Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes.

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

Application of artificial intelligence in endoscopic image analysis for the diagnosis of a gastric cancer pathogen-Helicobacter pylori infection.

Helicobacter pylori (H. pylori) infection is the principal cause of chronic gastritis, gastric ulcers, duodenal ulcers, and gastric cancer. In clinical practice, diagnosis of H. pylori infection by a gastroenterologists' impression of endoscopic images is inaccurate and cannot be used for the management of gastrointestinal diseases. The aim of this study was to develop an artificial intelligence classification system for the diagnosis of H. pylori infection by pre-processing endoscopic images and machine learning methods. Endoscopic images of the gastric body and antrum from 302 patients receiving endoscopy with confirmation of H. pylori status by a rapid urease test at An Nan Hospital were obtained for the derivation and validation of an artificial intelligence classification system. The H. pylori status was interpreted as positive or negative by Convolutional Neural Network (CNN) and Concurrent Spatial and Channel Squeeze and Excitation (scSE) network, combined with different classification models for deep learning of gastric images. The comprehensive assessment for H. pylori status by scSE-CatBoost classification models for both body and antrum images from same patients achieved an accuracy of 0.90, sensitivity of 1.00, specificity of 0.81, positive predictive value of 0.82, negative predicted value of 1.00, and area under the curve of 0.88. The data suggest that an artificial intelligence classification model using scSE-CatBoost deep learning for gastric endoscopic images can distinguish H. pylori status with good performance and is useful for the survey or diagnosis of H. pylori infection in clinical practice.

Intraday exposure to ambient ozone and emergency department visits among children: a case-crossover study in southern China.

Most existing studies have investigated short-term associations between ozone exposure and acute disease events among children at a daily timescale, which might neglect risk effects happening within several hours after ozone exposure. In this research, we aimed to depict intraday associations between pediatric emergency department visits (PEDVs) and exposure to ozone in order to better detect ultra-short-term effects of ozone exposure on children. We obtained hourly data of all-cause PEDVs, air pollutants, and meteorological factors in Shenzhen and Guangzhou, China, 2015-2018. We applied time-stratified case-crossover design and conditional logistic regression models to estimate odds ratios per 10-µg/m3 rise of ozone concentrations at various exposure periods (e.g., 0-3, 4-6, 7-12, 13-24, 25-48, and 49-72 h) prior to PEDVs, controlling for hourly relative humidity and temperature. Subgroup analyses divided by gender, age, and season were undertaken to identify the potential susceptible population and period. A total of 358,285 cases of PEDVs were included in two cities, and hourly average concentration of ozone was 45.5 µg/m3 in Guangzhou and 58.9 µg/m3 in Shenzhen, respectively. Increased risks of PEDVs occurred within a few hours (0-3 h) after exposure to ozone and remained up to 48 h. Population risks for PEDVs increased by 0.8% (95% confidence interval, 0.6 to 1.0) in Shenzhen and 0.7% (0.5 to 0.9) in Guangzhou for a 10-µg/m3 increase in ozone concentrations at lag 4-6 h and lag 7-12 h, respectively. These findings were robust to co-exposure adjustments in our sensitivity analyses. Significantly greater ozone-associated risks were consistently observed during cold months (October to March of the following year) in both cities, while we did not identify evidence for effect modification of children's age and gender. This study provided novel evidence for increased risks of acute disease events among children within several hours after ozone exposure, highlighting the significant implications for policymakers to establish hourly air quality standards for better protecting children's health.

Plant growth-promoting effect and genomic analysis of the P. putida LWPZF isolated from C. japonicum rhizosphere.

Plant growth-promoting rhizobacteria are a type of beneficial bacteria which inhabit in the rhizosphere and possess the abilities to promote plant growth. Pseudomonas putida LWPZF is a plant growth-promoting bacterium isolated from the rhizosphere soil of Cercidiphyllum japonicum. Inoculation treatment with LWPZF could significantly promote the growth of C. japonicum seedlings. P. putida LWPZF has a variety of plant growth-promoting properties, including the ability to solubilize phosphate, synthesize ACC deaminase and IAA. The P. putida LWPZF genome contained a circular chromosome (6,259,530 bp) and a circular plasmid (160,969 bp) with G+C contents of 61.75% and 58.25%, respectively. There were 5632 and 169 predicted protein-coding sequences (CDSs) on the chromosome and the plasmid respectively. Genome sequence analysis revealed lots of genes associated with biosynthesis of IAA, pyoverdine, ACC deaminase, trehalose, volatiles acetoin and 2,3-butanediol, 4-hydroxybenzoate, as well as gluconic acid contributing phosphate solubilization. Additionally, we identified many heavy metal resistance genes, including arsenate, copper, chromate, cobalt-zinc-cadmium, and mercury. These results suggest that P. putida LWPZF shows strong potential in the fields of biofertilizer, biocontrol and heavy metal contamination soil remediation. The data presented in this study will allow us to better understand the mechanisms of plant growth promotion, biocontrol, and anti-heavy metal of P. putida LWPZF.

