Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment.

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella.

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.

Working from Home in Urban China during the COVID-19 Pandemic: Assemblages of Work-Family Interference.

During the COVID-19 pandemic, millions of workers globally have been forced to work from home. Empirical data from Chinese cities in the Hubei province reveal work productivity decreased among many respondents working from home in 2020, primarily due to family interference with work. Such interference stems not only from the domain of daily life but also from other family members' e-working and e-learning. Conversely, respondents' work interferes with family; thus, interference operates bi-directionally. This article proposes an analytical framework of work-family interference along three dimensions: work-daily life, work-work, work-study, and each dimension can be understood through four distinct aspects: temporality, physicality, vocality, digitality. Remote workers encounter 'assemblages of work-family interference', consisting of a heterogeneous mixture of these dimensions and aspects. Furthermore, some factors (e.g., living patterns, work culture, digital infrastructure) constrain effective work-family boundary management among urban households.

Attribution of Community Emergency Volunteer Behaviour During the COVID-19 Pandemic: A Study of Community Residents in Shanghai, China.

Based on grounded theory, the present study summarizes the transcripts from 32 in-depth interviews with Chinese community emergency volunteers to uncover the attributions of community emergency volunteering in China during the COVID-19 pandemic. Community emergency volunteering in China is affected by four main factors: inner awareness, the external environment, national policy, and publicity and advocacy. Among these factors, inner awareness and the external environment are the internal and social psychological attributions, respectively, of emergency volunteering. In addition, publicity and advocacy also play a role in both inner awareness and the external environment and, together with national policies, act on community emergency volunteering. Finally, the high level of trust of some volunteers in their ruling party and government is a deep-seated driving force of their volunteering, a factor that has not been emphasized in past studies.

Filamentous prophage capsid proteins contribute to superinfection exclusion and phage defence in Pseudomonas aeruginosa.

Filamentous prophages in Pseudomonas aeruginosa PAO1 are converted to superinfective phage virions during biofilm development. Superinfection exclusion is necessary for the development of resistance against superinfective phage virions in host cells. However, the molecular mechanisms underlying the exclusion of superinfective Pf phages are unknown. In this study, we found that filamentous prophage-encoded structural proteins allow exclusion of superinfective Pf phages by interfering with type IV pilus (T4P) function. Specifically, the phage minor capsid protein pVII inhibits Pf phage adsorption by interacting with PilC and PilJ of T4P, and overproduction of pVII completely abrogates twitching motility. The minor capsid protein pIII provides partial superinfection exclusion and interacts with the PilJ and TolR/TolA proteins. Furthermore, pVII provides full host protection against infection by pilus-dependent lytic phages, and pIII provides partial protection against infection by pilus-independent lytic phages. Considering that filamentous prophages are common in clinical Pseudomonas isolates and their induction is often activated during biofilm formation, this study suggests the need to rethink the strategy of using lytic phages to treat P. aeruginosa biofilm-related infections.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.

OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Dissemination and loss of a biofilm-related genomic island in marine Pseudoalteromonas mediated by integrative and conjugative elements.

Acquisition of genomic islands (GIs) plays a central role in the diversification and adaptation of bacteria. Some GIs can be mobilized in trans by integrative and conjugative elements (ICEs) or conjugative plasmids if the GIs carry specific transfer-related sequences. However, the transfer mechanism of GIs lacking such elements remains largely unexplored. Here, we investigated the transmissibility of a GI found in a coral-associated marine bacterium. This GI does not carry genes with transfer functions, but it carries four genes required for robust biofilm formation. Notably, this GI is inserted in the integration site for SXT/R391 ICEs. We demonstrated that acquisition of an SXT/R391 ICE results in either a tandem GI/ICE arrangement or the complete displacement of the GI. The GI displacement by the ICE greatly reduces biofilm formation. In contrast, the tandem integration of the ICE with the GI in cis allows the GI to hijack the transfer machinery of the ICE to excise, transfer and re-integrate into a new host. Collectively, our findings reveal that the integration of an ICE into a GI integration site enables rapid genome dynamics and a new mechanism by which SXT/R391 ICEs can augment genome plasticity.

Ultra-bright γ-ray flashes and dense attosecond positron bunches from two counter-propagating laser pulses irradiating a micro-wire target.

