ccdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid-gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

The sprT Gene of Bacillus velezensis FZB42 Is Involved in Biofilm Formation and Bacilysin Production.

Bacillus velezensis FZB42, a representative strain of plant-growth-promoting rhizobacteria (PGPR), can form robust biofilm and produce multiple antibiotics against a wild range of phytopathogens. In this study, we observed different biofilm morphology of the mutant Y4, derived from a TnYLB-1 transposon insertion library of B. velezensis FZB42. We identified that the transposon was inserted into the sprT gene in Y4. Our bioinformatics analysis revealed that the SprT protein is an unstable hydrophilic protein located in the cytoplasm. It is highly conserved in Bacillus species and predicted to function as a metalloprotease by binding zinc ions. We also demonstrated that ΔsprT significantly reduced the swarming ability of FZB42 by ~5-fold and sporulation capacity by ~25-fold. In addition, the antagonistic experiments showed that, compared to the wild type, the ΔsprT strain exhibited significantly reduced inhibition against Staphylococcus aureus ATCC-9144 and Phytophthora sojae, indicating that the inactivation of sprT led to decreased production of the antibiotic bacilysin. The HPLC-MS analysis confirmed that bacilysin was indeed decreased in the ΔsprT strain, and qPCR analysis revealed that ΔsprT down-regulated the expression of the genes for bacilysin biosynthesis. Our results suggest that the sprT gene plays a regulatory role in multiple characteristics of B. velezensis FZB42, including biofilm formation, swarming, sporulation, and antibiotic production.

Transcriptional Profiling and Transposon Mutagenesis Study of the Endophyte Pantoea eucalypti FBS135 Adapting to Nitrogen Starvation.

The research on plant endophytes has been drawing a lot of attention in recent years. Pantoea belongs to a group of endophytes with plant growth-promoting activity and has been widely used in agricultural fields. In our earlier studies, Pantoea eucalypti FBS135 was isolated from healthy-growing Pinus massoniana and was able to promote pine growth. P. eucalypti FBS135 can grow under extremely low nitrogen conditions. To understand the mechanism of the low-nitrogen tolerance of this bacterium, the transcriptome of FBS135 in the absence of nitrogen was examined in this study. We found that FBS135 actively regulates its gene expression in response to nitrogen deficiency. Nearly half of the number (4475) of genes in FBS135 were differentially expressed under this condition, mostly downregulated, while it significantly upregulated many transportation-associated genes and some nitrogen metabolism-related genes. In the downregulated genes, the ribosome pathway-related ones were significantly enriched. Meanwhile, we constructed a Tn5 transposon library of FBS135, from which four genes involved in low-nitrogen tolerance were screened out, including the gene for the host-specific protein J, RNA polymerase σ factor RpoS, phosphoribosamine-glycine ligase, and serine acetyltransferase. Functional analysis of the genes revealed their potential roles in the adaptation to nitrogen limitation. The results obtained in this work shed light on the mechanism of endophytes represented by P. eucalypti FBS135, at the overall transcriptional level, to an environmentally limited nitrogen supply and provided a basis for further investigation on this topic.

Annulment of Bacterial Antagonism Improves Plant Beneficial Activity of a Bacillus velezensis Consortium.

