In vitro characterization of alternative l-threonate and d-erythronate catabolic pathways.

l-threonate is the metabolite of vitamin C, while d-erythronate is the metabolite of N-acetyl-d-glucosamine, the nutritional supplement for joint health. They are widely distributed in the environment and human biofluids. Nevertheless, the catabolisms of l-threonate and d-erythronate are sparsely reported. Here we explored the functional diversity of an acid sugar kinase family (Pfam families PF07005-PF17042), and discovered a novel 2-oxo-tetronate kinase. The conserved genome neighborhood of the 2-oxo-tetronate kinase encodes members of class-II fructose-bisphosphate aldolase family (F_bP_aldolase, PF01116) and a dehydrogenase family (PF03446-PF14833). Instructed by this analysis, we experimentally verified that these enzymes are capable of degrading l-threonate into dihydroxyacetone phosphate (DHAP) in Arthrobacter sp. ZBG10, Clostridium scindens ATCC 35704, and Pseudonocardia dioxanivorans ATCC 55486. Meanwhile, a convergent catabolic pathway for d-erythronate was characterized in P. dioxanivorans ATCC 55486. Moreover, the phylogenetic distribution analysis indicates that the biological range of the identified l-threonate and d-erythronate catabolic pathways appears to extend mostly to members of the Actinomycetota, Cyanobacteriota, Bacillota, Pseudomonadota, and Bacteroidota phyla.

Transcriptomic and metabolomic analyses to study the key role by which Ralstonia insidiosa induces Listeria monocytogenes to form suspended aggregates.

Ralstonia insidiosa can survive in a wide range of aqueous environments, including food processing areas, and is harmful to humans. It can induce Listeria monocytogenes to form suspended aggregates, resulting from the co-aggregation of two bacteria, which allows for more persistent survival and increases the risk of L. monocytogenes contamination. In our study, different groups of aggregates were analyzed and compared using Illumina RNA sequencing technology. These included R. insidiosa under normal and barren nutrient conditions and in the presence or absence of L. monocytogenes as a way to screen for differentially expressed genes (DEGs) in the process of aggregate formation. In addition, sterile supernatants of R. insidiosa were analyzed under different nutrient conditions using metabolomics to investigate the effect of nutrient-poor conditions on metabolite production by R. insidiosa. We also undertook a combined analysis of transcriptome and metabolome data to further investigate the induction effect of R. insidiosa on L. monocytogenes in a barren environment. The results of the functional annotation analysis on the surface of DEGs and qPCR showed that under nutrient-poor conditions, the acdx, puuE, and acs genes of R. insidiosa were significantly upregulated in biosynthetic processes such as carbon metabolism, metabolic pathways, and biosynthesis of secondary metabolites, with Log2FC reaching 4.39, 3.96, and 3.95 respectively. In contrast, the Log2FC of cydA, cyoB, and rpsJ in oxidative phosphorylation and ribosomal pathways reached 3.74, 3.87, and 4.25, respectively. Thirty-one key components were identified while screening for differential metabolites, which mainly included amino acids and their metabolites, enriched to the pathways of biosynthesis of amino acids, phenylalanine metabolism, and methionine metabolism. Of these, aminomalonic acid and Proximicin B were the special components of R. insidiosa that were metabolized under nutrient-poor conditions.

High-affinity SOAT1 ligands remodeled cholesterol metabolism program to inhibit tumor growth.

BACKGROUND: Although cholesterol metabolism is a common pathway for the development of antitumor drugs, there are no specific targets and drugs for clinical use. Here, based on our previous study of sterol O-acyltransferase 1 (SOAT1) in hepatocelluar carcinoma, we sought to screen an effective targeted drug for precise treatment of hepatocelluar carcinoma and, from the perspective of cholesterol metabolism, clarify the relationship between cholesterol regulation and tumorigenesis and development. METHODS: In this study, we developed a virtual screening integrated affinity screening technology for target protein drug screening. A series of in vitro and in vivo experiments were used for drug activity verification. Multi-omics analysis and flow cytometry analysis were used to explore antitumor mechanisms. Comparative analysis of proteome and transcriptome combined with survival follow-up information of patients reveals the clinical therapeutic potential of screened drugs. RESULTS: We screened three compounds, nilotinib, ABT-737, and evacetrapib, that exhibited optimal binding with SOAT1. In particular, nilotinib displayed a high affinity for SOAT1 protein and significantly inhibited tumor activity both in vitro and in vivo. Multi-omics analysis and flow cytometry analysis indicated that SOAT1-targeting compounds reprogrammed the cholesterol metabolism in tumors and enhanced CD8+ T cells and neutrophils to suppress tumor growth. CONCLUSIONS: Taken together, we reported several high-affinity SOAT1 ligands and demonstrated their clinical potential in the precision therapy of liver cancer, and also reveal the potential antitumor mechanism of SOAT1-targeting compounds.