We propose a novel scheme to generate ultra-bright ultra-short γ-ray flashes and high-energy-density attosecond positron bunches by using multi-dimensional particle-in-cell simulations with quantum electrodynamics effects incorporated. By irradiating a 10 PW laser pulse with an intensity of 1023 W/cm2 onto a micro-wire target, surface electrons are dragged-out of the micro-wire and are effectively accelerated to several GeV energies by the laser ponderomotive force, forming relativistic attosecond electron bunches. When these electrons interact with the probe pulse from the other side, ultra-short γ-ray flashes are emitted with an ultra-high peak brightness of 1.8 × 1024 photons s-1mm-2mrad-2 per 0.1%BW at 24 MeV. These photons propagate with a low divergence and collide with the probe pulse, triggering the Breit-Wheeler process. Dense attosecond e-e+ pair bunches are produced with the positron energy density as high as 1017 J/m3 and number of 109. Such ultra-bright ultra-short γ-ray flashes and secondary positron beams may have potential applications in fundamental physics, high-energy-density physics, applied science and laboratory astrophysics.

New quantitative classification of the anatomical relationship between impacted third molars and the inferior alveolar nerve.

BACKGROUND: Before extracting impacted lower third molars, dentists must first identify the spatial relationship between the inferior alveolar nerve (IAN) and an impacted lower third molar to prevent nerve injury from the extraction. Nevertheless, the current method for describing the spatial relationship between the IAN and an impacted lower third molar is deficient. Therefore, the objectives of this study were to: (1) evaluate the relative position between impacted lower third molars and the IAN; and (2) investigate the relative position between impacted lower third molars and the IAN by using a cylindrical coordinate system. METHODS: From the radiology department's database, we selected computed tomography images of 137 lower third molars (from 75 patients) requiring removal and applied a Cartesian coordinate system by using Mimics, a medical imaging software application, to measure the distribution between impacted mandibular third molars and the IAN. In addition, the orientation of the lower third molar to the IAN was also measured, but by using a cylindrical coordinate system with the IAN as the origin. RESULTS: According to the Cartesian coordinate system, most of the IAN runs through the inferior side of the third molar (78.6 %), followed by the lingual side (11.8 %), and the buccal side (8.9 %); only 0.7 % is positioned between the roots. Unlike the Cartesian coordinate system, the cylindrical coordinate system clearly identified the relative position, r and θ, between the IAN and lower third molar. CONCLUSIONS: Using the cylindrical coordinate system to present the relationship between the IAN and lower third molar as (r, θ) might provide clinical practitioners with a more explicit and objective description of the relative position of both sites. However, comprehensive research and cautious application of this system remain necessary.

Study on the photoaging process and metal ion release of plastic films with two kinds of structures in marine environment: Aliphatic and aromatic polymers.

The prevalence of plastics in the oceans has significantly intensified microplastic pollution, contributing to broader marine secondary pollution issues. This paper examines how plastic structure affects the aging characteristics of plastics and the release of metal ions, to better understand this secondary source of marine pollution. This study simulate the photoaging of plastics in natural environments, focusing on aliphatic and aromatic polymers. The results showed that the photodegradation degree was higher for aliphatic than aromatic polymers. All polymers contained thirteen detectable metals, with their release increasing over time due to photoaging, The release dynamics of these metal ions correlated more strongly with the level of polymer degradation rather than with the polymer structure itself, adhering to a second-order kinetic model driven by surface and intraparticle diffusion processes. The results will help control and treat marine plastic pollution.

Tumor-infiltrating immune cells and survival in head and neck squamous cell carcinoma: a retrospective computational study.

The immune infiltration profiles of the tumor microenvironment have effects on the prognosis of head and neck squamous cell carcinoma (HNSCC). Whereas, HNSCC is a heterogeneous group of tumors, but past work has not taken this into consideration. Herein, we investigate the associations between survival and the function of immune cells in different tumorigenic sites of HNSCC. 1149 samples of HNSCC were collected from publicly accessible databases. Based on gene expression data, CIBERSORTx was applied to determine the proportion of 22 immune cell subpopulations. In the Cox regression model, the associations between overall survival, disease-free survival, and immune cells were examined, modeling gene expression and immune cell proportion as quartiles. Consensus cluster analysis was utilized to uncover immune infiltration profiles. Regardless of tumor sites, CD8+ T cells and activated CD4 memory T cells were associated with favorable survival, while eosinophils were the opposite. The survival of the hypopharynx, oral cavity, and larynx subsites was somewhat affected by immune cells, while the survival of the oropharynx subsite potentially was the most impacted. High expression of TIGIT, CIITA, and CXCR6 was linked to better survival, mainly in the oropharynx subsite. Immune cell clusters with four distinct survival profiles were discovered, of which the cluster with a high CD8+ T cell content had a better prognosis. The immune-infiltration pattern is related to the survival of HNSCC to varying degrees depending on the tumor sites; forthcoming studies into immune-mediated infiltration profiles will lay the groundwork for treating HNSCC with precision therapy.

