Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a.

Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE Enterococcus faecium is increasingly associated with hard-to-treat antibiotic-resistant infections. The ability to generate clean genomic alterations is the first step in generating a complete mechanistic understanding of how E. faecium acquires pathogenic traits and causes disease. Here, we show that CRISPR-Cas12a can be used to quickly (under 2 weeks) and cheaply delete or insert genes into the E. faecium genome. This substantial improvement over current methods should speed up research on this important opportunistic pathogen.

Escherichia fergusonii, an Underrated Repository for Antimicrobial Resistance in Food Animals.

A total of 1,400 samples of food animals (pigs, chickens, and ducks) were collected between July and September 2019 in China to uncover the prevalence of E. fergusonii and its potential role in the evolution of antimicrobial resistance (AMR). An isolation of E. fergusonii was performed and pulsed-field gel electrophoresis (PFGE) was used to uncover the genetic relationship. The AMR of E. fergusonii isolates was comprehensively characterized using broth microdilution-based antimicrobial susceptibility testing, S1-PFGE, southern hybridization, whole-genome sequencing, and in-depth bioinformatics analysis. As a result, a total of 133 E. fergusonii isolates were obtained. These isolates could be grouped into 41 PFGE subclades, suggesting a diverse genetic relationship. The resistance phenotypes of sulfafurazole (97.74%) and tetracycline (94.74%) were the most frequently found. Of the E. fergusonii isolates, 51.88% were extended spectrum beta-lactamase (ESBL)-positive. Forty-three different AMR genes were revealed based on 25 genome sequences harboring mcr-1. Briefly, aph(6)-Id, aph(3'')-Ib and tet(A) genes were the most frequently observed, with the highest rate being 76.00% (19/25). Three mcr-1-harboring plasmids were identified after Nanopore sequencing, including pTB31P1 (IncHI2-IncHI2A, 184,652 bp), pTB44P3 (IncI2, 62,882 bp), and pTB91P1 (IncHI2-IncHI2A, 255,882 bp). Additionally, 25 E. fergusonii isolates harboring mcr-1 were clustered together with other E. fergusonii isolates from different regions and sources available in GenBank, suggesting a possible random process of mcr-1 transmission in E. fergusonii. In conclusion, E. fergusonii is widespread in food animals in China and might be an important reservoir of AMR genes, especially mcr-1, and facilitate the evolution of AMR. IMPORTANCEE. fergusonii, a member of the genus Escherichia, has been reported to transmit via the food chain and cause diseases in humans. However, the prevalence of multidrug-resistant E. fergusonii, especially mcr-1-positive E. fergusonii isolates, has rarely been reported. Here, we collected 1,400 samples from food animals in three provinces of China and obtained 133 E. fergusonii isolates (9.5%). We found that the prevalence of E. fergusonii isolates was diverse, with high levels of antimicrobial resistance. Among them, 18.8% E. fergusonii isolates carried the colistin resistance gene mcr-1. Thus, E. fergusonii may facilitate the evolution of colistin resistance as a reservoir of mcr-1. As far as we know, the prevalence and AMR of E. fergusonii in the food animals in this study was first reported in China. These findings increase our understanding of the role of E. fergusonii in public health and the evolution of antibiotic resistance.

Characterization of Genetic Elements Carrying mcr-1 Gene in Escherichia coli from the Community and Hospital Settings in Vietnam.

