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A novel tartronic acid decorated hexa-CeIII-incorporated phospho(III)tungstate aggregate (C4H12NO)6Na18H2[(HPW8O31)2[W11O39]2(H2TAD)4(H2O)4W4Ce6H2P2O14]·84H2O (1, H3TAD = tartronic acid) was synthesized by a one-step assembly strategy. Its main skeleton is constructed from two [W11O39]12- fragments, two [HPIIIW8O31]10- segments and one H2TAD--ornamented dodecanuclear heterometallic [W4Ce6H2PIII2O14(H2TAD)4(H2O)4]18+ cluster. In the structure, the [HPIIIO3]2- groups not only work as the heteroatom template to induce the formation of lacunary [HPIIIW8O31]10- segments but also function as the connector to bridge Ce3+ cations. With the help of a reaction strategy of combining ultrasonication treatment with the continuous ion layer adsorption method, the 1/CdS composite was constructed and exhibits prominent photoelectrochemical activity. The 1/CdS composite was used as a photoelectrochemical sensor for oxytetracycline detection at 0 V (vs Ag/AgCl), which displays excellent properties with quick response and low limit of detection (0.042 nM). This work can provide some helpful references in the construction of novel PIII-induced polyoxometalates consisting of different building blocks and can extend the applications of polyoxometalate-based nanocomposites into photoelectrochemical detection for antibiotics as well as biomolecules.
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An attractive isonicotinic acid-ornamented octa-CeIII-inserted phospho(III)tungstate [H2N(CH3)2]6Na8[Ce8(H2O)30W8Na2O20(INA)4][HPIIIW4O17]2[HPIIIW9O33]4·30H2O (1-Ce) (HINA = isonicotinic acid) has been isolated through the deliberately designed one-step assembly strategy, in which the HPO32- heteroanion template was introduced into the Ce3+/WO42- system in the presence of HINA. The polyoxoanion of 1-Ce consists of two identical [Ce4(H2O)15W4NaO10(INA)2][HPIIIW4O17][HPIIIW9O33]2}7- subunits linked by Ce-O-W bonds. The polyoxoanion exhibits three kinds of polyoxotungstate building blocks [W4NaO20(INA)2]17-, [HPIIIW4O17]6-, and [HPIIIW9O33]8-, in which [W4NaO20(INA)2]17- and [HPIIIW4O17]6- building units can be considered as seeds driven by the coordination of additional Ce3+ ions to induce aggregation of [HPIIIW9O33]8- fragments. Furthermore, 1-Ce possesses high peroxidase-like activity and can oxidize 3,3',5,5'-tetramethylbenzidine in the presence of H2O2 with a turnover rate of 6.20 × 10-3 s-1. Because l-cysteine (l-Cys) can reduce oxTMB to TMB, the detection of l-Cys was established based on the 1-Ce-based H2O2 colorimetric biosensing platform with the linear range of 5-100 µM and the limit of detection of 4.28 µM. This work not only can expand the scientific research studies on coordination chemistry and materials chemistry of rare-earth-inserted polyoxotungstates but also can provide practical application possibility in clinical diagnosis using liquid biopsy.
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Cério , Cisteína , Cisteína/química , Cério/química , Peróxido de Hidrogênio/química , Peroxidase/química , ColorimetriaRESUMO
Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness and visual impairment. This study focuses on the function of lysine acetyltransferase 1 (KAT1) in the progression of DR and the epigenetic mechanism. A mouse model with DR was induced by streptozotocin (STZ). Abundantly expressed genes in STZ-induced mice were analyzed. KAT1 was found to be significantly downregulated in the retinal tissues of model mice. Retinal microvascular endothelial cells (RMECs) and retinal Müller cells (rMCs) were cultured in high-glucose medium for in vitro studies. Upregulation of KAT1 suppressed inflammation, neovascularization, and vascular leakage in mouse retinal tissues, and it reduced the activity and inflammatory responses in rMCs, as well as the proliferation and metastatic potential of RMECs. KAT1 activated the transcription activity of YTHDF2 through histone acetylation of the promoter, and YTHDF2 triggered the instability of ITGB1 mRNA to induce mRNA degradation in an m6A manner. The activities of rMCs and RMECs were increased by sh-YTHDF2 but suppressed by sh-ITGB1. The FAK/PI3K/AKT signaling pathway was suppressed upon ITGB1 silencing. Collectively, this study demonstrated that KAT1 triggers YTHDF2-mediated ITGB1 mRNA instability to alleviate the progression of DR.