Bacillus sp. strains that are beneficial to plants are widely used in commercial biofertilizers and biocontrol agents for sustainable agriculture. Generally, functional Bacillus strains are applied as single-strain communities since the principles of synthetic microbial consortia constructed with Bacillus strains remain largely unclear. Here, we demonstrated that the mutual compatibility directly affects the survival and function of two-member consortia composed of Bacillus velezensis SQR9 and FZB42 in the rhizosphere. A mutation in the global regulator Spo0A of SQR9 markedly reduced the boundary phenotype (appearance of a visible boundary line at the meeting point of two swarms) with wild-type FZB42, and the combined use of the SQR9(△spo0A) mutant and FZB42 improved biofilm formation, root colonization, and the production of secondary metabolites that are beneficial to plants. Furthermore, alleviation of antagonistic interactions of two-member Bacillus consortia improved its beneficial effects to cucumber in a greenhouse experiment. Our results provide evidence that social interactions among bacteria could be an influencing factor for achieving a desired community-level function. IMPORTANCE Bacillus velezensis is one of the most widely applied bacteria in biofertilizers in China and Europe. Additionally, the molecular mechanisms of plant growth promotion and disease suppression by representative model strains are well established, such as B. velezensis SQR9 and FZB42. However, it remains extremely challenging to design efficient consortia based on these model strains. Here, we showed that swarm encounter phenotype is one of the major determinants that affects the performance of two-member Bacillus consortia in vitro and in the rhizosphere. Deletion in global regulatory gene spo0A of SQR9 reduced the strength of boundary formation with FZB42 and resulted in the improved plant growth promotion performance of the dual consortium. This knowledge provides new insights into efficient probiotics consortia design in Bacillus spp.

Stanniocalcin1 knockdown induces ferroptosis and suppresses glycolysis in prostate cancer via the Nrf2 pathway.

Stanniocalcin1 (STC1) is a secreted glycoprotein, which is highly expressed in prostate cancer cells. However, the biological functions of STC1 in modulating ferroptosis and glycolysis in prostate cancer are still not clear. The viability of PC-3 and DU145 cells was detected by CCK-8 assay. The relative Fe2+ level was detected by an Iron Assay Kit. MDA level was detected by Lipid Peroxidation MDA Assay Kit. Glucose uptake and lactate product were measured by Glycolysis Assay Kit and Lactate Assay Kit. In this study, STC1 was highly expressed in prostate cancer tissue specimens and cells. STC1 knockdown suppressed prostate cancer cell proliferation, and upregulated Fe2+ level, reduced glutathione (GSH) level, downregulated GPX4 and SLC7A11 protein expressions in PC-3 cells and DU145 cells. Besides, STC1 knockdown decreased glucose uptake, lactate product, and ATP level, as well as downregulated glycolysis-related protein HK2 and LDHA protein expressions. In addition, STC1 knockdown repressed the Nrf2/HO-1/NQO1 pathway. Nrf2 pathway activator, Oltipraz, upregulated Nrf2, total NQO1, and HO-1 expressions in PC-3 cells and DU145 cells. Moreover, Nrf2 pathway activator Oltipraz reversed the effect of STC1 knockdown on Fe2+ level and GPX4, SLC7A11, HK2, LDHA protein expressions in PC-3 cells and DU145 cells. Finally, STC1 knockdown restrained the tumor volume, tumor weight, and glycolysis in prostate cancer in vivo. Thus, STC1/Nrf2 pathway is a vital pathway to induce ferroptosis and suppress glycolysis in prostate cancer.

The Plant-Beneficial Rhizobacterium Bacillus velezensis FZB42 Controls the Soybean Pathogen Phytophthora sojae Due to Bacilysin Production.