Colistin is widely used in agriculture and aquaculture as prophylaxis, particularly in Asia. Recently, mcr-1 and other mobilizable genes conferring colistin resistance have spread globally in community and hospital populations. Characterizing mcr-1 mobile genetic elements and host genetic background is important to understand the transmission of this resistance mechanism. We conducted whole-genome sequencing of 94 mcr-1-positive Escherichia coli isolates (Mcr1-Ec isolates) from human and animal feces, food, and water in a community cohort (N = 87) and from clinical specimens from a referral hospital (N = 7) in northern Vietnam. mcr-1 was plasmid-borne in 71 and chromosomally carried in 25 (2 isolates contain one copy on chromosome and one copy on a plasmid) of 94 E. coli isolates from the community and hospital settings. All seven clinical isolates carried mcr-1 on plasmids. Replicon types of mcr-1-carrying plasmids included IncI2, IncP, IncX4, and IncFIA single replicons and combinations of IncHI2, IncN, and IncX1 multireplicons. Alignment of a long-read sequence of an IncI2 plasmid from animal feces with short-read sequences of IncI2 plasmids from a healthy human, water, and hospitalized patients showed highly similar structures (query cover from 90% to 98%, overall identity of >81%). We detected the potential existence of multireplicon plasmids harboring mcr-1 regardless of sample setting, confirming 10/71 with long-read sequencing. An intact/conserved Tn6330 transposon sequence or its genetic context variants were found in 6/25 Mcr1-Ec isolates with chromosomally carried mcr-1. The dissemination of mcr-1 is facilitated by a high diversity of plasmid replicon types and a high prevalence of the chromosomal Tn6330 transposon. IMPORTANCE The article presented advances our understanding of genetic elements carrying mcr-1 in Escherichia coli in both community and hospital settings. We provide evidence to suggest that diverse plasmid types, including multireplicon plasmids, have facilitated the successful transmission of mcr-1 in different reservoirs. The widespread use of colistin in agriculture, where a high diversity of bacteria are exposed, has allowed the selection and evolution of various transmission mechanisms that will make it a challenge to get rid of. Colocalization of mcr-1 and other antibiotic resistance genes (ARGs) on multireplicon plasmids adds another layer of complexity to the rapid dissemination of mcr-1 genes among community and hospital bacterial populations and to the slow pandemic of antimicrobial resistance (AMR) in general.

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species.

Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D ß-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most ß-lactam antibiotics in the fight against these resistant isolates.

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering.

BACKGROUND: The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked. RESULTS: In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold. CONCLUSIONS: Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Plasmid Fusion and Recombination Events That Occurred during Conjugation of poxtA-Carrying Plasmids in Enterococci.

Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA-carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA-carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA-carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA-carrying plasmid pE843-27. Moreover, both poxtA-carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS1216E-mediated plasmid integration in E. lactis, involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA-carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis. During both the fusion events, the poxtA-carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.

Biomimetic MOF Nanoparticles Delivery of C-Dot Nanozyme and CRISPR/Cas9 System for Site-Specific Treatment of Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) of unknown etiology affecting the colon and rectum. Previous studies have found that reactive oxygen species (ROS) overproduction and transmembrane glycoprotein CD98 (encoded by SLC3A2) upregulation played important roles in the initiation and progression of UC. On the basis of this, a biomimetic pH-responsive metal organic framework (MOF) carrier was constructed to deliver carbon nanodot-SOD nanozyme and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system for site-specific treatment of UC. In this system, carbon nanodots (C-dots) and CD98 CRISPR/Cas9 plasmid were successfully encapsulated into MOF carrier (ZIF-8 nanoparticles) by a one-pot approach (formed as CCZ), and then camouflaged with macrophage membrane (formed as CCZM). It was worth noting that the C-dot nanozyme showed excellent superoxide dismutase (SOD) enzymatic activity, which could scavenge ROS effectively. As expected, this biomimetic system exhibited pH-responsive, immune escape, and inflammation targeting capability simultaneously. In vitro experiments showed that ROS was significantly eliminated, and CD98 was downregulated by CCZM. In the dextran sulfate sodium salt (DSS)-induced UC model, administration of CCZM significantly ameliorated the inflammation symptoms of mice, including the colon length and pathological parameters such as epithelium integrity and inflammation infiltration. In addition, both in vitro and in vivo results demonstrated that biomimetic nanoparticles effectively reduced the expression of pro-inflammatory cytokines. Overall, this study would provide a promising approach for the precise treatment of UC.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

Genetic Characteristics and Microbiological Profile of Hypermucoviscous Multidrug-Resistant Klebsiella variicola Coproducing IMP-4 and NDM-1 Carbapenemases.

