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The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium Myxococcus xanthus harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the M. xanthus genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.

Operating system (OS) security has been considered as a critical layer within the comprehensive security framework of computer information systems. Mandatory access control (MAC) mechanisms such as SELinux and AppArmor have been employed to strengthen OS security. However, significant challenges have been encountered in the application of MAC in practice, primarily involving the complexity of security policy configuration, which has demanded specialized expertise and often resulted in coarse-grained protection measures. Aimed at enhancing the process of generating security policies for AppArmor, an automated access control policy generation method was proposed based on large language models (LLM). The process began with a static analysis of the target application to extract preliminary security policy rules. Subsequently, the application was extensively executed to gather logs that achieved maximum code coverage. Large models, in conjunction with the gathered log information, static analysis outcomes, and few-shot learning, were utilized to autonomously generate security policies for applications. This approach significantly reduced the dependence on security experts, lowered manual labor costs, and diminished the subjectivity and complexity associated with manual security policy configurations. The efficacy of this methodology was demonstrated through a comparative analysis between the AppArmor security policies generated by this framework and the default policies, focusing on the policies’ correctness, completeness, and succinctness. The experimental findings reveal that the generated policies are not only comprehensive and succinct but also do not impede the normal operation of the application. These results underscore the effectiveness of the proposed method in streamlining the security policy generation process and enhancing the quality of the policies.

Myxobacteria are well known for multicellular social behaviors and valued for biosynthesis of natural products. Myxobacteria social behaviors such as clumping growth severely hamper strain cultivation and genetic manipulation. Using Myxococcus xanthus DK1622, we engineered Hu04, which is deficient in multicellular behavior and pigmentation. Hu04, while maintaining nutritional growth and a similar metabolic background, exhibits improved dispersed growth, streamlining operational procedures. It achieves high cell densities in culture and is promising for synthetic biology applications.

ABSTRACT The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery.IMPORTANCEServing as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14–37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0–9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited β-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Al/TiB2-SiC composites were prepared based on TiB2-SiC porous ceramic preforms. The advantages of this work were characterized by the in-situ synthesized TiB2 in porous ceramic preforms which directly formed interconnected pores as well as TiB2-SiC ceramic skeletons during the one-step ceramic sintering. By controlling in-situ synthesized TiB2 volume fraction, it is possible to tailor TiB2-SiC preform structures so as to determine the structures and mechanical properties of Al/TiB2-SiC composites. The porous ceramic preforms with 20 vol% TiB2 addition achieved the highest porosity, the biggest pore size, but the lowest flexural strength. Al/TiB2-SiC composite with 20 vol% TiB2 addition showed the optimum mechanical properties. The improvement of mechanical properties of Al/TiB2-SiC composites was mainly ascribed to TiB2 additions as well as dense composite structures which were fully infiltrated with Al. This special interpenetrating structural feature and TiB2 addition also constituted an improved toughness through complex crack patterns such as branching, bridging, and deflection.

Two major obstacles for the practical application of lithium–sulfur batteries are sluggish redox kinetics and the shuttle effect of lithium polysulfides (LiPSs). Herein, MoN nanolayer-decorated multilayer graphene is fabricated via magnetron sputtering then serves as a multifunctional interlayer in Li–S batteries to suppress the shuttle effect and enhance redox kinetics. It is revealed that after the initial discharge process, the MoN layers break up into independent microreaction units consisting of MoN bodies and MoS2 edges, forming a heterogeneous composite catalyst in situ. The MoN bodies not only have high sulfur affinity to trap LiPSs but also enhance their redox kinetics. At the same time, the MoS2 edge weakens the mobility of LiPSs via the anchoring effect. As a result, Li–S cells using the interlayer present superior cycling stability under a high sulfur loading of 4.8 ​mg ​cm−2. This work may open a new avenue for developing high-performance Li–S batteries.

Merging the education model of clinical-type medical graduates into a double-track system, medical colleges and teaching hospitals are facing challenges of more requirements on cultivating graduates. In order to help the graduates to join in standardized resident training in an active attitude in order to strengthen their capacity building for learning and scientific research, this article discussed the development of integrated capacity during the training of clinical-type medical graduates from three aspects as clinical capacity, innovation spirit and personal quality.

Abstract Rational design hybrid nanostructure photocatalysts with efficient charge separation and transfer, and good solar light harvesting ability have critical significance for achieving high solar‐to‐chemical conversion efficiency. Here a highly active and stable composite photocatalyst is reported by integrating ultrathin ZnIn2S4 nanosheets on surface of hollow CdS cube to form the cube‐in‐cube structure. Experimental results combined with density functional theory calculations confirm that the Z‐scheme ZnIn2S4/CdS heterojunction is formed, which highly boosts the charge separation and transfer under the local‐electric‐field at semiconductor/semiconductor interface, and thus prolongs their lifetimes. Moreover, such a structure affords the highly enhanced light‐harvesting property. The optimized ZnIn2S4/CdS nanohybrids exhibit superior H2 generation rate under visible‐light irradiation (λ ≥ 420 nm) with excellent photochemical stability during 20 h continuous operation.