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Retinopatia Diabética/enzimologia , Células Endoteliais/enzimologia , Células Ependimogliais/enzimologia , Histona Acetiltransferases/metabolismo , Integrina beta1/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/patologia , Células Ependimogliais/patologia , Quinase 1 de Adesão Focal/metabolismo , Histona Acetiltransferases/genética , Integrina beta1/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , EstreptozocinaRESUMO
OBJECTIVE: Clostridioides difficile may colonize healthy infants and young children asymptomatically and for the long-term. C. difficile genotypes and the rate and determinants of colonization differ substantially and vary among countries and regions. A 1-year follow-up study was performed to determine the incidence, kinetics and influencing factors of C. difficile intestinal colonization. METHODS: Twenty-nine healthy infants (14 girls and 15 boys) living at home with their parents in Handan City were followed by survey from birth to 1 year of age, specifically from October 2014 through December 2015. C. difficile isolates were typed by PCR ribotyping and analyzed for the presence of toxin genes. RESULTS: During the follow-up study period in the first year of life, 20 of the 29 total enrolled infants acquired C. difficile. A total of 437 fecal samples were obtained, and 111 (25.4%) samples contained C. difficile, including 79 (71.2%) toxigenic strains. The toxigenic isolates comprised six PCR ribotypes, and two PCR ribotypes were identified as nontoxigenic strains. CONCLUSION: Our study showed that C. difficile colonization increase with age during the 12-month period, and the dominant toxigenic types of C. difficile isolates in infants were those involved in long-term colonization. Feeding patterns may affect the dynamic progress of C. difficile colonization.
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Portador Sadio/epidemiologia , Clostridioides difficile/classificação , Clostridioides difficile/genética , Infecções por Clostridium/epidemiologia , Biodiversidade , China/epidemiologia , Clostridioides difficile/isolamento & purificação , DNA Bacteriano , Fezes/microbiologia , Comportamento Alimentar , Feminino , Seguimentos , Genótipo , Humanos , Incidência , Lactente , Recém-Nascido , Intestinos/microbiologia , Masculino , Reação em Cadeia da Polimerase , RNA Ribossômico 16S , RibotipagemRESUMO
Clostridioides difficile is a colonizer of the human gut; asymptomatic colonization has been reported to be more common in infants and is highly variable across regions even with no symptoms of diarrhea or death. Antibiotic treatment strategies might increase the antibiotic resistance of C. difficile. We performed a one-point study involving 1098 healthy infants (0-36 months) to address the deficiency of reports on C. difficile colonization in Chinese community infants. The C. difficile colonization rate was 22.8% (250/1098), and more than half of the strains (55.2%) were toxigenic isolates. Among the 138 toxigenic isolates, 111 were of the A+B+CDT- genotype, 26 strains were A-B+CDT-, and one strain was A+B+CDT+. Fifteen different PCR ribotypes were found among the 250 isolates, and PCR-ribotype HB03 appeared to be dominant type, accounting for 19.6% (49/250). High levels of resistance to antimicrobial agents were observed. Our study showed that age and hospitalization before stool collection were positively correlated with the C. difficile colonization rate, whereas the delivery term was negatively related to the colonization rate. Particular attention should be paid to the increasing resistance of C. difficile to rifamycin.
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Portador Sadio/epidemiologia , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/epidemiologia , Povo Asiático , Toxinas Bacterianas/genética , Portador Sadio/microbiologia , China/epidemiologia , Infecções por Clostridium/microbiologia , Genótipo , Voluntários Saudáveis , Humanos , Lactente , Epidemiologia Molecular , Reação em Cadeia da Polimerase , Prevalência , RibotipagemRESUMO
The promotion of early osseointegration is crucial for the success of biomedical titanium implants. Physical and chemical modifications to the material surface can significantly compensate for the lack of biocompatibility and early osseointegration of the implant. In this study, we implanted strontium onto titanium plates and analyzed the effect of strontium-doped materials on angiogenesis and biocompatibility in the human bone structure. Our findings demonstrated that strontium-loaded titanium sheet materials effectively promote human umbilical vein endothelial cell (HUVEC) biocompatibility and vascular differentiation ability, as evidenced by proliferation-apoptosis assays, RT-qPCR for vascular neogenesis markers, ELISA for vascular endothelial growth factor (VEGF) levels, and nitric oxide (NO) analysis. Mechanism studies based on RNAseq and Western blotting analysis revealed that strontium can promote titanium material biocompatibility with HUVEC cells and vascular neovascularization ability by activating the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Meanwhile, blocking the ERK1/2 signaling pathway could reverse the promotional effect of vascular formation. Overall, we have successfully fabricated a multifunctional biocompatible bone implant with better histocompatibility and angiogenesis compared to uncoated implants.
