Bridging Nickel-MOF and Copper Single Atoms/Clusters with H-Substituted Graphdiyne for the Tandem Catalysis of Nitrate to Ammonia.

Interfacial engineering of synergistic catalysts is one of the keys to achieving multiple proton-coupled electron transfer processes in nitrate-to-ammonia conversion. Herein, by joining ultrathin nickel-based metal-organic framework (denoted Ni-MOF) nanosheets with few-layered hydrogen-substituted graphdiyne-supported copper single atoms and clusters (denoted HsGDY@Cu), a tandem catalyst of Ni-MOFs@HsGDY@Cu with dual-active interfaces was developed for the concerted catalysis of nitrate-to-ammonia. In such a system, the sandwiched HsGDY layer could serve as a bridge to connect the coordinated unsaturated Ni2+ sites with Cu single atoms/clusters in a limited range of 0 to 3.6 nm. From Ni2+ to Cu, via the hydrogen spillover process, the hydrogen radicals (H·) generated at the unsaturated Ni2+ sites could migrate across HsGDY to the Cu sites to participate in the transformation of *HNO3 to NH3. From Cu to Ni2+, bypassing the higher reaction energy for *HNO3 formation on the Ni2+ sites, the NO2- detached from the Cu sites could diffuse onto the unsaturated Ni2+ sites to form NH3 as well. The combined results make this hybrid a tandem catalyst with dual active sites for the catalysis of nitrate-to-ammonia conversion with improved Faradaic efficiency at lower overpotentials.

Development of toluidine red particle agglutination-based turbidimetric immunoassay for anticardiolipin antibody detection in syphilis.

INTRODUCTION: Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Nontreponemal tests are used to monitor disease activity. Toluidine red unheated serum test (TRUST), as one of nontreponemal tests, is generally applicable to hospitals at different levels. However, accurate judgment of TRUST results is inseparable from an experienced and accurate operator. To reduce current shortcomings of manual TRUST method, we attempted to convert the manual TRUST test into automatic TRUST test, that is, to determine the degree of aggregation of toluidine red particles by detecting the absorbance value of serum after reaction with toluidine red particles. METHODS: 50 µL of serum sample and 80 µL toluidine red particles were added to 96-well plate. Then, the 96-well plate was placed on a microplate reader at medium grade for 8 min to mix. Then, plasma reagin reacted with toluidine red particles and promoted the aggregation of toluidine red particles to form a large clot, which eventually caused a decrease in the absorbance at 540 nm. RESULTS: The results showed that the specificity of the automatic TRUST test was 100%, the sensitivity was 87%. And this method showed 93.5% correlation with manual TRUST test. The developed method is simple and involves less subjectivity in reading results, opening new avenues for syphilis diagnostic testing. CONCLUSION: Turbidimetric immunoassay can avoid the shortcomings of subjective interpretation, time-consuming and manual operation of manual TRUST method, and is more suitable for large-scale screening in health examination.

Improving gene set enrichment analysis (GSEA) by using regulation directionality.

To infer the biological meaning from transcriptome data, it is useful to focus on genes that are regulated by the same regulator, i.e., regulons. Unfortunately, current gene set enrichment analysis (GSEA) tools do not consider whether a gene is activated or repressed by a regulator. This distinction is crucial when analyzing regulons since a regulator can work as an activator of certain genes and as a repressor of other genes, yet both sets of genes belong to the same regulon. Therefore, simply averaging expression differences of the genes of such a regulon will not properly reflect the activity of the regulator. What makes it more complicated is the fact that many genes are regulated by different transcription factors, and current transcriptome analysis tools are unable to indicate which regulator is most likely responsible for the observed expression difference of a gene. To address these challenges, we developed the gene set enrichment analysis program GINtool. Additional features of GINtool are novel graphical representations to facilitate the visualization of gene set analyses of transcriptome data, the possibility to include functional categories as gene sets for analysis, and the option to analyze expression differences within operons, which is useful when analyzing prokaryotic transcriptome and also proteome data.IMPORTANCEMeasuring the activity of all genes in cells is a common way to elucidate the function and regulation of genes. These transcriptome analyses produce large amounts of data since genomes contain thousands of genes. The analysis of these large data sets is challenging. Therefore, we developed a new software tool called GINtool that can facilitate the analysis of transcriptome data by using prior knowledge of gene sets controlled by the same regulator, the so-called regulons. An important novelty of GINtool is that it can take into account the directionality of gene regulation in these analyses, i.e., whether a gene is activated or repressed, which is crucial to assess whether a regulon or functional category is affected. GINtool also includes new graphical methods to facilitate the visual inspection of regulation events in transcriptome data sets. These and additional analysis methods included in GINtool make it a powerful software tool to analyze transcriptome data.

