Miro-mediated mitochondrial transport: A new dimension for disease-related abnormal cell metabolism?

Mitochondria are versatile and highly dynamic organelles found in eukaryotic cells that play important roles in a variety of cellular processes. The importance of mitochondrial transport in cell metabolism, including variations in mitochondrial distribution within cells and intercellular transfer, has grown in recent years. Several studies have demonstrated that abnormal mitochondrial transport represents an early pathogenic alteration in a variety of illnesses, emphasizing its significance in disease development and progression. Mitochondrial Rho GTPase (Miro) is a protein found on the outer mitochondrial membrane that is required for cytoskeleton-dependent mitochondrial transport, mitochondrial dynamics (fusion and fission), and mitochondrial Ca2+ homeostasis. Miro, as a critical regulator of mitochondrial transport, has yet to be thoroughly investigated in illness. This review focuses on recent developments in recognizing Miro as a crucial molecule in controlling mitochondrial transport and investigates its roles in diverse illnesses. It also intends to shed light on the possibilities of targeting Miro as a therapeutic method for a variety of diseases.

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563 µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255 kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915 mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.

Preconcentration of Cinnamic Acid Derivatives in Traditional Chinese Medicines by an Effective Dispersive Liquid-Liquid Microextraction Based on a Hydrophobic Deep Eutectic Solvent.

A dispersive liquid-liquid microextraction based on hydrophobic deep eutectic solvent (hDES) was developed for the extraction and quantification of four cinnamic acid derivatives in traditional Chinese medicines coupled with high-performance liquid chromatography-ultraviolet detection. In this method, a hDES (tetrabutylammonium chloride-hexanoic acid, molar ratio of 1:2) was prepared as the extractant. It only took 15 s to handle multiple samples simultaneously by hand-assisted dispersion. The use of a narrow-bore tube reduced the amount of the hydrophobic extractant with easier recovery. The approach was influenced by several key parameters, including the composition and consumption of the DES, sample phase pH, salt amount, extraction time, and centrifugation time, all of which had been investigated and optimized. Moreover, the formation of the DES was characterized by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Under the optimal conditions, enrichment factors of the target analytes ranged from 135 to 220. Satisfactory linearities (r ≥ 0.9977), detection limits (0.2-0.4 ng/mL), precision (<8.5%), and accuracy (recoveries: 90.0%-104.6%) were obtained. The method has been successfully applied to the simultaneous extraction and preconcentration of four cinnamic acid derivatives in Chinese medicinal samples with rapidness, high efficiency, and convenience.

A Mixture Fluency model using responses and response times with cognitive diagnosis model framework.

Random guessing behaviors are frequently observed in low-stakes assessments, often attributed to factors such as test-takers lacking motivation or experiencing time constraints and fatigue. Existing research suggests that responses stemming from random guessing behaviors introduce biases into the constructs and relationships of interest. This is particularly problematic when estimating the relationship between speed and ability. This study introduces a Mixture Fluency model designed to account for random guessing behaviors while utilizing valid response accuracy and response time to uncover students' latent attribute profiles. The model directly addresses a limitation present in the Fluency cognitive diagnostic model (Wang & Chen, Psychometrika, 85, 600-629, (2020), which assumes that test-takers consistently employ solution behaviors when answering questions. To investigate the effectiveness of the proposed Mixture Fluency model, we conducted a simulation study encompassing various simulation conditions. Results from this study not only confirm the model's ability to detect potential random guessing behaviors but also demonstrate its capacity to enhance the inference of targeted latent constructs within the assessment. Additionally, we showcase the practical utility of the proposed model through an application to real data.

Insights into effects of thermotolerant nitrifying and sulfur-oxidizing inoculants on nitrogen-sulfur co-metabolism in sewage sludge composting.