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Estrôncio , Titânio , Humanos , Titânio/farmacologia , Titânio/química , Estrôncio/farmacologia , Estrôncio/química , Fator A de Crescimento do Endotélio Vascular , Proteína Quinase 3 Ativada por Mitógeno , Angiogênese , Sistema de Sinalização das MAP Quinases , Propriedades de SuperfícieRESUMO
Different degrees of roasting result in differences in the quality and flavor of large-leaf yellow tea. The current sensory evaluation and chemical detection methods cannot meet the requirement of online differentiation of LYT roasting degree, so an accurate and comprehensive assessment method needs to be developed urgently. First, the two aroma sensing technologies were compared. Two variable screening methods and three recognition algorithms were employed to build discriminant models. The results showed that the discrimination rate of the colorimetric sensor array (CSA) in the prediction set reached 91.89 %, outperforming that of the E-nose. Subsequently, three fusion strategies were applied to improve the discrimination accuracy. The discrimination rate of the middle fusion strategy resulted in an optimal resolution of 94.59 %. The results obtained from the homologous fusion were able to evaluate the roasting degree comprehensively and accurately, which provides a new method and idea for tea aroma quality.
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Aroma affects the quality of black tea, and the rapid evaluation of aroma quality is the key to realize the intelligent processing of black tea. A simple colorimetric sensor array coupled with a hyperspectral system was proposed for the rapid quantitative detection of key volatile organic compounds (VOCs) in black tea. Feature variables were screened based on competitive adaptive reweighted sampling (CARS). Furthermore, the performance of the models for VOCs quantitative prediction was compared. For the quantitative prediction of linalool, benzeneacetaldehyde, hexanal, methyl salicylate, and geraniol, the CARS-least-squares support vector machine model's correlation coefficients were 0.89, 0.95, 0.88, 0.80, and 0.78, respectively. The interaction mechanism of array dyes with VOCs was based on density flooding theory. The optimized highest occupied molecular orbital levels, lowest unoccupied molecular orbital energy levels, dipole moments, and intermolecular distances were determined to be strongly correlated with interactions between array dyes and VOCs.
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Camellia sinensis , Compostos Orgânicos Voláteis , Chá/química , Odorantes/análise , Colorimetria , Camellia sinensis/química , Compostos Orgânicos Voláteis/análise , Análise Espectral , CorantesRESUMO
To develop a comprehensive evaluation method for Keemun black tea, we used micro-near-infrared spectroscopy, computer vision, and colorimetric sensor array to collect data. We used support vector machine, least-squares support vector machine (LS-SVM), extreme learning machine, and partial least squares discriminant analysis algorithms to qualitatively discriminate between different grades of tea. Our results indicated that the LS-SVM model with mid-level data fusion attained an accuracy of 98.57% in the testing set. To quantitatively determine flavour substances in black tea, we used support vector regression. The correlation coefficient for the predicted sets of gallic acid, caffeine, epigallocatechin, catechin, epigallocatechin gallate, epicatechin, gallocatechin gallate and total catechins were 0.84089, 0.94249, 0.94050, 0.83820, 0.81111, 0.82670, 0.93230, and 0.93608, respectively. Furthermore, all compounds exhibited residual predictive deviation values exceeding 2. Hence, combining spectral, shape, colour, and aroma data with mid-level data can provide a rapid and comprehensive assessment of Keemun black tea quality.