Soybean root rot caused by the oomycete Phytophthora sojae is a serious soilborne disease threatening soybean production in China. Bacillus velezensis FZB42 is a model strain for Gram-positive plant growth-promoting rhizobacteria and is able to produce multiple antibiotics. In this study, we demonstrated that B. velezensis FZB42 can efficiently antagonize P. sojae. The underlying mechanism for the inhibition was then investigated. The FZB42 mutants deficient in the synthesis of lipopeptides (bacillomycin D and fengycin), known to have antifungal activities, and polyketides (bacillaene, difficidin, and macrolactin), known to have antibacterial activities, were not impaired in their antagonism toward P. sojae; in contrast, mutants deficient in bacilysin biosynthesis completely lost their antagonistic activities toward P. sojae, indicating that bacilysin was responsible for the activity. Isolated pure bacilysin confirmed this inference. Bacilysin was previously shown to be antagonistic mainly toward prokaryotic bacteria rather than eukaryotes. Here, we found that bacilysin could severely damage the hyphal structures of P. sojae and lead to the loss of its intracellular contents. A device was invented allowing interactions between P. sojae and B. velezensis FZB42 on nutrient agar. In this manner, the effect of FZB42 on P. sojae was studied by transcriptomics. FZB42 significantly inhibited the expression of P. sojae genes related to growth, macromolecule biosynthesis, pathogenicity, and ribosomes. Among them, the genes for pectate lyase were the most significantly downregulated. Additionally, we showed that bacilysin effectively prevents soybean sprouts from being infected by P. sojae and could antagonize diverse Phytophthora species, such as Phytophthora palmivora, P. melonis, P. capsici, P. litchi, and, most importantly, P. infestans. IMPORTANCEPhytophthora spp. are widespread eukaryotic phytopathogens and often extremely harmful. Phytophthora can infect many types of plants important to agriculture and forestry and thus cause large economic losses. Perhaps due to inappropriate recognition of Phytophthora as a common pathogen in history, research on the biological control of Phytophthora is limited. This study shows that B. velezensis FZB42 can antagonize various Phytophthora species and prevent the infection of soybean seedlings by P. sojae. The antibiotic produced by FZB42, bacilysin, which was already known to have antibacterial effectiveness, is responsible for the inhibitory action against Phytophthora. We further showed that some Phytophthora genes and pathways may be targeted in future biocontrol studies. Therefore, our data provide a basis for the development of new tools for the prevention and control of root and stem rot in soybean and other plant diseases caused by Phytophthora.

Ultrathin layer TAFC on BiVO4 with ligand-to-metal charge transfer enhances built-in electric field for boosting photoelectrochemical water oxidation.

To reveal the mechanism of charge transfer between interfaces of BiVO4-based heterogeneous materials in photoelectrochemical water splitting system, the cocatalyst was grown in situ using tannic acid (TA) as a ligand and Fe and Co ions as metal centers (TAFC), and then uniformly and ultra-thinly coated on BiVO4 to form photoanodes. The results show that the BiVO4/TAFC achieves a superior photocurrent density (4.97 mA cm-2 at 1.23 VRHE). The charge separation and charge injection efficiencies were also significantly higher, 82.0 % and 78.9 %, respectively. From XPS, UPS, KPFM, and density functional theory calculations, Ligand-to-metal charge transfer (LMCT) acts as an electron transport highway in TAFC ultrathin layer to promote the concentration of electrons towards metal center, leading to an increase in the work function, which enhances the built-in electric field and further improves the charge transport. This study demonstrated that the LMCT pathway on TA-metal complexes enhances the built-in electric field in BiVO4/TAFC to promote charge transport and thus enhance water oxidation, providing a new understanding of the performance improvement mechanism for the surface-modified composite photoanodes.

A metal-organic framework-based fluorescence resonance energy transfer nanoprobe for highly selective detection of Staphylococcus Aureus.

Survival and infection of pathogenic bacteria, such as Staphylococcus aureus (S. aureus), pose a serious threat to human health. Efficient methods for recognizing and quantifying low levels of bacteria are imperiously needed. Herein, we introduce a metal-organic framework (MOF)-based fluorescence resonance energy transfer (FRET) nanoprobe for ratiometric detection of S. aureus. The nanoprobe utilizes blue-emitting 7-hydroxycoumarin-4-acetic acid (HCAA) encapsulated inside zirconium (Zr)-based MOFs as the energy donor and green-emitting fluorescein isothiocyanate (FITC) as the energy acceptor. Especially, vancomycin (VAN) is employed as the recognition moiety to bind to the cell wall of S. aureus, leading to the disassembly of VAN-PEG-FITC from MOF HCAA@UiO-66. As the distance between the donor and acceptor increases, the donor signal correspondingly increases as the FRET signal decreases. By calculating the fluorescence intensity ratio, S. aureus can be quantified with a dynamic range of 1.05 × 103-1.05 × 107 CFU mL-1 and a detection limit of 12 CFU mL-1. Due to the unique high affinity of VAN to S. aureus, the nanoprobe shows high selectivity and sensitivity to S. aureus, even in real samples like lake water, orange juice, and saliva. The FRET-based ratiometric fluorescence bacterial detection method demonstrated in this work has a prospect in portable application and may reduce the potential threat of pathogens to human health.