Isolation, Characterization, and Tea Growth-Promoting Analysis of JW-CZ2, a Bacterium With 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Activity Isolated From the Rhizosphere Soils of Tea Plants.

One of the major mechanisms underlying plant growth-promoting rhizobacteria (PGPR) is the lowering of ethylene level in plants by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) in the environment. In the present study, using ACC as the sole nitrogen source, we screened seven ACC deaminase-producing bacterial strains from rhizosphere soils of tea plants. The strain with the highest ACC deaminase activity was identified as Serratia marcescens strain JW-CZ2. Inoculation of this strain significantly increased shoot height and stem diameter of tea seedlings, displaying significant promotive effects. Besides, S. marcescens strain JW-CZ2 displayed high ACC deaminase activities in wide ranges of ACC concentration, pH, and temperature, suggesting the applicable potential of JW-CZ2 as a biofertilizer. Genome sequencing indicated that clusters of orthologous groups of proteins (COG) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of JW-CZ2 mainly included amino acid transport and metabolism, transcription, carbohydrate transport and metabolism, inorganic ion transport and metabolism, and membrane transport. Moreover, genes in relation to phosphate solubilization, indole acetic acid (IAA) production, and siderophore were observed in the genome of JW-CZ2, and further experimental evidence demonstrated JW-CZ2 could promote solubilization of inorganic phosphate, inhibit growth of pathogenic fungi, and produce IAA and siderophore. These aspects might be major reasons underlying the plant growth-promoting function of JW-CZ2. Overall, this study provides a new S. marcescens strain, which has applicable potential as a promising biofertilizer.

Thermal degradation of cyanidin-3-O-glucoside: Mechanism and toxicity of products.

The thermal degradation behavior of cyanidin-3-O-gluoside (Cy3G) in nitrogen and air was studied using thermogravimetric analysis (TGA), thermogravimetry-Fourier transform infrared spectroscopy (TG-FTIR) and pyrolysis-gas chromatography/mass spectrometry (Py-GCMS). The results show that the thermal degradation of Cy3G in nitrogen and in air can be divided into three steps. The total degradation rate was 63.09% in nitrogen and 99.42% in air, and the total activation energy (Ea) was 65.85 and 80.98 kJ·mol-1, respectively. The TG-FTIR analysis showed that Cy3G is significantly decomposed at 200-300 °C. The Py-GCMS analysis shows that the first step in the thermal degradation of Cy3G in nitrogen is the cleavage of glycosidic bonds to give cyanidin and glucoside. The glucoside and cyanidin then degrade further to give mainly low molecular weight compounds, together with furan derivatives, pyran derivatives and aromatic compounds. The phenols and furans found in the pyrolysis products are known to have a degree of toxicity.

Structural basis for a bacterial Pip system plant effector recognition protein.

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors.

Volatile Organic Compounds of Endophytic Burkholderia pyrrocinia Strain JK-SH007 Promote Disease Resistance in Poplar.

Volatile organic compounds (VOCs) play important roles in the regulation of plant growth and pathogen resistance. However, little is known about the influence of VOCs released from endophytic strains (Burkholderia pyrrocinia strain JK-SH007) on controlling pathogens or inducing systemic resistance in poplar. In this study, we found that VOCs produced by strain JK-SH007 inhibit three poplar canker pathogens (Cytospora chrysosperma, Phomopsis macrospora, and Fusicoccum aesculi) and promote defense enzyme activity and malondialdehyde (MDA) and total phenol (TP) accumulation. Thirteen kinds of VOC components were identified using the solid-phase microextraction combined with gas chromatography-mass spectrometry method. Dimethyl disulfide (DMDS) accounted for the largest proportion of these VOCs. Treatments of poplar seedlings with different volumes of VOC standards (DMDS, benzothiazole, dimethylthiomethane, and phenylacetone) showed that DMDS had the greatest effects on various defense enzyme activities and MDA and TP accumulation. We also found that the inhibitory effect of the VOCs on the three pathogens was gradually enhanced with increasing standard volume. Moreover, the treatment of samples with DMDS significantly reduced the severity and development of the disease caused by three poplar canker pathogens. Comparative transcriptomics analysis of poplar seedlings treated with DMDS showed that there were 1,586 differentially expressed genes in the leaves and stems, and quantitative PCR showed that the gene expression trends were highly consistent with the transcriptome sequencing results. The most significant transcriptomic changes induced by VOCs were related to hormone signal transduction, transcriptional regulation of plant-pathogen interactions, and energy metabolism. Moreover, 137 transcription factors, including members of the ethylene response factor, NAC, WRKY, G2-like, and basic helix-loop-helix protein families, were identified to be involved in the VOC-induced process. This study elucidates the resistance induced by Burkholderia pyrrocinia strain JK-SH007 to poplar canker at the molecular level and can make possible a new method for the comprehensive prevention and control of poplar disease.