Biosynthesis of Platinum Nanoparticles with Cordyceps Flower Extract: Characterization, Antioxidant Activity and Antibacterial Activity.

The aim of this work is to develop a green route for platinum nanoparticles (PtNPs) biosynthesized using Cordyceps flower extract and to evaluate their antioxidant activity and antibacterial activity. Different characterization techniques were utilized to characterize the biosynthetic PtNPs. The results showed that PtNPs were spherical particles covered with Cordyceps flower extract. The average particle size of PtNPs in Dynamic Light Scattering was 84.67 ± 5.28 nm, while that of PtNPs in Transmission Electron Microscope was 13.34 ± 4.06 nm. Antioxidant activity of PtNPs was evaluated by DPPH free radical scavenging ability test. The results showed that the antioxidant activity was positively correlated with the concentration of PtNPs, the DPPH scavenging efficiency of PtNPs (0.50-125.00 µg/mL) was 27.77-44.00%. In addition, the morphological changes of four kinds of bacteria (Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Staphylococcus aureus) exposed to PtNPs were observed by scanning electron microscope. The results showed that the antibacterial activity of PtNPs against Gram-negative bacteria was stronger than that of Gram-positive bacteria.

Identification of catabolic pathway for 1-deoxy-D-sorbitol in Bacillus licheniformis.

1-Deoxy-D-sorbitol, the 1-deoxy analogue of D-sorbitol, has been detected in human urine as well as in natural herbs and spices. Although there are sporadic reports about 1-deoxy-D-sorbitol dehydrogenase, the complete catabolic pathway of 1-deoxy-D-sorbitol remains unsolved. Informed by the promiscuous activities of fructose-6-phosphate aldolase (FSA) which is involved in the sorbitol (glucitol) utilization (gut) operon and guided by the large scale bioinformatics analysis, we predicted and then experimentally verified the gut operon encoded by Bacillus licheniformis ATCC14580 is responsible for the catabolism of both D-sorbitol and 1-deoxy-D-sorbitol by in vitro activity assays of pathway enzymes, in vivo growth phenotypes, and transcriptomic studies. Moreover, the phylogenetic distribution analysis suggests that the D-sorbitol and 1-deoxy-D-sorbitol catabolic gene cluster is mostly conserved in members of Firmicutes phylum.

In Vitro Reconstitution of the Pantothenic Acid Degradation Pathway in Ochrobactrum anthropi.

Pantothenic acid is an essential metabolite found throughout all branches of life. Although the enzymes responsible for pantothenic biosynthesis have been characterized, those leading to its biodegradation remain poorly understood. In the study described herein, we showed that use of a "genomic enzymology" strategy enabled identification of four biodegradation pathway genes, which were then confirmed by using kinetic analysis of the purified recombinant enzymes encoded in Ochrobactrum anthropi. The reconstituted pathway converts pantothenic acid to ß-alanine and (R)-pantoate, and then (R)-pantoate to aldopentoate, which is transformed to (R)-3,3-dimethylmalate and hence to α-ketoisovalerate. The pathway genes are common to Proteobacterial genomes in which they are not colocated.

Formation of Multispecies Biofilms and Their Resistance to Disinfectants in Food Processing Environments: A Review.