Sustained anaerobic degradation of 4-chloro-2-methylphenoxyacetic acid by acclimated sludge in a continuous-flow reactor.

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a widely used herbicide across the world. MCPA is persistent and easily transports into anoxic environment, such as groundwater, sediments and deep soils. However, little research on anaerobic microbial degradation of MCPA was carried out. The functional microorganisms as well as the catabolic pathway are still unknown. In this research, an anaerobic MCPA-degrading bacterial consortium was enriched from the river sediment near a pesticide-manufacturing plant. After about 6 months' acclimation, the MCPA transformation rate of the consortium reached 4.32 µmol g-1 day-1, 25 times faster than that of the original sludge. 96% of added MCPA (2.5 mM) was degraded within 9 d of incubation. Three metabolites including 4-chloro-2-methylphenol (MCP), 2-methylphenol (2-MP) and phenol were identified during the anaerobic degradation of MCPA. An anaerobic catabolic pathway was firstly proposed: firstly, MCPA was transformed to MCP via the cleavage of the aryl ether, then MCP was reductively dechlorinated to 2-MP which was further demethylated to phenol. The 16S rRNA gene amplicon sequencing revealed a substantial shift in the bacterial community composition after the acclimation. SBR1031, Acidaminococcaceae, Aminicenantales, Syntrophorhabdus, Acidaminobacter, Bacteroidetes_vadinHA17, Methanosaeta, Bathyarchaeia, KD4-96, Anaeromyxobacter, and Dehalobacter were significantly increased in the enriched consortium after acclimation, and positively correlated with the anaerobic degradation of MCPA as suggested by heat map correlation analysis. This study provides a basis for further elucidation of the anaerobic catabolism of MCPA, and contributes to developing efficient and low-cost anaerobic treatment technologies for MCPA pollution.

Adjusting the valence band center of Co-Ni-bimetallic sulfides through lattice expansion and stacking faults triggered by strain engineering to boost oxygen evolution reaction.

Stress engineering can improve catalytic performance by straining the catalyst lattice. An electrocatalyst, Co3S4/Ni3S2-10%Mo@NC, was prepared with abundant lattice distortion to boost oxygen evolution reaction (OER). With the assistance of the intramolecular steric hindrance effect of metal-organic frameworks, slow dissolution by MoO42- of the Ni substrate and recrystallization of Ni2+ was observed in the process of Co(OH)F crystal growth with mild temperature and short time reaction. The lattice expansion and stacking faults created defects inside the Co3S4 crystal, improved the material conductivity, optimized the valence band electron distribution of the material, and promoted the rapid conversion of the reaction intermediates. The presence of reactive intermediates of the OER under catalytic conditions was investigated using operando Raman spectroscopy. The electrocatalysts exhibited super high performance, a current density of 10 mA cm-2 at an overpotential of 164 mV and 100 mA cm-2 at 223 mV, which were comparable to those of integrated RuO2. Our work for the first time demonstrates that the dissolution-recrystallization triggered by strain engineering is a good modulation approach to adjust the structure and surface activity of catalyst, suggesting promising industrial application.

Transcriptomic profiling of Bacillus amyloliquefaciens FZB42 in response to maize root exudates.

