NEC early and develop reasonable treatment plans. IMPACT: PVG is an important and common imaging manifestation of NEC. Controversy exists regarding whether PVG is an indication for surgical intervention and predicts poor prognosis. Our study suggested that patients with PVG had a greater risk of surgical intervention or death compared with patients without PVG. PVG, IMV, PLT and pH <7.35 at the onset of NEC are independent risk factors for severe NEC. A prediction nomogram model based on PVG and other risk factors may help physicians identify severe NEC early and develop reasonable treatment plans.

6.
Nano Lett ; 23(16): 7516-7523, 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37540083

RESUMO

Gate tunable two-dimensional (2D) superconductors offer significant advantages in studying superconducting phase transitions. Here, we address superconductivity in exfoliated 1T'-MoTe2 monolayers with an intrinsic band gap of ∼7.3 meV using field effect doping. Despite large differences in the dispersion of the conduction and valence bands, superconductivity can be achieved easily for both electrons and holes. The onset of superconductivity occurs near 7-8 K for both charge carrier types. This temperature is much higher than that in bulk samples. Also the in-plane upper critical field is strongly enhanced and exceeds the BCS Pauli limit in both cases. Gap information is extracted using point-contact spectroscopy. The gap ratio exceeds multiple times the value expected for BCS weak-coupling. All of these observations suggest a strong enhancement of the pairing interaction.

7.
J Sci Food Agric ; 103(13): 6219-6232, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37145100

RESUMO

BACKGROUND: Mycoplasma hyorhinis is a prevalent respiratory pathogen in swine, causing significant economic loss to pig producers. There is growing evidence that respiratory pathogen infections have a large impact on intestinal microecology. To study the effect of M. hyorhinis infection on gut microbial composition and metabolome profile, pigs were infected with M. hyorhinis. Metagenomic sequencing analysis was performed of fecal samples and a liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gut digesta was made. RESULTS: Pigs infected with M. hyorhinis had enriched Sutterella and Mailhella, and depleted Dechloromonas, Succinatimonas, Campylobacter, Blastocystis, Treponema, and Megasphaera. The pigs infected with M. hyorhinis also had greater abundances of bacterium_0_1xD8_71, Ruminococcus_sp__CAG_353, Firmicutes_bacterium_CAG_194, Firmicutes_bacterium_CAG_534, bacterium_1xD42_87, and lower abundances of Chlamydia_suis, Megasphaera_elsdenii, Treponema_porcinum, Bacteroides_sp__CAG_1060, Faecalibacterium_prausnitzii. Metabolomic analysis revealed that some lipids and lipid-like molecules increased in the small intestine, whereas most lipids and lipid-like molecule metabolites decreased in the large intestine. These altered metabolites induce changes in intestinal sphingolipid metabolism, amino acid metabolism, and thiamine metabolism. CONCLUSION: These findings demonstrate that infection with M. hyorhinis can alter the gut microbial composition and metabolite structure in pigs, which may further affect amino acid metabolism and lipid metabolism in the intestine. © 2023 Society of Chemical Industry.


Assuntos
Microbioma Gastrointestinal , Infecções por Mycoplasma , Mycoplasma hyorhinis , Doenças dos Suínos , Suínos , Animais , Cromatografia Líquida , Espectrometria de Massas em Tandem , Metaboloma , Aminoácidos , Lipídeos
8.
Plant J ; 105(6): 1495-1506, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33300184

RESUMO

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.


Assuntos
Genoma de Planta/genética , Vitis/genética , Resposta ao Choque Frio/genética , Congelamento , Perfilação da Expressão Gênica , Genes de Plantas/genética , Estudo de Associação Genômica Ampla , Fosfoglicerato Quinase/genética , Filogenia , Proteínas de Plantas/genética , Análise de Sequência de DNA , Vitis/metabolismo , Vitis/fisiologia
9.
Vet Res ; 53(1): 95, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36397177

RESUMO

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson's index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.


Assuntos
Biofilmes , Mycoplasma hyopneumoniae , Doenças dos Suínos , Animais , Genótipo , Tipagem de Sequências Multilocus/veterinária , Mycoplasma hyopneumoniae/genética , Suínos , Doenças dos Suínos/microbiologia , Virulência
10.
Infect Immun ; 88(10)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747599

RESUMO

Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniaein vitro Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.