In this study, high temperature thermotolerant nitrifying bacteria (TNB) and high temperature thermotolerant sulfide oxidizing bacteria (TSOB) were obtained from compost samples and inoculated into sewage sludge (SS) compost. The effects of inoculation on physical and chemical parameters, ammonia and hydrogen sulfide release, nitrogen form and sulfur compound content change and physical-chemical properties during nitrogen and sulfur conversion were studied. The results showed that inoculation of TNB and TSOB increased the temperature, pH, OM degradation, C/N ratio and germination index (GI) of compost. Compared with the control treatment (CK), the addition of inoculants reduced the release of NH3 and H2S, and transformed them into nitrogen and sulfur compounds, the hydrolysis of polymeric ferrous sulfate was promoted, resulting in relatively high content of sulfite and sulfate. At the same time, the physical and chemical properties of SS have a strong correlation with nitrogen and sulfur compounds.

Mechanism of sulfur-oxidizing inoculants and nitrate on regulating sulfur functional genes and bacterial community at the thermophilic compost stage.

The emission of H2S odors predominantly occurred at the thermophilic phase of composting, which could cause odorous gas pollution and reduce the fertilizer value of composting products. And sulfur-oxidizing bacteria (SOB) possess oxidative capacities for inorganic sulfur compounds with nitrate applied as electron acceptors. Therefore, this study aimed to assess the effectiveness of combined additives (SOB inoculants and nitrate) on the bacterial community diversity, sulfur-oxidizing gene abundances, and metabolic function prediction at the thermophilic stage of sewage sludge composting. The highest sulfate contents were increased by 1.02-1.34 folds, and the abundances of the sulfur-oxidizing genes (sqr, pdo, sox, and sor) were also enhanced by adding the combined additives. Network patterns revealed a strengthened interaction of inoculants and sulfur functional genes. Microbial functional pathways predicted higher metabolic levels of carbohydrate and amino acid metabolisms with the addition of combined additives, and the predicted relative abundances of sulfur metabolism and nitrogen metabolism were increased by 19.3 ± 2.5% and 24.7 ± 4.1%, respectively. Heatmap analysis showed that the SOB might have a competitive advantage over the indigenous denitrifying bacteria in using nitrate for biochemical reactions. Correlation analyses suggested that sulfur-oxidizing efficacy could be indirectly affected by the environmental parameters through changing the structure of bacterial community. These findings provide new insights toward an optimized inoculation strategy of using SOB and nitrate to enhance sulfur preservation and modulate the bacterial communities at the thermophilic phase of sewage sludge composting.

Characterization of sulfide oxidation and optimization of sulfate production by a thermophilic Paenibacillus naphthalenovorans LYH-3 isolated from sewage sludge composting.

The sulfur-containing odor emitted from sludge composting could be controlled by sulfide oxidizing bacteria, yet mesophilic strains show inactivation during the thermophilic stage of composting. Aimed to investigate and characterize the thermotolerant bacterium that could oxidize sulfide into sulfate, a heterotrophic strain was isolated from sewage sludge composting and identified as Paenibacillus naphthalenovorans LYH-3. The effects of various environmental factors on sulfide oxidation capacities were studied to optimize the sulfate production, and the highest production rate (27.35% ± 0.86%) was obtained at pH 7.34, the rotation speed of 161.14 r/min, and the inoculation amount of 5.83% by employing Box-Behnken design. The results of serial sulfide substrates experiments indicated that strain LYH-3 could survive up to 400 mg/L of sulfide with the highest sulfide removal rate (88.79% ± 0.35%) obtained at 50 mg/L of sulfide. Growth kinetic analysis presented the maximum specific growth rate µm (0.5274 hr-1) after 22 hr cultivation at 50°C. The highest enzyme activities of sulfide quinone oxidoreductase (0.369 ± 0.052 U/mg) and sulfur dioxygenase (0.255 ± 0.014 U/mg) were both obtained at 40°C, and the highest enzyme activity of sulfite acceptor oxidoreductase (1.302 ± 0.035 U/mg) was assessed at 50°C. The results indicated that P. naphthalenovorans possessed a rapid growth rate and efficient sulfide oxidation capacities under thermophilic conditions, promising a potential application in controlling sulfur-containing odors during the thermophilic stage of sludge composting.