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This study synthesized stable and sensitive hemp spherical AgNPs as the SERS substrate for the simultaneous and rapid detection of sunset yellow, lemon yellow, carmine and erythrosine adulteration in black tea. With R6G as the probe molecule, the AgNPs were determined to have satisfactory stability over 60 days with an enhancement factor of 108. The effects of three variable screening methods on model performance were compared. Among them, CARS-PLS exhibited superior performance for the quantification of all the four colorants, with prediction set correlation coefficients of 0.95, 0.97, 0.99 and 0.88, respectively. The differentiation of the mixed colorants was also achieved, with recoveries ranging from 91.87 % to 106.5 % with RSD value <1.97 %, demonstrating the high accuracy and precision of the proposed method. The results indicate that AgNPs-based SERS is an effective method and has substantial potential for application in the identification and quantification of colorant in tea.
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Camellia sinensis , Cannabis , Camellia sinensis/química , Carmim , Eritrosina , Análise Espectral Raman/métodos , Chá/químicaRESUMO
The mechanism through which solar withering (SW) affects the quality of white tea is unclear. To address this gap in the literature, in this study, we used metabolomics and transcriptomics to investigate the effect of SW on the quality of WT. WT that underwent SW was slightly more bitter and astringent than WT that underwent natural withering (control group). Specifically, SW considerably increased the concentration of astringent flavonoids and flavone glycosides in WT. This increase was mainly attributed to the upregulated expression of key genes in the shikimic acid, phenylpropanoid, and flavonoid biosynthesis pathways, such as shikimate kinase, chalcone synthase, and flavonol synthase. In addition, SW experienced considerable heat and light stress. The levels of glycerophosphatidylcholine and carbohydrates increased in response to the stress, which also affected the taste of WT. The results of this study indicate the mechanism through which SW affects the quality of WT.
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Adstringentes , Transcriptoma , Metabolômica , Paladar , CháRESUMO
Rapid monitoring of fermentation quality has been the key to realizing the intelligent processing of black tea. In our study, mixing ratios, sensing array components and reaction times were optimized before an optimal solution phase colorimetric sensor array was constructed. The characteristic spectral information of the array was obtained by UV-visible spectroscopy and subsequently combined with machine learning algorithms to construct a black tea fermentation quality evaluation model. The competitive adaptive reweighting algorithms (CARS)-support vector machine model discriminated the black tea fermentation degree with 100% accuracy. For quantification of catechins and four theaflavins (TF, TFDG, TF-3-G, and TF-3'-G), the correlation coefficients of the CARS least square support vector machine model prediction set were 0.91, 0.86, 0.76, 0.72 and 0.79, respectively. The results obtained within 2 min enabled accurate monitoring of the fermentation quality of black tea, which provides a new method and idea for intelligent black tea processing.
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Camellia sinensis , Catequina , Catequina/análise , Fermentação , Espectrofotometria Ultravioleta , Espectroscopia de Luz Próxima ao Infravermelho , CháRESUMO
The geographical origin and processing month of green tea greatly affect its economic value and consumer acceptance. This study investigated the feasibility of combining near-infrared hyperspectral imaging (NIR-HSI) with chemometrics for the identification of green tea. Tea samples produced in three regions of Chongqing (southeastern Chongqing, northeastern Chongqing, and western Chongqing) for four months (from May to August 2020) were collected. Principal component analysis (PCA) was used to reduce data dimensionality and visualize the clustering of samples in different categories. Linear partial least squares-discriminant analysis (PLS-DA) and nonlinear support vector machine (SVM) algorithms were used to develop discriminant models. The PCA-SVM models based on the first four and first five principal components (PCs) achieved the best accuracies of 97.5% and 95% in the prediction set for geographical origin and processing month of green tea, respectively. This study demonstrated the feasibility of HSI in the identification of green tea species, providing a rapid and nondestructive method for the evaluation and control of green tea quality.