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

BACKGROUND: The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-ß-xylanase XynA on the B. subtilis transcriptome using RNA-seq. RESULTS: Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%. CONCLUSIONS: Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant.

IMPORTANCE: WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

Interfacial Engineering of Bimetallic Ni/Co-MOFs with H-Substituted Graphdiyne for Ammonia Electrosynthesis from Nitrate.

The electrochemical synthesis of ammonia is highly dependent on the coupling reaction between nitrate and water, for which an electrocatalyst with a multifunctional interface is anticipated to promote the deoxygenation and hydrogenation of nitrate with water. Herein, by engineering the surface of bimetallic Ni/Co-MOFs (NiCoBDC) with hydrogen-substituted graphdiyne (HsGDY), a hybrid nanoarray of NiCoBDC@HsGDY with a multifunctional interface has been achieved toward scale-up of the nitrate-to-ammonia conversion. On the one hand, a partial electron transfers from Ni2+ to the coordinatively unsaturated Co2+ on the surface of NiCoBDC, which not only promotes the deoxygenation of *NO3 on Co2+ but also activates the water-dissociation to *H on Ni2+. On the other hand, the conformal coated HsGDY facilitates both electrons and NO3- ions gathering on the interface between NiCoBDC and HsGDY, which moves forward the rate-determining step from the deoxygenation of *NO3 to the hydrogenation of *N with both *H on Ni2+ and *H2O on Co2+. As a result, such a NiCoBDC@HsGDY nanoarray delivers high NH3 yield rates with Faradaic efficiency above 90% over both wide potential and pH windows. When assembled into a galvanic Zn-NO3- battery, a power density of 3.66 mW cm-2 is achieved, suggesting its potential in the area of aqueous Zn-based batteries.

Hybrid nanoarrays of Cu-MOFs@H-substituted graphdiyne with various levels of Lewis acidity for nitrate electroreduction.

To mimic enzymes in nature, a set of hybrid nanoarrays of Cu-MOFs sealed in hydrogen-substituted graphdiyne has been developed in order to serve as Lewis-acid-promoted catalysts. By regulating the electron-withdrawing capability of the ligands bridging Cu2+ sites, these Cu-MOFs provided different levels of Lewis acidity toward nitrate affinity, a feature crucial for nitrate-to-ammonia conversion.

Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA.

Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.

A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

Different Resource Allocation in a Bacillus subtilis Population Displaying Bimodal Motility.

To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially. This is one reason why bimodal differentiation is primarily observed from the onset of the stationary phase, when exponential growth ceases. An exception is the bimodal induction of motility in Bacillus subtilis, which occurs early during exponential growth. Several mechanisms have been put forward to explain this, including double-negative feedback regulation and the stability of the mRNA molecules involved. In this study, we used fluorescence-assisted cell sorting (FACS) to compare the transcriptomes of motile and nonmotile cells and noted that expression of ribosomal genes is lower in motile cells. This was confirmed using an unstable green fluorescent protein (GFP) reporter fused to the strong ribosomal rpsD promoter. We propose that the reduction in ribosomal gene expression in motile cells is the result of a diversion of cellular resources to the synthesis of the chemotaxis and motility systems. In agreement with this, single-cell microscopic analysis showed that motile cells are slightly shorter than nonmotile cells, an indication of slower growth. We speculate that this growth rate reduction can contribute to the bimodal induction of motility during exponential growth. IMPORTANCE To cope with sudden environmental changes, bacteria can use a bet-hedging strategy and generate different types of cells within a population-so-called bimodal differentiation. For example, a Bacillus subtilis culture can contain both motile and nonmotile cells. In this study, we compared the gene expression between motile and nonmotile cells. It appeared that motile cells express fewer ribosomes. To confirm this, we constructed a ribosomal promoter fusion that enabled us to measure expression of this promoter in individual cells. This reporter fusion confirmed our initial finding. The reallocation of cellular resources from ribosome synthesis toward synthesis of the motility apparatus results in a reduction in growth. Interestingly, this growth reduction has been shown to stimulate bimodal differentiation.