ABSTRACT: In food processing environments, various microorganisms can adhere and aggregate on the surface of equipment, resulting in the formation of multispecies biofilms. Complex interactions among microorganisms may affect the formation of multispecies biofilms and resistance to disinfectants, which are food safety and quality concerns. This article reviews the various interactions among microorganisms in multispecies biofilms, including competitive, cooperative, and neutral interactions. Then, the preliminary mechanisms underlying the formation of multispecies biofilms are discussed in relation to factors, such as quorum-sensing signal molecules, extracellular polymeric substances, and biofilm-regulated genes. Finally, the resistance mechanisms of common contaminating microorganisms to disinfectants in food processing environments are also summarized. This review is expected to facilitate a better understanding of interspecies interactions and provide some implications for the control of multispecies biofilms in food processing.

Whole genome sequencing and genome annotation of Dermacoccus abyssi strain HZAU 226 isolated from spoiled eggs.

Dermacoccus abyssi strain HZAU 226 is a spoilage bacterium isolated from eggs. So far, there are still few genomic resources available on the Dermacoccus abyssi. Here, we reported the complete genome sequence of Dermacoccus abyssi strain HZAU 226. High-quality DNA was extracted using the Qiagen kit, then single-molecule sequencing was performed by GridION sequencer. The raw data was quality-controlled and assembled to obtain the final genome, which consisted of a complete genome of 2,992,060 bp circular chromosome and a 64,524 bp plasmid. The structural and functional annotations of the genome were achieved through the analysis of different available databases, including antibiotic resistance genes, secondary metabolite synthesis genes and stress-related genes. Meanwhile, comparative genomic analyses of the strains were also performed. This is the first report on the complete genome of Dermacoccus abyssi, which will provide genomic resources for the study of spoilage bacteria in eggs.

Transcriptome Analysis of Gene Expression in Dermacoccus abyssi HZAU 226 under Lysozyme Stress.

Lysozyme acts as a kind of cationic antimicrobial protein and effectively hydrolyzes bacterial peptidoglycan to have a bactericidal effect, which also plays an important role in protecting eggs from microbial contamination. Dermacoccus abyssi HZAU 226, a Gram-positive bacterium isolated from spoiled eggs, has egg white and lysozyme tolerance, but its survival mechanism is unknown, especially from a transcriptomics point of view. In this study, the high lysozyme tolerance of D. abyssi HZAU 226 was characterized by three independent experiments, and then the Illumina RNA-seq was used to compare the transcriptional profiles of this strain in Luria-Bertani (LB) medium with and without 5 mg/mL lysozyme to identify differentially expressed genes (DEGs); 1024 DEGs were identified by expression analysis, including 544 up-regulated genes and 480 down-regulated genes in response to lysozyme treatment. The functional annotation analysis results of DEGs showed that these genes were mainly involved in glutathione biosynthesis and metabolism, ion transport, energy metabolism pathways, and peptidoglycan biosynthesis. This study is the first report of bacterial-related lysozyme RNA-seq, and our results help in understanding the lysozyme-tolerance mechanism of bacteria from a new perspective and provide transcriptome resources for subsequent research in related fields.

Differential Effects of Growth Medium Salinity on Biofilm Formation of Two Salmonella enterica Strains.

ABSTRACT: Salmonella enterica is a prominent foodborne pathogen, including diverse serotypes that are prolific biofilm formers. Its ability to form biofilm can be affected by multiple environmental factors. In this study, the effect of salinity on biofilm formation by S. enterica was evaluated by using two recently isolated strains of Salmonella serotypes Enteritidis and Newport. Although supplementing the growth medium with a low concentration (0.5 to 2%) of sodium chloride (NaCl) slightly enhanced biofilm formation for the strain S. enterica serovar Enteritidis 110, it sharply reduced or abolished biofilm formation by the strain S. enterica serovar Newport 193. This differential effect of salinity on S. enterica strains of different serotypes was poorly correlated with inhibition of planktonic growth but strongly correlated with cell motility. Examining genes known to affect biofilm formation showed that the expression of adrA, csgD, and fliC, which encode proteins required for surface adhesion and cell motility, was significantly downregulated with salinity increase in Salmonella Newport 193 but not in Salmonella Enteritidis 110. Therefore, it is plausible that the differential effect of salinity on biofilm formation by Salmonella Enteritidis 110 and Salmonella Newport 193 resulted from the differential regulation to genes required for cell adherence and motility.