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

We sequenced and assembled eight complete plastid genomes from three closely related pleurocarpous moss families: Amblystegium serpens, Campyliadelphus stellatus, Cratoneuron filicinum, Drepanocladus aduncus, and Leptodictyum humile (Amblystegiaceae), Calliergon sarmentosum and Warnstorfia exannulata (Calliergonaceae), and Calliergonella cuspidata (Pylaisiaceae). The newly generated plastid genomes range from 124,256 to 124,819 bp, with two inverted repeat regions (9,624–9,696 bp) separated by a large single-copy region (86,422–86,924 bp) and a small single-copy region (18,430–18,514 bp). All these plastid genomes encode 116 unique genes including 82 protein-coding genes, 30 tRNA genes and four rRNAgenes. The overall GC content is between 28.6%–29.3%. Phylogenetic analysis showed that all Amblystegiaceae species Amblystegium serpens, Campyliadelphus stellatus, Cratoneuron filicinum, Drepanocladus aduncus, Leptodictyum humile, and Sanionia uncinata clustered in one clade, which is sister to the Pylaisiaceae species Calliergonella cuspidata. The two Calliergonaceae species Calliergon sarmentosum and Warnstorfia exannulata form a clade and is sister to Amblystegiaceae and Pylaisiaceae.

Summary Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti‐tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC‐subbranch with 160 GT sequences. We demonstrated that the YjiC‐subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC‐subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high‐active GTs (Q66 and P77) but changed in two low‐efficient GTs. Site‐directed residues swapping at the two positions in the two low‐active GTs (BssGT and BamGT) and the high‐active GT BsGT‐1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

Abstract Electrochemical irreversibility and sluggish mobility of Na+ in the cathode materials result in poor cycle stability and rate capability for sodium‐ion batteries. Herein, a new strategy of introducing Mg ions into the hinging sites of Mn‐based tunnel‐structured cathode material is designed. Highly reversible electrochemical reaction and phase transition in this cathode are realized. The resulted Na0.44Mn0.95Mg0.05O2 with Mg2+ in the hinging Mn‐O5 square pyramidal exhibits promising cycle stability and rate capability. At a current density of 2 C, 67% of the initial discharge capacity is retained after 800 cycles (70% at 20 C), much improved than the undoped Na0.44MnO2. The improvement is attribute to the enhanced Na+ diffusion kinetics and the lowered desodiation energy after Mg doping. Highly reversible charge compensation and structure evolution are proved by synchrotron‐based X‐ray techniques. Differential charge density and atom population analysis of the average electron number of Mn indicate that Na0.44Mn0.95Mg0.05O2 is more electron‐abundant in Mn 3d orbits near the Fermi level than that in Na0.44MnO2, leading to higher redox participation of Mn ions.

Exploring new anode materials is critical for the development of Sodium-ion batteries (SIBs). Herein, a binary-metal selenide NiCo2Se4 was synthesized and investigated as a new anode material for SIBs. After compositing with conductive carbon, the NiCo2Se4@C composite delivers a reversible capacity of 603.2 mAh g−1 with a high initial coulombic efficiency of 85.79% at 0.5 A g−1. At an ultrahigh current density of 2 A g−1, a reversible capacity of 377.5 mAh g−1 can still be obtained after 600 cycles. The detailed Na storage mechanism for NiCo2Se4 is revealed. After discharge, Na2Se, Ni and Co nanoparticles are formed and highly dispersed in Na2Se matrix. After recharge, NiCo2Se4 phase can be regenerated with small amount of CoSe2 and NiSe phases. The multi-phase coexistence after recharge is responsible for the initial capacity loss and the excellent cycle performance in subsequent cycles. Keywords: Sodium-ion batteries, Anode, Selenide, Reaction mechanism

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388), with unknown functional differentiation. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation but that the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth but significantly decreased the UV-radiation survival, homologous recombination (HR) ability, and induction of LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and increased the sensitivity to DNA damage caused by hydrogen peroxide but did not change the UV-radiation resistance or SOS gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange (DSE) and LexA autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved mainly to regulate gene expression for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for DNA HR and genome sophistication.

Abstract Background The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. Results In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. Conclusions Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

Abstract Background Microbial secondary metabolism is regulated by a complex and mostly-unknown network of global and pathway-specific regulators. A dozen biosynthetic gene clusters for secondary metabolites have been reported in myxobacteria, but a few regulation factors have been identified. Results We identified a transcription regulator Esi for the biosynthesis of epothilones. Inactivation of esi promoted the epothilone production, while overexpression of the gene suppressed the production. The regulation was determined to be resulted from the transcriptional changes of epothilone genes. Esi was able to bind, probably via the N-terminus of the protein, to an inverted repeat sequence in the promoter of the epothilone biosynthetic gene cluster. The Esi-homologous sequences retrieved from the RefSeq database are all of the Proteobacteria. However, the Esi regulation is not universal in myxobacteria, because the esi gene exists only in a few myxobacterial genomes. Conclusions Esi binds to the epothilone promoter and down-regulates the transcriptional level of the whole gene cluster to affect the biosynthesis of epothilone. This is the first transcription regulator identified for epothilone biosynthesis.

To investigate the correlations between SIRT1 gene polymorphisms and diabetic kidney disease (DKD). There were 150 patients with DKD in the observation group (urinary albumin excretion rate (UAER) ≥ 300 mg 24 h−1), and 160 patients with a more than 10 year history of type 2 diabetes but without retinopathy and DKD (UAER