Immuno-Stimulatory Activity of Escherichia coli Mutants Producing Kdo2-Monophosphoryl-Lipid A or Kdo2-Pentaacyl-Monophosphoryl-Lipid A.

Lipid A is the active center of lipopolysaccharide which also known as endotoxin. Monophosphoryl-lipid A (MPLA) has less toxicity but retains potent immunoadjuvant activity; therefore, it can be developed as adjuvant for improving the strength and duration of the immune response to antigens. However, MPLA cannot be chemically synthesized and can only be obtained by hydrolyzing lipopolysaccharide (LPS) purified from Gram-negative bacteria. Purifying LPS is difficult and time-consuming and can damage the structure of MPLA. In this study, Escherichia coli mutant strains HWB01 and HWB02 were constructed by deleting several genes and integrating Francisella novicida gene lpxE into the chromosome of E. coli wild type strain W3110. Compared with W3110, HWB01 and HWB02 synthesized very short LPS, Kdo2-monophosphoryl-lipid A (Kdo2-MPLA) and Kdo2-pentaacyl-monophosphoryl-lipid A (Kdo2-pentaacyl-MPLA), respectively. Structural changes of LPS in the outer membranes of HWB01 and HWB02 increased their membrane permeability, surface hydrophobicity, auto-aggregation ability and sensitivity to some antibiotics, but the abilities of these strains to activate the TLR4/MD-2 receptor of HKE-Blue hTLR4 cells were deceased. Importantly, purified Kdo2-MPLA and Kdo2-pentaacyl-MPLA differed from wild type LPS in their ability to stimulate the mammalian cell lines THP-1 and RAW264.7. The purification of Kdo2-MPLA and Kdo2-pentaacyl-MPLA from HWB01 and HWB02, respectively, is much easier than the purification of LPS from W3110, and these lipid A derivatives could be important tools for developing future vaccine adjuvants.

Structure characterization of phospholipids and lipid A of Pseudomonas putida KT2442.

Pseudomonas putida KT2442 is an important bacterium for producing various types of polyhydroxyalkanoate polymers. Phospholipids and lipid A in membranes of P. putida play important roles in stress responses, but detailed structural information of these lipids is not known. In this study, phospholipids and lipid A were isolated from P. putida KT2442, and their structures were analyzed using thin layer chromatography, high performance liquid chromatography, and electrospray ionization/mass spectrometry. Major phospholipids in P. putida KT2442 were phosphatidylethanolamine (79.9%), phosphatidylglycero1 (12.7%), and cardiolipin (7.4%), with C16:1 and/or C18:1 acyl chains. Four lipid A species were found in P. putida KT2442: two are hexa-acylated, and the other two are penta-acylated. Compared with lipid A of P. aeruginosa, P. putida lipid A has less hydroxylation on the secondary acyl chains and less modification. Therefore, P. putida lipid A could be used as a base structure to investigate lipid A modification of P. aeruginosa for understanding its pathogenesis.

Evaluation the quality characteristics of wheat flour and shelf-life of fresh noodles as affected by ozone treatment.

In this study, the effects of ozone treatment on the microorganism mortality in wheat flour and shelf-life of fresh noodles were investigated, as well as the physicochemical properties of wheat flour and textural qualities of cooked noodles. Results showed that the total plate count (TPC) can be largely reduced in wheat flour exposed to ozone gas for 30 min and 60 min. Whiteness of flour and noodle sheet, dough stability, and peak viscosity of wheat starch were all increased by ozone treatment. Free cysteine content in wheat flour was shown to decrease significantly (P<0.05) as the treatment time increased and remarkable protein aggregates were observed in both reduced and non-reduced SDS-PAGE patterns. In addition, ozone treated noodles were generally higher in firmness, springiness, and chewiness, while lower in adhesiveness. Microbial growth and darkening rate of fresh noodles made from ozone treated flour were delayed significantly.