Functional assignment of multiple catabolic pathways for D-apiose.

Colocation of the genes encoding ABC, TRAP, and TCT transport systems and catabolic pathways for the transported ligand provides a strategy for discovering novel microbial enzymes and pathways. We screened solute-binding proteins (SBPs) for ABC transport systems and identified three that bind D-apiose, a branched pentose in the cell walls of higher plants. Guided by sequence similarity networks (SSNs) and genome neighborhood networks (GNNs), the identities of the SBPs enabled the discovery of four catabolic pathways for D-apiose with eleven previously unknown reactions. The new enzymes include D-apionate oxidoisomerase, which catalyzes hydroxymethyl group migration, as well as 3-oxo-isoapionate-4-phosphate decarboxylase and 3-oxo-isoapionate-4-phosphate transcarboxylase/hydrolase, which are RuBisCO-like proteins (RLPs). The web tools for generating SSNs and GNNs are publicly accessible ( http://efi.igb.illinois.edu/efi-est/ ), so similar 'genomic enzymology' strategies for discovering novel pathways can be used by the community.

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.

Members of a Novel Kinase Family (DUF1537) Can Recycle Toxic Intermediates into an Essential Metabolite.

DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-l-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5'-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate l-4-hydroxythreonine (4HT) into 4-phosphohydroxy-l-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms.

Fabrication and Photovoltaic Characteristics of Silicon Nanoscrew and Nanohole Based Solar Cells.

A simple fabrication technology for both nanoscrews and nanoholes by Cesium Chloride (CsCI) self-assembly lithography and dry etching on silicon substrates is demonstrated. The porous Al template ranging from 400 nm to 2 µm average diameter is formed by lift-off the CsCl nanoislands in DI water as the ICP etching masks for nanoholes. Nanoscrews and nanoholes of desired height/depth from 1.2 to 4 µm are obtained by this method. The reflectance of nanoscrew and nanohole structures can achieve below 5% from wavelength of 400 to 1000 nm which is much lower than that of pyramid but the performance of pyramid solar cells is the best. Results show that the performance of nanoscrew and nanohole solar cells is strongly affected by some other factors such as surface passivation and electrode-contact property. Therefore, not only the antireflection advantage but also the surface passivation and improving the electrode-contact property should be considered together to improve nanostructure solar cells photovoltaic performance.

A unique cis-3-hydroxy-l-proline dehydratase in the enolase superfamily.

The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8 ). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7 ; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9 ; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ(1)-pyrroline-2-carboxylate (ß-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.

Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

An efficient identity-based key management scheme for wireless sensor networks using the Bloom filter.

With the rapid development and widespread adoption of wireless sensor networks (WSNs), security has become an increasingly prominent problem. How to establish a session key in node communication is a challenging task for WSNs. Considering the limitations in WSNs, such as low computing capacity, small memory, power supply limitations and price, we propose an efficient identity-based key management (IBKM) scheme, which exploits the Bloom filter to authenticate the communication sensor node with storage efficiency. The security analysis shows that IBKM can prevent several attacks effectively with acceptable computation and communication overhead.

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks.

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ∼85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

Synthesis of benzoxazoles via an amine-catalyzed [4 + 1] annulation.

An unprecedented simple pyrrolidine catalyzed [4 + 1] annulation reaction of ynals with N-protected-2-aminophenols is reported. The utilization of the unique property and reactivity of the C≡C triple bond in ynals leads to two consecutive conjugate addition reactions at the same ß-position with pyrrolidine via iminium activation. The powerful cascade process affords a new alternative approach to biologically and synthetically important benzoxazoles in high yields (83-95%).

Quinine-thiourea catalyzed enantioselective hydrophosphonylation of trifluoromethyl 2(1H)-quinazolinones.

An organocatalytic enantioselective addition reaction of cyclic ketoimines with phosphites has been developed for the first time. The process, catalyzed by Soós' quinine thiourea, affords synthetically and medicinally interesting enantioenriched trifluoromethyl dihydroquinazolinones in high yields and with high enantioselectivities.