Comparison between spleen and liver stiffness measurements by sound touch elastography for diagnosing cirrhosis at different aminotransferase levels: a prospective multicenter study.

OBJECTIVES: To compare the performance of spleen stiffness measurement (SSM) and liver stiffness measurement (LSM) by sound touch elastography (STE) for the diagnosis of cirrhosis at different alanine aminotransferase (ALT) levels, and to compare the applicability and repeatability of SSM with LSM performed by STE, a new two-dimensional shear wave elastography technology. METHODS: This prospective multicenter study included 25 centers and recruited chronic hepatitis B (CHB) patients with liver biopsy between May 2018 and November 2019. All patients underwent LSM and SSM by STE. Success and reliability rates were calculated and compared. Intra-observer agreement was assessed using intraclass correlation coefficients (ICCs). Differences between areas under the receiver operating characteristic curves (AUCs) of LSMs and SSMs at different ALT levels were compared using the Delong test. RESULTS: Among 603 CHB patients, the success and reliability rates of SSM were 94.53% (570/603) and 85.74% (517/603), respectively, which were similar to those of LSM (p > 0.05), respectively. The ICC for intra-observer agreements of SSM was 0.964 (p < 0.001). In the total cohort, ALT ≤ 2 × upper limit of normal (ULN) group, and A0-1 group, the AUCs of SSMs were significantly lower than those of LSMs for the diagnosis of cirrhosis (p < 0.001). In the ALT > 2 × ULN group and A2-3 group, the AUC of SSM improved and was not significantly different from that of LSM (p = 0.342, p = 0.510, respectively). CONCLUSIONS: SSM by STE achieved applicability and repeatability equivalent to those of LSM. SSM might be a good substitute to LSM in patients with high ALT levels. KEY POINTS: • Spleen stiffness measurement performed by sound touch elastography was proven to have similar applicability and repeatability to liver stiffness measurement in this prospective multicenter study. • Spleen stiffness measurement demonstrated a poorer diagnostic performance for cirrhosis compared with liver stiffness measurement in the total cohort and low ALT level group, yet it showed a similar diagnostic performance to liver stiffness measurement in patients with high ALT levels.

Population-based projections of blood supply and demand, China, 2017-2036.

OBJECTIVE: To estimate the long-term effect of the changing demography in China on blood supply and demand. METHODS: We developed a predictive model to estimate blood supply and demand during 2017-2036 in mainland China and in 31 province-level regions. Model parameters were obtained from World Population Prospects, China statistical yearbook 2016, China's report on blood safety and records from a large tertiary hospital. Our main assumptions were stable age-specific per capita blood supply and demand over time. FINDINGS: We estimated that the change in demographic structure between 2016 (baseline year) and 2036 would result in a 16.0% decrease in blood supply (from 43.2 million units of 200 mL to 36.3 million units) and a 33.1% increase in demand (from 43.2 million units to 57.5 million units). In 2036, there would be an estimated shortage of 21.2 million units. An annual increase in supply between 0.9% and 1.8% is required to maintain a balance in blood supply and demand. This increase is not enough for every region as regional differences will increase, e.g. a blood demand/supply ratio ≥ 1.45 by 2036 is predicted in regions with large populations older than 65 years. Sensitivity analyses showed that increasing donations by 4.0% annually by people aged 18-34 years or decreasing the overall blood discard rate from 5.0% to 2.0% would not offset but help reduce the blood shortage. CONCLUSION: Multidimensional strategies and tailored, coordinated actions are needed to deal with growing pressures on blood services because of China's ageing population.

Cell subpopulation deconvolution reveals breast cancer heterogeneity based on DNA methylation signature.