Molecular basis of the phenotypic variants arising from a Pseudoalteromonas lipolytica mutator.

Bacterial deficiencies in the DNA repair system can produce mutator strains that promote adaptive microevolution. However, the role of mutator strains in marine Pseudoalteromonas, capable of generating various gain-of-function genetic variants within biofilms, remains largely unknown. In this study, inactivation of mutS in Pseudoalteromonas lipolytica conferred an approximately 100-fold increased resistance to various antibiotics, including ciprofloxacin, rifampicin and aminoglycoside. Furthermore, the mutator of P. lipolytica generated variants that displayed enhanced biofilm formation but reduced swimming motility, indicating a high phenotypic diversity within the ΔmutS population. Additionally, we observed a significant production rate of approximately 50 % for the translucent variants, which play important roles in biofilm formation, when the ΔmutS strain was cultured on agar plates or under shaking conditions. Using whole-genome deep-sequencing combined with genetic manipulation, we demonstrated that point mutations in AT00_17115 within the capsular biosynthesis cluster were responsible for the generation of translucent variants in the ΔmutS subpopulation, while mutations in flagellar genes fliI and flgP led to a decrease in swimming motility. Collectively, this study reveals a specific mutator-driven evolution in P. lipolytica, characterized by substantial genetic and phenotypic diversification, thereby offering a reservoir of genetic attributes associated with microbial fitness.

Genomic Island-Encoded Diguanylate Cyclase from Vibrio alginolyticus Regulates Biofilm Formation and Motility in Pseudoalteromonas.

Many bacteria use the second messenger c-di-GMP to regulate exopolysaccharide production, biofilm formation, motility, virulence, and other phenotypes. The c-di-GMP level is controlled by the complex network of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that synthesize and degrade c-di-GMP. In addition to chromosomally encoded DGCs, increasing numbers of DGCs were found to be located on mobile genetic elements. Whether these mobile genetic element-encoded DGCs can modulate the physiological phenotypes in recipient bacteria after horizontal gene transfer should be investigated. In our previous study, a genomic island encoding three DGC proteins (Dgc137, Dgc139, and Dgc140) was characterized in Vibrio alginolyticus isolated from the gastric cavity of the coral Galaxea fascicularis. Here, the effect of the three DGCs in four Pseudoalteromonas strains isolated from coral Galaxea fascicularis and other marine environments was explored. The results showed that when dgc137 is present rather than the three DGC genes, it obviously modulates biofilm formation and bacterial motility in these Pseudoalteromonas strains. Our findings implied that mobile genetic element-encoded DGC could regulate the physiological status of neighboring bacteria in a microbial community by modulating the c-di-GMP level after horizontal gene transfer.

Multicolor biosensor for hyaluronidase based on target-responsive hydrogel and etching of gold nanorods by H2O2.

Hyaluronidase (HAase) is a potential tumor biomarker for diseases of the digestive tract and nervous system, the development of simple and sensitive techniques for HAase determination is urgent needed. Gold nanorods (Au NRs) can be etched by H2O2 with high efficiency and display color changing. In this work, a HAase-responsive hydrogel system had been designed and the amount of H2O2 spilled from the system had a close relationship with the amount of HAase, then the spilled H2O2 had been applied to etch Au NRs. The color change of the solution was used to realize semi-quantitative determination of HAase. Furthermore, the longitudinal peak shift of Au NRs had a linear correlation with the concentration of HAase in the range of 10-60 U/mL (within 40 min) and the limit of detection (LOD) was 3.8 U/mL (S/N = 3), which can be used to realize accurate quantitative analysis of HAase. The proposed method has been applied to monitor HAase in serum of pancreatic cancer patients with satisfied results.

Comparative genomic insights into habitat adaptation of coral-associated Prosthecochloris.

Green sulfur bacteria (GSB) are a distinct group of anoxygenic phototrophic bacteria that are found in many ecological niches. Prosthecochloris, a marine representative genus of GSB, was found to be dominant in some coral skeletons. However, how coral-associated Prosthecochloris (CAP) adapts to diurnal changing microenvironments in coral skeletons is still poorly understood. In this study, three Prosthecochloris genomes were obtained through enrichment culture from the skeleton of the stony coral Galaxea fascicularis. These divergent three genomes belonged to Prosthecochloris marina and two genomes were circular. Comparative genomic analysis showed that between the CAP and non-CAP clades, CAP genomes possess specialized metabolic capacities (CO oxidation, CO2 hydration and sulfur oxidation), gas vesicles (vertical migration in coral skeletons), and cbb 3-type cytochrome c oxidases (oxygen tolerance and gene regulation) to adapt to the microenvironments of coral skeletons. Within the CAP clade, variable polysaccharide synthesis gene clusters and phage defense systems may endow bacteria with differential cell surface structures and phage susceptibility, driving strain-level evolution. Furthermore, mobile genetic elements (MGEs) or evidence of horizontal gene transfer (HGT) were found in most of the genomic loci containing the above genes, suggesting that MGEs play an important role in the evolutionary diversification between CAP and non-CAP strains and within CAP clade strains. Our results provide insight into the adaptive strategy and population evolution of endolithic Prosthecochloris strains in coral skeletons.