We report here a hypermucoviscous, New Delhi metallo-ß-lactamase 1 (NDM-1) and imipenemase 4 (IMP-4) carbapenemases-coproducing Klebsiella variicola isolate obtained from a pediatric patient. This strain was resistant to carbapenems and most other ß-lactams. Although hypermucoviscous, this strain possessed attenuated virulence according to serum killing assay and Galleria mellonella infection model. Notably, two copies of blaNDM-1 were contained on two tandem ISCR1 elements and coexisted with blaIMP-4 in a novel hybrid multidrug resistance plasmid. This is the first description of the coexistence of blaNDM-1 and blaIMP-4 in a single plasmid of hypermucoviscous K. variicola. IMPORTANCE As an important member of the Klebsiella pneumoniae complex, Klebsiella variicola is poorly studied as an emerging human pathogen. We, for the first time, report a unique K. variicola isolated from a pediatric patient in China. This isolate exhibited hypermucoviscosity, a classic hypervirulence characteristic of K. pneumoniae, and contained multiple carbapenem-resistant genes, including blaIMP-1 and blaNDM-1. Interestingly, these antimicrobial resistance genes were located on a novel hybrid plasmid, and our results suggested that this plasmid might have been introduced from K. pneumoniae and undergone a series of integration and recombination evolutionary events. Overall, our study provides more insight into K. variicola and highlights its superior capability to acquire and maintain foreign resistance genes.

A comparison between MBP- and NT* as N-terminal fusion partner for recombinant protein production in E. coli.

Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this step is still a bottleneck in biophysical studies of many proteins. Often, fusion proteins are used to increase expression levels, solubility, or both. Here, we compare a recently reported fusion tag, NT*, with Maltose Binding Protein (MBP), a well-known fusion tag and solubility enhancer. NT* shows high expression and solubility when used as an N-terminal fusion partner for several aggregation-prone peptides. Its efficacy in enhancing the solubility of aggregation-prone globular proteins has, however, not been tested. We find here that although the overall expression levels for NT* fusions are much higher than those for the MBP fusion, MBP was far superior for enhancing the solubility of the passenger protein. Nevertheless, the effective yield after purification from the soluble fraction of both MBP-fusion and NT*-fusion was comparable, mainly due to higher expression levels in NT*-fusion and a smaller fraction of the passenger protein net weight being locked in the fusion protein. We conclude that NT* is an excellent fusion tag to improve the overall expression of globular proteins but does not increase the passenger protein's solubility compared to MBP. Proteins that are partially soluble or can be refolded in-vitro will significantly benefit from N-terminal NT* fusions. MBP, however, still remains one of the very few options for an N-terminal fusion if the solubility of the protein after expression is critical for preserving its proper fold or activity.

Expression, purification, characterization of DNA binding activity and crystallization of a putative type II DNA Cytosine-5-methyltransferase from Microcystis aeruginosa.

DNA 5-methylcytosine modification plays an important role in the regulation of a variety of biological functions in both prokaryotic and eukaryotic organisms. Previous studies show that DNA Cytosine-5-methylation is predominantly associated with restriction-modification system in bacteria. IPF4390 is deduced to be a putative type II DNA Cytosine-5 methyltransferase from a fresh water cyanobacterium, Microcystis aeruginosa. Both its substrate sequence specificity and catalytic mechanism need to be revealed. In this study, the cloning, expression, purification, DNA binding assays and crystallization of IPF4390 are reported. Results of DNA binding assays demonstrate that IPF4390 can specifically recognize and bind two double-stranded DNAs containing GGNCC (N = A, T, C or G) sequences (HgiBI: 5'-ATAAGGACCAATA-3'; TdeIII: 5'-ATAAGGGCCAATA-3'). Therefore, IPF4390 is probably capable of blocking endonuclease cleavage once restriction sites containing these sequences. Moreover, the crystal of IPF4390 in the presence of TdeIII was obtained, and its X-ray diffraction data were collected and scaled to a maximum resolution of 2.46 Å.