BACKGROUND: Plant root exudates have been shown to play an important role in mediating interactions between plant growth-promoting rhizobacteria (PGPR) and their host plants. Most investigations were performed on Gram-negative rhizobacteria, while much less is known about Gram-positive rhizobacteria. To elucidate early responses of PGPR to root exudates, we investigated changes in the transcriptome of a Gram-positive PGPR to plant root exudates. RESULTS: Bacillus amyloliquefaciens FZB42 is a well-studied Gram-positive PGPR. To obtain a comprehensive overview of FZB42 gene expression in response to maize root exudates, microarray experiments were performed. A total of 302 genes representing 8.2% of the FZB42 transcriptome showed significantly altered expression levels in the presence of root exudates. The majority of the genes (261) was up-regulated after incubation of FZB42 with root exudates, whereas only 41 genes were down-regulated. Several groups of the genes which were strongly induced by the root exudates are involved in metabolic pathways relating to nutrient utilization, bacterial chemotaxis and motility, and non-ribosomal synthesis of antimicrobial peptides and polyketides. CONCLUSIONS: Here we present a transcriptome analysis of the root-colonizing bacterium Bacillus amyloliquefaciens FZB42 in response to maize root exudates. The 302 genes identified as being differentially transcribed are proposed to be involved in interactions of Gram-positive bacteria with plants.

Acrylamide causes neurotoxicity by inhibiting glycolysis and causing the accumulation of carbonyl compounds in BV2 microglial cells.

Acrylamide is a potent neurotoxin produced during industrial production or food processing at high temperatures, and its effect on glycolytic processes may be critical in triggering neurotoxicity. Our work investigated the neurotoxic effects of acrylamide based on glycolysis using immortalized mouse microglia cell line BV2. The results showed that 1.5 mM acrylamide significantly inhibited the expression and activity of triphosphate isomerase and 3-phosphoglyceraldehyde dehydrogenase. Moreover, acrylamide limited the expression of pyruvate kinase and the decrease of pyruvate and lactate content of glycolysis products. In addition, acrylamide could increase intracellular methylglyoxal content and further affect its detoxification system. Not only that, subsequent cellular responses resulting from methylglyoxal accumulation, such as oxidative stress, activation of ERK, upregulation of NF-κB inflammatory signaling pathway, and elevated pro-inflammatory factor TNF-α, were found in the acrylamide-treated cell model. Therefore, we suggest that acrylamide's perturbation of the glycolytic process leads to methylglyoxal accumulation, which may be responsible for its key to neurotoxicity.

Application and Development of Biocontrol Agents in China.

While the growing population in the world has a large demand for food, agriculture and forestry are currently facing severe challenges due to phytopathogens and pests along with global warming. For half a century chemical pesticides and fertilizers have made a great contribution to agricultural production. However, the excessive use of chemical agents has caused obvious side effects on the environment and the sustainable development of agriculture in the long term. China has recorded one of the fastest economic growths for more than 20 years but at the cost of a seriously polluted environment. Since a decade ago, China has paid increasing attention to environment protection and taken intensified measures for pollution control and ecological restoration. In this context, the biocontrol agent industry in China has experienced a golden decade of rapid development. In this minireview, we will introduce the application and development of microorganism-based biocontrol agents in China over the past two decades.

Population differentiation and epidemic tracking of Bursaphelenchus xylophilus in China based on chromosome-level assembly and whole-genome sequencing data.

BACKGROUND: Bursaphelenchus xylophilus, the pinewood nematode, kills millions of pine trees worldwide every year, and causes enormous economic and ecological losses. Despite extensive research on population variation, there is little understanding of the population-wide variation spectrum in China. RESULTS: We sequenced an inbred B. xylophilus strain using Pacbio+Illumina+Bionano+Hi-C and generated a chromosome-level assembly (AH1) with six chromosomes of 77.1 Mb (chromosome N50: 12 Mb). The AH1 assembly shows very high continuity and completeness, and contains novel genes with potentially important functions compared with previous assemblies. Subsequently, we sequenced 181 strains from China and the USA and found ~7.8 million single nucleotide polymorphisms (SNPs). Analysis shows that the B. xylophilus population in China can be divided into geographically bounded subpopulations with severe cross-infection and potential migrations. In addition, distribution of B. xylophilus is dominated by temperature zones while geographically associated SNPs are mainly located on adaptation related GPCR gene families, suggesting the nematode has been evolving to adapt to different temperatures. A machine-learning based epidemic tracking method has been established to predict their geographical origins, which can be applied to any other species. CONCLUSION: Our study provides the community with the first high-quality chromosome-level assembly which includes a comprehensive catalogue of genetic variations. It provides insights into population structure and effective tracking method for this invasive species, which facilitates future studies to address a variety of applied, genomic and evolutionary questions in B. xylophilus as well as related species.

Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons.

The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42(T) (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42(T) to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7(T) but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42(T), formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7(T). DNA-DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7(T) and FZB42(T) yielded relatedness values of 63.7-71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7(T) and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42(T) ( = DSM 23117(T) = BGSC 10A6(T)), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7(T)( = ATCC 23350(T) = Fukumoto Strain F(T)), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies.

Relationship of the Pine Growth Promoting Pantoea eucalypti FBS135 with Type Strains P. eucalypti LMG 24197T and P. vagans 24199T.

Endophytes in woody plants are much less understood. Pantoea strain FBS135 is an endophytic bacterium isolated from Pinus massoniana with the ability to promote pine growth significantly. In this study, we demonstrated that FBS135 has the astonishing ability of low nitrogen tolerance but no ability of nitrogen fixation. To exactly determine the phylogenetic status of FBS135, we sequenced the whole genomes of P. eucalypti LMG 24197T and P. vagans 24199T, type strains of two Pantoea species, which are evolutionarily closest to FBS135. P. eucalypti LMG 24197T contained a single chromosome of 4,035,995 bp (C+G, 54.6%) plus three circular plasmids while LMG 24199T comprises a single circular chromosome of 4,050,173 bp (C+G, 55.6%) and two circular plasmids. With the genomic information, FBS135 was finally identified as a P. eucalypti strain, although it showed some different physiological traits from the two type strains. Comparative genomic analyses were performed for the three strains, revealing their common molecular basis associated with plant lifecycle as well as the differences in their gene arrangements relating to nitrogen utilization.

Annexin A8 regulated by lncRNA-TUG1/miR-140-3p axis promotes bladder cancer progression and metastasis.

Bladder cancer is the ninth most diagnosed cancer in the world. This study aims to investigate the role and mechanisms of the taurine-upregulated gene 1 (TUG1)/miR-140-3p/annexin A8 (ANXA8) axis in bladder cancer. Western blotting and qRT-PCR determined the expression levels of ANXA8, miR-140-3p, TUG1, and epithelial-mesenchymal transition (EMT) markers. RNA immunoprecipitation (RIP), luciferase assay, and RNA pull-down assay validated the association among ANXA8, miR-140-3p, and TUG1. The biological functions were determined by colony formation, Annexin V-fluorescein isothiocyanate (FITC)/propidium (PI) staining, and transwell assays. Xenograft tumorigenesis detected tumor growth and metastasis in vivo. Pathological analysis was examined by hematoxylin and eosin (H&E) and immunohistochemistry (IHC) analyses. ANXA8 was elevated in bladder tumors and cells. Knockdown of ANXA8 suppressed cell growth, migration, invasion, and EMT in UMUC-3 and T24 cells. ANXA8 was determined as a miR-140-3p target gene. Overexpression of miR-140-3p suppressed cell proliferation, migration, invasion, and EMT via targeting ANXA8. TUG1 promoted ANXA8 expression via sponging miR-140-3p. Silencing of miR-140-3p or ANXA8 overexpression abrogated the tumor-suppressive effects of TUG1 silencing on bladder cancer cell growth and metastasis. The TUG1/miR-140-3p/ANXA8 axis was also implicated in tumor growth and lung metastasis in vivo. TUG1 promotes bladder cancer progression and metastasis through activating ANXA8 by sponging miR-140-3p, which sheds light on the mechanisms of bladder cancer pathogenesis.

Two Lysine Sites That Can Be Malonylated Are Important for LuxS Regulatory Roles in Bacillus velezensis.