Tumour heterogeneity describes the coexistence of divergent tumour cell clones within tumours, which is often caused by underlying epigenetic changes. DNA methylation is commonly regarded as a significant regulator that differs across cells and tissues. In this study, we comprehensively reviewed research progress on estimating of tumour heterogeneity. Bioinformatics-based analysis of DNA methylation has revealed the evolutionary relationships between breast cancer cell lines and tissues. Further analysis of the DNA methylation profiles in 33 breast cancer-related cell lines identified cell line-specific methylation patterns. Next, we reviewed the computational methods in inferring clonal evolution of tumours from different perspectives and then proposed a deconvolution strategy for modelling cell subclonal populations dynamics in breast cancer tissues based on DNA methylation. Further analysis of simulated cancer tissues and real cell lines revealed that this approach exhibits satisfactory performance and relative stability in estimating the composition and proportions of cellular subpopulations. The application of this strategy to breast cancer individuals of the Cancer Genome Atlas's identified different cellular subpopulations with distinct molecular phenotypes. Moreover, the current and potential future applications of this deconvolution strategy to clinical breast cancer research are discussed, and emphasis was placed on the DNA methylation-based recognition of intra-tumour heterogeneity. The wide use of these methods for estimating heterogeneity to further clinical cohorts will improve our understanding of neoplastic progression and the design of therapeutic interventions for treating breast cancer and other malignancies.

An analysis about heterogeneity among cancers based on the DNA methylation patterns.

BACKGROUND: It is generally believed that DNA methylation, as one of the most important epigenetic modifications, participates in the regulation of gene expression and plays an important role in the development of cancer, and there exits epigenetic heterogeneity among cancers. Therefore, this study tried to screen for reliable prognostic markers for different cancers, providing further explanation for the heterogeneity of cancers, and more targets for clinical transformation studies of cancer from epigenetic perspective. METHODS: This article discusses the epigenetic heterogeneity of cancer in detail. Firstly, DNA methylation data of seven cancer types were obtained from Illumina Infinium HumanMethylation 450 K platform of TCGA database. Then, differential methylation analysis was performed in the promotor region. Secondly, pivotal gene markers were obtained by constructing the DNA methylation correlation network and the gene interaction network in the KEGG pathway, and 317 marker genes obtained from two networks were integrated as candidate markers for the prognosis model. Finally, we used the univariate and multivariate COX regression models to select specific independent prognostic markers for each cancer, and studied the risk factor of these genes by doing survival analysis. RESULTS: First, the cancer type-specific gene markers were obtained by differential methylation analysis and they were found to be involved in different biological functions by enrichment analysis. Moreover, specific and common diagnostic markers for each type of cancer was sorted out and Kaplan-Meier survival analysis showed that there was significant difference in survival between the two risk groups. CONCLUSIONS: This study screened out reliable prognostic markers for different cancers, providing a further explanation for the heterogeneity of cancer at the DNA methylation level and more targets for clinical conversion studies of cancer.

Comparison of three-phase hollow fiber liquid-phase microextraction based on reverse micelle with conventional two-phase hollow fiber liquid-phase microextraction and their applications for analysis of cinnamic acids in traditional Chinese medicines.

A novel three-phase hollow fiber liquid-phase microextraction was developed based on reverse micelle as extraction solvent and acceptor phase, and compared with conventional two-phase hollow fiber liquid-phase microextraction. Both procedures were used in the extraction and concentration of four cinnamic acids (caffeic acid, p-hydroxycinnamic acid, ferulic acid, and cinnamic acid) in traditional Chinese medicines prior to high-performance liquid chromatography analysis. Parameters affecting the two procedures were investigated and optimized to obtain the optimum enrichment factors. The mechanism of the developed procedure was explored and elucidated by comparison with conventional two-phase hollow fiber liquid-phase microextraction. Under the optimized conditions, the analytes' enrichment factors were between 50 and 118 for the proposed procedure, and 31-96 for conventional two-phase mode. Satisfactory linear ranges (r2  ≥ 0.99), detection limits (0.1-0.6 ng/mL), precisions (<9.2%), and accuracies (recoveries: 80-123.1%) were observed for the two procedures. The results showed that the enrichment capacity of the proposed procedure for the cinnamic acids is better than that of conventional two-phase procedure, and both are eco-friendly, simple, and effective for the enrichment and detection of cinnamic acids in traditional Chinese medicines.

DiseaseMeth version 2.0: a major expansion and update of the human disease methylation database.