Hybrids of Quinoline and Anilinopyrimidine: Novel EGFRT790M Inhibitors with Antiproliferative Activity against Non-Small Cell Lung Cancer Cell Lines.

BACKGROUND: The third-generation irreversible Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKIs) inhibit the T790M mutation while sparing EGFRWT. However, the C797S point mutation confers resistance to existing irreversible EGFRT790M inhibitors. OBJECTIVE: Novel EGFRT790M inhibitors were designed through hybridization of quinoline and anilinopyrimidine, and biologically evaluated their antiproliferative activity against Non-Small Cell Lung Cancer (NSCLC) cell lines. METHODS: The target compounds 11a-h were synthesized and structurally characterized with 1H, 13C Nuclear Magnetic Resonance (NMR) spectroscopy and High-Resolution Mass Spectrometry (HRMS). Their inhibitory effects on tumor cell proliferation and EGFR kinase were biologically evaluated. Additionally, molecular docking studies were also performed on the representative typical EGFRT790M inhibitor. RESULTS: Most of the evaluated compounds displayed moderate antiproliferative activity on H1975 cells with EGFRL858R/T790M. However, compound 11a (IC50 = 2.235 ± 0.565µM) showed stronger inhibition than gefitinib (IC50 = 8.830 ± 0.495µM) in concentration- and time-dependent manner. Moreover, compound 11a exhibited weaker inhibitory activities on cells with EGFRWT. Specifically, compound 11a strongly suppressed EGFRL858R/T790M (IC50 = 0.515 ± 0.011µM) relative to EGFRWT (IC50 = 0.913 ± 0.068µM). Furthermore, molecular docking studies demonstrated its strong binding contacts with the EGFRT790M enzyme through hydrogen bonds and other non-bonded interactions. CONCLUSION: Taken together, these results indicate that the hybrid of quinoline and anilinopyrimidine 11a, could be a potential inhibitor of EGFRT790M in NSCLC, which warrants further in-depth studies.

Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR.

Pseudomonas brassicacearum GS20 is an antagonistic strain of bacteria recently isolated from the rhizosphere of Codonopsis pilosula. No validated reference gene has been indentified from P. brassicacearum to use in the normalization of real-time quantitative reverse transcription-PCR data. Therefore, in this study, nine candidate reference genes (recA, gyrA, rpoD, proC, gmk, rho, 16S, ftsz, and secA) were assessed at different growth phases and under various growth conditions. The expression stability of these candidate genes was evaluated using BestKeeper, NormFinder and GeNorm. In general, the results showed rho, rpoD and gyrA were the most suitable reference genes for P. brassicacearum GS20. The relative expression of iron-regulated gene (fhu) was normalized to verify the reliability of the proposed reference genes under iron-replete and iron-limited conditions. The trend in relative expression was consistent with the change in siderophore production under different iron conditions. This study presents reliable reference genes for transcriptional studies in P. brassicacearum GS20 under the chosen experimental conditions.

A Novel Cold-Adaptive Endo-1,4-ß-Glucanase From Burkholderia pyrrocinia JK-SH007: Gene Expression and Characterization of the Enzyme and Mode of Action.

The efficient industrial conversion of plant-derived cellulose to simple sugars and other value-added chemicals requires various highly stable and reactive enzymes. Industrial processes especially synchronous saccharification and fermentation (SSF)-based production of cellulosic bio-ethanol require enzymes that are active at lower temperatures. In this study, we have identified, characterized, and expressed the cold-adaptive endo-1,4-ß-glucanase (BpEG) isolated from the Burkholderia pyrrocinia JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The BpEG without the signal peptide was cloned into the expression vector pET32a and significantly expressed in Escherichia coli BL21 (DE3) competent cells. The SDS-PAGE and Western blot analysis of BpEG revealed that the recombinant BpEG was approximately 60 kDa. Purified recombinant BpEG exhibited hydrolytic activity against carboxymethyl cellulose (CMC) and phosphoric acid swollen cellulose (PASC), but not crystalline cellulose and xylan substrates. High performance, anion exchange, chromatography-pulsed amperometric detector (HPAEC-PAD) analysis of the enzymatic products obtained from depolymerization of 1,4-ß-linked biopolymers of different lengths revealed an interesting cutting mechanism employed by endoglucanases. The recombinant BpEG exhibited 6.0 of optimum pH and 35°C of optimum temperature, when cultured with CMC substrate. The BpEG enzyme exhibited stable activity between pH 5.0 and 9.0 at 35°C. Interestingly, BpEG retained about 42% of its enzymatic activity at 10°C compared to its optimal temperature. This new cold-adaptive cellulase could potentially achieve synchronous saccharification and fermentation (SSF) making BpEG a promising candidate in the fields of biofuel, biorefining, food and pharmaceutical industries.