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Chá , Imageamento Hiperespectral , Análise dos Mínimos Quadrados , Análise de Componente Principal , Espectroscopia de Luz Próxima ao Infravermelho , Máquina de Vetores de SuporteRESUMO
Grafting is widely used worldwide because of its obvious advantages, especially in solanaceous vegetable crops. However, the molecular mechanisms underlying graft formation are unknown. In this study, internode tissues from above and below the graft junction were harvested, and we performed weighted gene co-expression network analysis (WGCNA) to describe the temporal and spatial transcriptional dynamics that occur during graft formation in tomato. The wounding stress response involved in JA, ETH, and oxylipins mainly occurred at 1â¯h after grafting (HAG). From 3 to 12 HAG, the biological processes of snRNA and snoRNA modification and the gibberellin-mediated signaling pathway functioned both above and below the graft junction. However, auxin transport and signaling, DNA replication, and xylem and phloem pattern formation were restricted to the scion, whereas the cytokinin-activated signaling pathway and the cellular response to sucrose starvation was restricted to the rootstock. At 24-72 HAG, cell division occurred above the graft junction, and photosynthesis-related pathways were activated below the graft junction. The levels of auxin and cytokinin reached their maxima above and below the graft junction at 12 HAG, respectively. Exogenous application of certain concentrations of IAA and 6-BA will promote xylem and phloem transport capacity. The current work has analyzed the stage-specific events and hub genes during the developmental progression of tomato grafting. We found that auxin and cytokinin levels respond to grafting, above and below the graft junction, respectively, to promote the formation of xylem and phloem patterning. In addition, the accumulation of auxin above the graft junction induced cells to prepare for mitosis and promoted the formation of callus. In short, our work provides an important reference for theoretical research and production application of tomato grafting in the future.
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Citocininas/fisiologia , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Solanum lycopersicum/crescimento & desenvolvimento , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/metabolismo , Solanum lycopersicum/fisiologia , Microscopia , Floema/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/metabolismo , Transcriptoma , Xilema/crescimento & desenvolvimentoRESUMO
Many plants can successfully join root and shoot sections at cut surfaces when severed at the stem. Graft healing is complex and conserved in diverse taxonomic groups with different vascular structures. Herein, we compared transcriptome data from autografted and separated stem sections of Arabidopsis thaliana and tomato (Solanum lycopersicum) to explore changes related to graft healing. Using orthologous gene pairs identified between the two species, temperal expression patterns of evolutionary associated genes in grafted top and bottom, separated top and bottom, and intact stems were exhibited. Genes with expression preference indicate functional diversification of genes related to anatomical structure and cellular development in the two species. Expression profiles of the variable genes revealed common pathways operating during graft healing, including phenylpropanoid metabolism, response to oxygen-containing compounds, xylan, and cell wall biogenesis, mitosis and the cell cycle, carboxylic acid catabolism, and meristem structural organization. In addition, vascular differentiation related NAC domain transcription factors and genome-wide members in Arabidopsis and tomato were used for phylogenetic and expression analysis. Expression differences were largely consistent with sequence differences, reflecting high similarity for protein-coding and regulatory regions of individual clades. NAC proteins mainly clustered in accordance with their reported functions in xylem differentiation or cambium formation. The putative conserved mechanisms suggested by conserved genes and functions could help to expand graft healing theory to a wider range of species, and temporal fluctuations in common pathways imply conserved biological processes during graft healing.
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BACKGROUND/OBJECTIVE: Although lots of long non-coding RNAs (lncRNAs) have been demonstrated to be involved in carcinogenesis, the functions of numerous of lncRNAs remain unknown. Bioinformatics online database showed that lncRNA LOC100132707 was highly expressed in metastatic melanoma tissues, and its expression predicted a lower overall survival rate in melanoma patients. However, LOC100132707 function in uveal melanoma (UM) progression still remains unclear. In the present study, we aimed to elucidate the role and molecular mechanisms underlying LOC100132707 in UM. METHODS: RT-PCR was used to detect the levels of LOC100132707 in UM cells. Cell migration, invasion and tumorigenesis were tested by using the transwell chamber assay and in vivo assay. RESULTS: LOC100132707 expression in metastatic UM cell line MM28 was significantly higher than that of the non-metastatic UM cell lines, MP38, MP46 and MP65, as well as the expressions of LOC100132707-related genes, including XRN1, PARP14, JAK2, DDX60, BUB1 and SAMD9L. LOC100132707 downregulation significantly repressed cell migration and invasion abilities, whereas overexpressing JAK2 rescued these effects. Consistently, upregulation of LOC100132707 induced significant increases in cell migration and invasion abilities via upregulating JAK2. In addition, silencing of LOC100132707 significantly repressed the in vivo tumor formation ability in UM cells. CONCLUSION: This study reveals that silence of LOC100132707 represses the migration of UM via downregulating JAK2. The LOC100132707/JAK2 axis might serve as a potent target for the prevention and treatment of UM metastasis.