The human disease methylation database (DiseaseMeth, http://bioinfo.hrbmu.edu.cn/diseasemeth/) is an interactive database that aims to present the most complete collection and annotation of aberrant DNA methylation in human diseases, especially various cancers. Recently, the high-throughput microarray and sequencing technologies have promoted the production of methylome data that contain comprehensive knowledge of human diseases. In this DiseaseMeth update, we have increased the number of samples from 3610 to 32 701, the number of diseases from 72 to 88 and the disease-gene associations from 216 201 to 679 602. DiseaseMeth version 2.0 provides an expanded comprehensive list of disease-gene associations based on manual curation from experimental studies and computational identification from high-throughput methylome data. Besides the data expansion, we also updated the search engine and visualization tools. In particular, we enhanced the differential analysis tools, which now enable online automated identification of DNA methylation abnormalities in human disease in a case-control or disease-disease manner. To facilitate further mining of the disease methylome, three new web tools were developed for cluster analysis, functional annotation and survival analysis. DiseaseMeth version 2.0 should be a useful resource platform for further understanding the molecular mechanisms of human diseases.

Additive hazards model with auxiliary subgroup survival information.

The semiparametric additive hazards model is an important way for studying the effect of potential risk factors for right-censored time-to-event data. In this paper, we study the additive hazards model in the presence of auxiliary subgroup [Formula: see text]-year survival information. We formulate the known auxiliary information in the form of estimating equations, and combine them with the conventional score-type estimating equations for the estimation of the regression parameters based on the maximum empirical likelihood method. We prove that the new estimator of the regression coefficients follows asymptotically a multivariate normal distribution with a sandwich-type covariance matrix that can be consistently estimated, and is strictly more efficient, in an asymptotic sense, than the conventional one without incorporation of the available auxiliary information. Simulation studies show that the new proposal has substantial advantages over the conventional one in terms of standard errors, and with the accommodation of more informative information, the proposed estimator becomes more competing. An AIDS data example is used for illustration.

Liver Fibrosis with Two-dimensional US Shear-Wave Elastography in Participants with Chronic Hepatitis B: A Prospective Multicenter Study.

Purpose To investigate the diagnostic performance of two-dimensional (2D) shear-wave elastography (SWE) in chronic hepatitis B. Materials and Methods This prospective multicenter study from January 2015 to January 2016 was conducted at 12 hospitals and included 654 participants with chronic hepatitis B who had undergone liver biopsy and 2D SWE examination. Participants were divided into chronic infection and chronic hepatitis groups. The diagnostic performance of 2D SWE was compared with the aspartate amino transferase-to-platelet ratio index (APRI), the Fibrosis-4 index (FIB-4), and transient elastography (TE) by using a DeLong test and was also compared between two subgroups. Dual cutoff values for cirrhosis were determined with multilevel likelihood ratio analysis. Results Overall, 402 participants with chronic hepatitis B were enrolled (154 with chronic infection and 248 with chronic hepatitis). The areas under the receiver operating characteristic curve of 2D SWE (0.87; 95% confidence interval [CI]: 0.83, 0.90) were higher than those of TE (0.80; 95% CI: 0.68, 0.88), APRI (0.70; 95% CI: 0.65, 0.74), and FIB-4 (0.73; 95% CI: 0.69, 0.78) in cirrhosis. The high area under the receiver operating characteristic curve (0.92; 95% CI: 0.87, 0.96) was achieved in the chronic infection group and was significantly higher than that of the chronic hepatitis group (0.84; 95% CI: 0.78, 0.88; P = .017). Dual cutoff values with the likelihood ratios below 0.1 and above 10 (8.4 kPa and 11.0 kPa to rule out and rule in a diagnosis of cirrhosis, respectively) were effectively determined in chronic infection; a total of 81.2% (125 of 154) participants with cirrhosis were definitively diagnosed. Conclusion The performance of two-dimensional (2D) shear-wave elastography (SWE) was higher than that of other noninvasive methods. 2D SWE was most effective in ruling in and ruling out cirrhosis in participants with chronic infection, which may prompt antiviral treatment. © RSNA, 2018 Online supplemental material is available for this article.