Assuntos
Translocação Bacteriana/fisiologia , Modelos Biológicos , Mycoplasma hyopneumoniae/fisiologia , Mucosa Respiratória/microbiologia , Animais , Aderência Bacteriana/fisiologia , Brônquios/citologia , Células Epiteliais , Depuração Mucociliar , Pneumonia Suína Micoplasmática/microbiologia , Pneumonia Suína Micoplasmática/patologia , Mucosa Respiratória/patologia , Suínos , Junções Íntimas/patologia
11.
Anal Chem ; 92(12): 8046-8050, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32449350

RESUMO

In this work, well-ordered platinum (Pt) nanocubes (NCs), with precise control on the size and the spatial arrangement, are synthesized from a microemulsion overgrowth in a block copolymer (BC) nanotemplate. The nanovials on this self-assembled BC template serve as microreactors for the reduction of the HCl/H2PtCl6 precursor and direct the ordered periodic arrangement of the reduced Pt nanoparticles (NPs). As the content of HCl increases from 0% to 25%, the Pt NPs evolve from quasi-spheres to NCs, for which the density functional theory (DFT) computation reveals that the different adsorption energies of Cl and HCl dominate this morphology transition. For their potential application in fuel cells, the electrochemical catalysis of the Pt NCs demonstrates a 2.8-fold mass activity in contrast to the commercial JM 40% catalyst at the same Pt loading in ethanol oxidation reaction (EOR) and a good stability of 2.2% ECSA loss over 10 000 CV cycling.

12.
Cell Mol Biol Lett ; 25: 36, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32528541

RESUMO

BACKGROUND: Rhomboid domain containing 1 (RHBDD1) plays a crucial role in tumorigenesis. Silibinin, which is a natural extract from milk thistle, has shown anti-tumor effects against various tumors. Here, we investigate whether silibinin affects the function of RHBDD1 in non-small cell lung cancer (NSCLC) cell proliferation, migration and invasion. METHODS: The Oncomine database and an immunohistochemistry (IHC) assay were used to determine the RHBDD1 expression levels in lung cancer tissues. The associations between RHBDD1 and overall survival rate or clinicopathological parameters were respectively assessed using the Kaplan-Meier overall survival analysis or Chi-squared test. CCK-8 and Transwell assays were applied to analyze cell proliferation, migration and invasion. A549 cells were incubated with increasing concentrations of silibinin. RHBDD1 knockdown and overexpression were achieved via transfection with si-RHBDD1 or RHBDD1 overexpression plasmid, respectively. Western blotting was performed to measure the expressions of epithelial-mesenchymal transition (EMT) markers. RESULTS: We found that overexpression of RHBDD1 in lung cancer tissues correlates with a poor prognosis of survival. Clinical specimen analysis showed that upregulation of RHBDD1 correlates remarkably well with TNM stage and lymph node metastasis. Silibinin suppresses A549 cell proliferation, migration, invasion and EMT in a dose-dependent manner. Importantly, RHBDD1 was downregulated in silibinin-treated A549 cells. RHBDD1 overexpression reversed the suppressive effects of silibinin on A549 cell proliferation, migration, invasion and EMT expression, while its knockdown enhanced them. CONCLUSIONS: These findings shown an anti-tumor impact of silibinin on NSCLC cells via repression of RHBDD1.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Serina Endopeptidases/genética , Silibina/farmacologia , Células A549 , Idoso , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Serina Endopeptidases/metabolismo , Taxa de Sobrevida
13.
BMC Plant Biol ; 19(1): 80, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777012

RESUMO

BACKGROUND: Gibberellins (GAs) and their regulator DELLA are involved in many aspects of plant growth and development and most of our current knowledge in the DELLA-facilitated GA signaling was obtained from the studies of annual species. To understand GA-DELLA signaling in perennial species, we created ten GA-insensitive transgenic grapevines carrying a DELLA mutant allele (Vvgai1) in the background of Vitis vinifera 'Thompson Seedless' and conducted comprehensive analysis of their RNA expression profiles in the shoot, leaf and root tissues. RESULTS: The transgenic lines showed varying degrees of dwarf stature and other typical DELLA mutant phenotypes tightly correlated with the levels of Vvgai1 expression. A large number of differentially expressed genes (DEGs) were identified in the shoot, leaf and root tissues of the transgenic lines and these DEGs were involved in diverse biological processes; many of the DEGs showed strong tissue specificity and about 30% them carried a DELLA motif. We further discovered unexpected expression patterns of several key flowering induction genes VvCO, VvCOL1 and VvTFL1. CONCLUSIONS: Our results not only confirmed many previous DELLA study findings in annual species, but also revealed new DELLA targets and responses in grapevine, including the roles of homeodomain transcription factors as potential co-regulators with DELLA in controlling the development of grapevine which uniquely possess both vegetative and reproductive meristems at the same time. The contrasting responses of some key flowering induction pathway genes provides new insights into the divergence of GA-DELLA regulations between annual and perennial species in GA-DELLA signaling.