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OBJECTIVE: Human uveal melanoma (UM) is a common ocular malignant tumor with a high risk of metastasis. Emerging evidence indicates that long non-coding RNAs (lncRNAs) are correlated with the development of UM. Here, we aimed to determine the biological significance of lncRNA growth arrest-specific transcript 5 (GAS5) in UM. METHODS: The expression levels of GAS5 and microRNA-21 (miR-21) in UM tissues and cells were detected by qRT-PCR analysis. CCK-8 assay was performed to investigate the viability of UM cells after cell transfections, and the migration and invasion of UM cells were determined by transwell assay. The protein expression levels were detected by Western blot assay. The relationship between miR-21 and GAS5 in UM cells was confirmed by bioinformatics prediction and luciferase report assay. RESULTS: Our experiments demonstrated that GAS5 was markedly downregulated in UM cells and clinical specimens. Overexpression of GAS5 inhibited, whereas knockdown of GAS5 promoted the viability, migration, and invasion of UM cells. The epithelial-to-mesenchymal transition (EMT) process of UM cells was also suppressed by upregulating of GAS5 and enhanced by downregulating of GAS5. Additionally, as a competitive endogenous RNA (ceRNA), GAS5 directly binded to the oncogenic miR-21 in UM cells, and overexpression of miR-21 attenuated the EMT-suppressing effect of GAS5. CONCLUSION: Taken together, our findings suggest that GAS5/miR-21 axis is implicated in the pathogenesis of UM and might serve as a potential therapeutic target.
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Carbon dioxide is one of the most important anthropogenic greenhouse gases. We previously confirmed that elevated [CO2] alleviated the negative consequences of drought stress to cucumber seedlings, but the physiological mechanism remains unknown. We investigated the morphological and physiological characteristics as well as iTRAQ-based proteomics analyses in this study under different combinations [CO2] (400 and (800⯱â¯20) µmol·mol-1) and water conditions (no, moderate and severe drought stress simulated by polyethylene glycol 6000). The results showed: (1) elevated [CO2] significantly increased plant height, stem diameter, leaf area and relative water content (RWC) under drought stress; (2) drought stress significantly increased J and K peaks of the chlorophyll a fluorescence transient, indicating the damage of photosynthetic electron transport chain, while elevated [CO2] decreased them especially under moderate drought condition; (3) iTRAQ-based proteomics analyses indicated that elevated [CO2] increased the abundance of psbJ and the PSI reaction center subunit VI-2 in seedlings exposed to moderate drought stress; (4) the abundance of uroporphyrinogen decarboxylase 2 and tetrapyrrole-binding protein decreased in response to elevated [CO2] under severe drought condition; (5) elevated [CO2] regulated the expression of chloroplast proteins such as those related to stress and defense response, redox homeostasis, metabolic pathways. In conclusion, elevated [CO2] enhanced the efficiency of photosynthetic electron transport, limited the absorption of excess light energy, enhanced the ability of antioxidant and osmotic adjustment, and alleviated the accumulation of toxic substances under drought stress. These findings provide new clues for understanding the molecular basis of elevated [CO2] alleviated plant drought stress.
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Dióxido de Carbono/metabolismo , Cucumis sativus/metabolismo , Secas , Proteômica/métodos , Cloroplastos/metabolismo , Cucumis sativus/fisiologia , Plântula/metabolismo , Plântula/fisiologiaRESUMO
Clostridium difficile is the causative pathogen for antibiotic-related nosocomial diarrhea. For epidemiological study and identification of virulent clones, a new binary typing method was developed for C. difficile in this study. The usefulness of this newly developed optimized 10-loci binary typing method was compared with two widely used methods ribotyping and multilocus sequence typing (MLST) in 189 C. difficile samples. The binary typing, ribotyping and MLST typed the samples into 53 binary types (BTs), 26 ribotypes (RTs), and 33 MLST sequence types (STs), respectively. The typing ability of the binary method was better than that of either ribotyping or MLST expressed in Simpson Index (SI) at 0.937, 0.892 and 0.859, respectively. The ease of testing, portability and cost-effectiveness of the new binary typing would make it a useful typing alternative for outbreak investigations within healthcare facilities and epidemiological research.
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Técnicas de Tipagem Bacteriana/métodos , Clostridioides difficile/classificação , Clostridioides difficile/isolamento & purificação , Tipagem de Sequências Multilocus/métodos , Ribotipagem/métodos , Proteínas de Bactérias/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Simulação por Computador , DNA Bacteriano/análise , Loci Gênicos , Genótipo , Humanos , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.