Systematic identification and annotation of human methylation marks based on bisulfite sequencing methylomes reveals distinct roles of cell type-specific hypomethylation in the regulation of cell identity genes.

DNA methylation is a key epigenetic mark that is critical for gene regulation in multicellular eukaryotes. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understand the complex regulatory network for cell fate determination. In this study, we proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757 887 genome segments. Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, we identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in cell type-specific manner. In particular, we observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation.

SEA: a super-enhancer archive.

Super-enhancers are large clusters of transcriptional enhancers regarded as having essential roles in driving the expression of genes that control cell identity during development and tumorigenesis. The construction of a genome-wide super-enhancer database is urgently needed to better understand super-enhancer-directed gene expression regulation for a given biology process. Here, we present a specifically designed web-accessible database, Super-Enhancer Archive (SEA, http://sea.edbc.org). SEA focuses on integrating super-enhancers in multiple species and annotating their potential roles in the regulation of cell identity gene expression. The current release of SEA incorporates 83 996 super-enhancers computationally or experimentally identified in 134 cell types/tissues/diseases, including human (75 439, three of which were experimentally identified), mouse (5879, five of which were experimentally identified), Drosophila melanogaster (1774) and Caenorhabditis elegans (904). To facilitate data extraction, SEA supports multiple search options, including species, genome location, gene name, cell type/tissue and super-enhancer name. The response provides detailed (epi)genetic information, incorporating cell type specificity, nearby genes, transcriptional factor binding sites, CRISPR/Cas9 target sites, evolutionary conservation, SNPs, H3K27ac, DNA methylation, gene expression and TF ChIP-seq data. Moreover, analytical tools and a genome browser were developed for users to explore super-enhancers and their roles in defining cell identity and disease processes in depth.

A Biocontrol Strain of Bacillus subtilis WXCDD105 Used to Control Tomato Botrytis cinerea and Cladosporium fulvum Cooke and Promote the Growth of Seedlings.

In this study, a strain named WXCDD105, which has strong antagonistic effects on Botrytis cinerea and Cladosporium fulvum Cooke, was screened out from the rhizosphere of healthy tomato plants. The tomato plants had inhibition diameter zones of 5.00 mm during the dual culture for four days. Based on the morphological and physiological characteristics, the 16S rDNA sequence, and the gyrB gene sequence analysis, the strain WXCDD105 was identified as Bacillus subtilis suBap. subtilis. The results of the mycelial growth test showed that the sterile filtrate of the strain WXCDD105 could significantly inhibit mycelial growth of Botrytis cinerea and Cladosporium fulvum Cooke. The inhibition rates were 95.28 and 94.44%, respectively. The potting experiment showed that the strain WXCDD105 made effective the control of tomato gray mold and tomato leaf mold. The control efficiencies were 74.70 and 72.07%. The antagonistic test results showed that the strain WXCDD105 had different degrees of inhibition on 10 kinds of plant pathogenic fungi and the average inhibition rates were more than 80%. We also found that the strain WXCDD105 stimulated both the seed germination and seedling growth of tomatoes. Using the fermentation liquid of WXCDD105 (108 cfu·mL−1) to treat the seeds, the germination rate and radicle length were increased. Under the treatment of the fermentation liquid of the strain WXCDD105 (106 cfu·mL−1), nearly all physiological indexes of tomato seedlings were significantly higher than that of the control groups. This could not only keep the nutritional quality of tomato fruits but also prevent them from rotting. This study provided us with an excellent strain for biological control of tomato gray mold, tomato leaf mold, and tomato growth promotion. This also laid the technical foundation for its application.

Biochemical characterization of recombinant Enterovirus 71 3C protease with fluorogenic model peptide substrates and development of a biochemical assay.

Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant kcat/Km of 11.8 ± 0.82 mM(-1) min(-1). Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z' factor of 0.87 ± 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC50] = 1.89 ± 0.25 µM) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC50] = 4.54 ± 0.51 µM; 50% cytotoxic concentration [CC50] > 100 µM). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.