Assuntos
Giberelinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Transdução de Sinais , Vitis/genética , Flores/genética , Flores/fisiologia , Redes Reguladoras de Genes , Especificidade de Órgãos , Fenótipo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Análise de Sequência de RNA , Fatores de Transcrição/genética , Vitis/fisiologia
14.
Int J Mol Sci ; 20(11)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174253

RESUMO

MADS-box transcription factors FLOWERING LOCUS C (FLC) and APETALA1 (AP1)/CAULIFLOWER (CAL) have an opposite effect in vernalization-regulated flowering in Arabidopsis. In woody plants, a functional FLC-like gene has not been verified through reverse genetics. To reveal chilling-regulated flowering mechanisms in woody fruit crops, we conducted phylogenetic analysis of the annotated FLC-like proteins of apple and found that these proteins are grouped more closely to Arabidopsis AP1 than the FLC group. An FLC3-like MADS-box gene from columnar apple trees (Malus domestica) (MdFLC3-like) was cloned for functional analysis through a constitutive transgenic expression. The MdFLC3-like shows 88% identity to pear's FLC-like genes and 82% identity to blueberry's CAL1 gene (VcCAL1). When constitutively expressed in a highbush blueberry (Vaccinium corymbosum L.) cultivar 'Legacy', the MdFLC3-like induced expressions of orthologues of three MADS-box genes, including APETALA1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, and CAL1. As a consequence, in contrast to the anticipated late flowering associated with an overexpressed FLC-like, the MdFLC3-like promoted flowering of transgenic blueberry plants under nonchilling conditions where nontransgenic 'Legacy' plants could not flower. Thus, the constitutively expressed MdFLC3-like in transgenic blueberries functioned likely as a blueberry's VcCAL1. The results are anticipated to facilitate future studies for revealing chilling-mediated flowering mechanisms in woody plants.


Assuntos
Mirtilos Azuis (Planta)/genética , Flores/genética , Proteínas de Domínio MADS/genética , Malus/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Mirtilos Azuis (Planta)/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas
15.
J Cell Physiol ; 233(12): 9763-9776, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30078190

RESUMO

Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.


Assuntos
Brônquios/citologia , Células Epiteliais/enzimologia , Cultura Primária de Células/métodos , Telomerase/genética , Animais , Brônquios/enzimologia , Proliferação de Células/genética , Circovirus/patogenicidade , Humanos , Cariótipo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Suínos , Telomerase/biossíntese
16.
BMC Cell Biol ; 19(1): 10, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29954317

RESUMO

BACKGROUND: Air-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined. RESULTS: In this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs. CONCLUSIONS: Ali-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Traqueia/citologia , Animais , Linhagem Celular Transformada , Citocinas/metabolismo , Impedância Elétrica , Células Epiteliais/ultraestrutura , Feminino , Mediadores da Inflamação/metabolismo , Mucinas/metabolismo , Sus scrofa , Junções Íntimas/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
17.
Vet Res ; 49(1): 114, 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30454073

RESUMO

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein-protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.


Assuntos
Aderência Bacteriana , Frutose-Bifosfato Aldolase/fisiologia , Mycoplasma hyopneumoniae/enzimologia , Animais , Aderência Bacteriana/fisiologia , Western Blotting/veterinária , Eletroforese em Gel Bidimensional/veterinária , Citometria de Fluxo/veterinária , Frutose-Bifosfato Aldolase/genética , Genoma Bacteriano/genética , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/patogenicidade , Pneumonia Suína Micoplasmática/microbiologia , Proteômica , Mucosa Respiratória/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Suínos/microbiologia , Traqueia/microbiologia , Virulência
18.
Tumour Biol ; 39(3): 1010428317695971, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28347229