Yield, Nutritional Content, and Antioxidant Activity of Pleurotus ostreatus on Corncobs Supplemented with Herb Residues.

Improper disposal of herb residues in China has caused severe problems to the surrounding environment and human safety. Three herb residues, i.e., compound Kushen injection residues (CKI) and part one and part two of Qizhitongluo Capsule residues (QC1 and QC2, respectively), were used for the cultivation of Pleurotus ostreatus. The effect of the supplementation of corncobs (CC) with different herb residues on yield, nutritional composition, and antioxidant activity of P. ostreatus was investigated. Compared to the control, the higher mycelial growth rate was observed on substrates CC +30% CKI and CC +30% QC1, while the higher yield was obtained from substrates CC +30% QC2 and CC +30% CKI. Moreover, chemical analysis of fruit bodies revealed that the addition of herb residues to CC significantly increased proteins, amino acids, ashes, minerals (Na and Ca), and total phenolic contents but significantly reduced carbohydrates and IC50 values of DPPH radicals. In addition, no heavy metals (Pb, Cd, and As) were detected in the fruiting bodies harvested from different substrate combinations. These results demonstrated that mixtures of CC with herb residues might be utilized as a novel, practical, and easily available substrate for the cultivation of P. ostreatus, which is beneficial for the effective management of herb residues.

Screening and functional analysis of the peroxiredoxin specifically expressed in Bursaphelenchus xylophilus--the causative agent of pine wilt disease.

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode.

[Optimization of biocontrol agent Burkholderia pyrrocinia strain JK-SH007 fermentation by response surface methodology].

In order to improve ferment efficiency of biocontrol agent Burkholderia pyrrocinia JK-SH007, the fermentation conditions of this strain were optimized. The optimal fermentation conditions were corn steep liquor (13.88 g/L) and glucose (3.37 g/L) by screening test, steepest ascent experiments and response surface analysis. The results showed that the cell density of JK-SH007 (1.18 x 10(9) CFU/mL) increased 1.35 times than before, and there was a 28.84% increase in antifungal activity.

[Phylogenetic application and analysis of horizontal transfer based on the prokaryote eno gene].

The phenomenon of conflicting gene trees has become a remarkable and difficult problem. Application of multiple genes has been a widespread practice to reconstruct phylogenies in phylogenetic studies. Enolase is a key glycolytic enzyme, The enzymes from a large variety of organisms, including archaebacteria, eubacteria and eukaryotes, were studied. We downloaded eno sequences from the genomes of bacteria and archaea that have been completely sequenced. The comprehensive homology search and phylogenetic analysis of the eno were used, and nineteen horizontally transferred genes were identified. The results of analysis showed lots of differences between the features of horizontal transferred genes and the ones of whole genomic genes, such as nucleotide composition, gene combination, codon usage bias, and selection pressure. These results reconfirmed that the horizontally transferred genes were exogenous. The result revealed that prokaryote eno genes were highly conserved, medium-sized, is a good material in the phylogenetic. This paper can provide a reference in study of life habit and evolutionary history of donor and receptor, and enolase structure and function.

[Screening, identification, and promoting effect of phosphate-solubilizing bacteria in rhizosphere of Taxus chinensis var. mairei].

OBJECTIVE: Phosphate-solubilizing bacteria (PSB) were isolated, screened and identified from the rhizosphere of Taxus chinensis var. mairei, and growth-promoting effects on T. chinensis var. mairei by high effective PSB were determined. METHODS: By using selective culture media, PSB were isolated from rhizospheric soil, the high effective PSB was further screened using NBRI-BPB medium, and the molybdenum-antimony anti-spectrophotometric method was applied to determine the phosphate-dissolving ability of the high effective PSB after four days fermentation in NBRIP medium. Bacteria were identified by the Biolog system combined with 16S rDNA gene sequence analysis and morphological, physiological and biochemical characteristics. The inoculation test in potted seedlings was carried out under the greenhouse. CONCLUSION: Four strains of high effective PSB were screened and identified as Pseudomonas fluorescens, Bacillus cereus, Sinorhizobium meliloti and Bacillus licheniformis, respectively. These strains had significant effects on improving the growth of the seedlings of T. chinensis var. mairei.