Microscopic dissolution process of cellulose in alkaline aqueous solvents and its application in CNFs extraction - Investigating temperature as a variable.

The target of this study is to gain a deeper understanding of the micro-dissolution process of cellulose in alkaline aqueous solutions and to develop a novel method for extracting cellulose nanofibrils (CNFs). Herein, the dissolution process of cellulose in alkaline aqueous solutions will be controlled by varying the temperature, and the undissolved cellulose will be analyzed to reveal the microscopic dissolution process of cellulose, and a novel process for extracting cellulose nanofibrils (CNFs) will be developed based on the findings. The crystalline structure of cellulose was gradually disrupted as the dissolution progressed, and the crystal form of cellulose changed gradually from cellulose I to cellulose II during the dissolution process, while all undissolved cellulose crystals remained as cellulose I. Cellulose, after its structure is disrupted during the dissolution process, will inevitably decompose into CNFs, and the microscopic dissolution process of cellulose follows a "top-down" dissolution sequence. The CNFs extraction method developed in this study can extract CNFs with high yield (>60 %) in a stable manner, as well as narrow particle size distribution, high crystallinity (>77 %), and good thermal stability. This study enhances the comprehension of the dissolution process of cellulose and paves a possible way for industrialization of CNFs production.

Gut microbial structural variation associates with immune checkpoint inhibitor response.

The gut microbiota may have an effect on the therapeutic resistance and toxicity of immune checkpoint inhibitors (ICIs). However, the associations between the highly variable genomes of gut bacteria and the effectiveness of ICIs remain unclear, despite the fact that merely a few gene mutations between similar bacterial strains may cause significant phenotypic variations. Here, using datasets from the gut microbiome of 996 patients from seven clinical trials, we systematically identify microbial genomic structural variants (SVs) using SGV-Finder. The associations between SVs and response, progression-free survival, overall survival, and immune-related adverse events are systematically explored by metagenome-wide association analysis and replicated in different cohorts. Associated SVs are located in multiple species, including Akkermansia muciniphila, Dorea formicigenerans, and Bacteroides caccae. We find genes that encode enzymes that participate in glucose metabolism be harbored in these associated regions. This work uncovers a nascent layer of gut microbiome heterogeneity that is correlated with hosts' prognosis following ICI treatment and represents an advance in our knowledge of the intricate relationships between microbiota and tumor immunotherapy.

The Secondary Metabolites of Bacillus subtilis Strain Z15 Induce Apoptosis in Hepatocellular Carcinoma Cells.

The lipopeptides produced by Bacillus subtilis have anti-cancer potential. We had previously identified a secondary metabolite of B. subtilis strain Z15 (BS-Z15), which has an operon that regulates lipopeptide synthesis, and also demonstrated that the fermentation products of this strain exerted antioxidant and pro-immune effects. The purpose of this study was to investigate in vitro and in vivo the anticancer effects of BS-Z15 secondary metabolites (BS-Z15 SMs) on hepatocellular carcinoma (HCC) cells. BS-Z15 SMs significantly inhibited H22 cell-derived murine xenograft tumor growth without any systemic toxicity. In addition, BS-Z15 SMs decreased the viability of H22 cells and BEL-7404 cells in vitro with respective IC50 values of 33.83 and 27.26 µg/mL. Consistent with this, BS-Z15 SMs induced apoptosis and G0/G1 phase arrest in the BEL-7404 cells, and the mitochondrial membrane potential was also significantly reduced in a dose-dependent manner. Mechanistically, BS-Z15 SMs upregulated the pro-apoptotic p53, Bax, cytochrome C, and cleaved-caspase-3/9 proteins and downregulated the anti-apoptotic Bcl-2. These findings suggest that the induction of apoptosis in HCC cells by BS-Z15 SMs may be related to the mitochondrial pathway. Thus, the secondary metabolites of B. subtilis strain Z15 are promising to become new anti-cancer drugs for the clinical treatment of liver cancer.