RESUMO

The aim of this study was to investigate the role of G-protein signaling modulator 2 in the carcinogenesis and progression of hepatocellular carcinoma. We previously showed that G-protein signaling modulator 2 was upregulated in hepatitis B virus-related hepatocellular carcinoma tissues through a hierarchical clustering analysis. With this study, we first assessed the expression pattern of G-protein signaling modulator 2 in hepatocellular carcinoma specimens and adjacent noncancerous tissues; clinical data were analyzed, along survival times, utilizing the Kaplan-Meier method. Moreover, the functions of G-protein signaling modulator 2 were examined using small-interfering RNAs in vitro. The results showed that G-protein signaling modulator 2 was clearly overexpressed in hepatocellular carcinoma tissues and cell lines and that the G-protein signaling modulator 2 expression level was related to tumor size and hepatitis B virus infection. Furthermore, G-protein signaling modulator 2 knockdown studies suggested that G-protein signaling modulator 2 accelerates cell growth, cell cycle, migration, and invasion and inhibits apoptosis, acting as an oncogene in hepatocellular carcinoma. Western blotting indicated that silencing of G-protein signaling modulator 2 in HepG2 and SMMC-7721 cells increased the expression levels of Bax, caspase-3, and E-cadherin, while notably suppressing the cyclin-dependent kinase 4, cyclin-dependent kinase 6, CyclinD1, Snail1, Vimentin, and matrix metallopeptidase 9 expression levels, compared with that in the control groups. In addition, we found that G-protein signaling modulator 2 can affect the expression of key proteins involved in protein kinase B activation. In conclusion, high expression of G-protein signaling modulator 2 was involved in the pathological processes of hepatocellular carcinoma through activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which may provide an attractive potential diagnostic biomarker and therapeutic target for treatment of hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/biossíntese , Adulto , Idoso , Apoptose/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais
19.
Phys Rev Lett ; 118(10): 107203, 2017 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-28339266

RESUMO

Kitaev interactions underlying a quantum spin liquid have long been sought, but experimental data from which their strengths can be determined directly, are still lacking. Here, by carrying out inelastic neutron scattering measurements on high-quality single crystals of α-RuCl_{3}, we observe spin-wave spectra with a gap of ∼2 meV around the M point of the two-dimensional Brillouin zone. We derive an effective-spin model in the strong-coupling limit based on energy bands obtained from first-principles calculations, and find that the anisotropic Kitaev interaction K term and the isotropic antiferromagnetic off-diagonal exchange interaction Γ term are significantly larger than the Heisenberg exchange coupling J term. Our experimental data can be well fit using an effective-spin model with K=-6.8 meV and Γ=9.5 meV. These results demonstrate explicitly that Kitaev physics is realized in real materials.

20.
BMC Genomics ; 17(1): 795, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27729006

RESUMO

BACKGROUND: Early ripening is an important desirable attribute for fruit crops. 'Kyoho' is a popular table grape cultivar in many Asian countries. 'Fengzao' is a bud mutant of 'Kyoho' and ripens nearly 30 days earlier than 'Kyoho'. To identify genes controlling early fruit development and ripening in 'Fengzao', RNA-Seq profiles of the two cultivars were compared at 8 different berry developmental stages in both berry peel and flesh tissues. METHODS: RNA-Seq profiling of berry development between 'Kyoho' and 'Fenzhao' were obtained using the Illumina HiSeq system and analyzed using various statistical methods. Expression patterns of several selected genes were validated using qRT-PCR. RESULTS: About 447 millions of RNA-Seq sequences were generated from 40 RNA libraries covering various different berry developmental stages of 'Fengzao' and 'Kyoho'. These sequences were mapped to 23,178 and 22,982 genes in the flesh and peel tissues, respectively. While most genes in 'Fengzao' and 'Kyoho' shared similar expression patterns over different berry developmental stages, there were many genes whose expression were detected only in 'Fengzao' or 'Kyoho'. We observed 10 genes in flesh tissue and 22 genes in peel tissue were differentially expressed at FDR ≤ 0.05 when the mean expression of 'Fengzao' and 'Kyoho' were compared. The most noticeable one was VIT_214s0030g00950 (a superoxide dismutase gene). This ROS related gene showed lower expression levels in 'Fengzao' than 'Kyoho' in both peel and flesh tissues across various berry developmental stages with the only exception at véraison. VIT_200s0238g00060 (TMV resistance protein n-like) and VIT_213s0067g01100 (disease resistance protein at3g14460-like) were the two other noticeable genes which were found differentially expressed between the two cultivars in both peel and flesh tissues. GO functional category and KEGG enrichment analysis of DEGs indicated that gene activities related to stress and ROS were altered between the two cultivars in both flesh and peel tissues. Several differentially expressed genes of interest were successfully validated using qRT-PCR. CONCLUSIONS: Comparative profiling analysis revealed a few dozens of genes which were differentially expressed in the developing berries of 'Kyoho' and its early ripening mutant 'Fengzao'. Further analysis of these differentially expressed genes suggested that gene activities related to ROS and pathogenesis were likely involved in contributing to the early ripening in 'Fengzao'.


Assuntos
Frutas/genética , Perfilação da Expressão Gênica , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma , Vitis/genética , Análise por Conglomerados , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Anotação de Sequência Molecular , Reprodutibilidade dos Testes
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