S-ribosylhomocysteine lyase (LuxS) has been shown to regulate bacterial multicellular behaviors, typically biofilm formation. However, the mechanisms for the regulation are still mysterious. We previously identified a malonylation modification on K124 and K130 of the LuxS in the plant growth-promoting rhizobacterium B. velezensis (FZB42). In this work, we investigated the effects of the two malonylation sites on biofilm formation and other biological characteristics of FZB42. The results showed that the K124R mutation could severely impair biofilm formation, swarming, and sporulation but promote AI-2 production, suggesting inhibitory effects of high-level AI-2 on the features. All mutations (K124R, K124E, K130R, and K130E) suppressed FZB42 sporulation but increased its antibiotic production. The double mutations generally had a synergistic effect or at least equal to the effects of the single mutations. The mutation of K130 but not of K124 decreased the in vitro enzymatic activity of LuxS, corresponding to the conservation of K130 among various Bacillus LuxS proteins. From the results, we deduce that an alternative regulatory circuit may exist to compensate for the roles of LuxS upon its disruption. This study broadens the understanding of the biological function of LuxS in bacilli and underlines the importance of the two post-translational modification sites.

Preparative isolation and purification of zearalenone from rice culture by combined use of macroporous resin column and high-speed counter-current chromatography.

Zearalenone is one of the most harmful mycotoxins found in grains and there is a large demand for zearalenone substrate for research purposes. A new separation method was developed for the preparative purification of zearalenone from rice culture of Fusarium graminearum by utilizing macroporous resin column combined with high-speed counter-current chromatography. Zearalenone was adsorpted on XAD-2 resin at 25 °C, neutral pH and a feed flow of 4 BV/h, followed by dynamic desorption by 60% ethanol solution. Further purification was achieved by high-speed counter-current chromatography using an optimized biphasic solvent system. A total of 267 mg of zearalenone crystal was obtained in one single run from 4.2 g of crude extract. The purity of the final product was 98.9% and the total recovery yield of zearalenone in this study was 73.9%. This dual-step purification procedure provided an effective way to obtain the costly mycotoxin for both toxicological and detoxification studies on zearalenone.

Mini-percutaneous nephrolithotomy for pediatric complex renal calculus disease: one-stage or two-stage?

OBJECTIVES: To compare two different treatment strategies, one-stage and two-stage multi-tract mini-percutaneous nephrolithotomy (mt-mPCNL), for pediatric complex renal calculus disease. METHODS: Between the period of July 2016 and July 2018, a total of 36 children aged 15 years and younger, with complex renal calculi disease, who underwent total ultrasound-guided mt-mPCNL by a single experienced urologist were enrolled in our study. All patients were assigned either to Group 1 (n = 18) who received one-stage mt-mPCNL or Group 2 (n = 18) who received planned two-stage mt-mPCNL. RESULTS: The demographic data were comparable between the two groups. There were no serious complications (Modified Clavien Grade ≥ III) observed in either group. The stone -free rate (SFR), operation time, postoperative creatinine increase, and perioperative complication rates were similar in both groups (P = 0.603, 0.818, 0.161, and 0.402, respectively). The postoperative hospital stay (5.8 days vs. 7.4 days) and cost (17373.3 CNY vs. 23717.1 CNY) were statistically less in Group 1. Group 2 had significantly less total estimated blood loss (70.6 ml vs. 130.0 ml, P < 0.001). The operation time of two cases in Group 1 with perioperative sepsis or systemic inflammatory response syndrome (SIRS) was more than two hours. CONCLUSIONS: Our preliminary results indicated that both one-stage and two-stage mt-mPCNL were safe and effective for pediatric complex renal calculi. Two-stage mt-mPCNL could significantly reduce blood loss; while one-stage mt-mPCNL could significantly decrease the length and costs of hospitalization. We also suggest that the planned two-stage mt-mPCNL should be applied in children with estimated operation time more than two hours.