Search Results (3,042)

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms. Full article

The global spread of antimicrobial resistance genes (ARGs) in Escherichia coli is a major public health concern. The aim of this study was to investigate the genomic characteristics of extended-spectrum β-lactamase (ESBL)-producing and third-generation cephalosporin-resistant E. coli from a previously obtained collection of 260 E. coli isolates from fecal samples of patients attending primary healthcare facilities in Addis Ababa and Hossana, Ethiopia. A total of 29 E. coli isolates (19 phenotypically confirmed ESBL-producing and 10 third-generation cephalosporin-resistant isolates) were used. Whole-genome sequencing (NextSeq 2000 system, Illumina) and bioinformatic analysis (using online available tools) were performed to identify ARGs, virulence-associated genes (VAGs), mobile genetic elements (MGEs), serotypes, sequence types (STs), phylogeny and conjugative elements harbored by these isolates. A total of 7 phylogenetic groups, 22 STs, including ST131, and 23 serotypes with different VAGs were identified. A total of 31 different acquired ARGs and 10 chromosomal mutations in quinolone resistance-determining regions (QRDRs) were detected. The isolates harbored diverse types of MGEs, with IncF plasmids being the most prevalent (66.7%). Genetic determinants associated with conjugative transfer were identified in 75.9% of the E. coli isolates studied. In conclusion, the isolates exhibited considerable genetic diversity and showed a high potential for transferability of ARGs and VAGs. Bioinformatic analyses also revealed that the isolates exhibited substantial genetic diversity in phylogenetic groups, sequence types (ST) and serogroups and were harboring a variety of virulence-associated genes (VAGs). Thus, the studied isolates have a high potential for transferability of ARGs and VAGs. Full article

Marburg hemorrhagic fever (MHF) is a fatal infectious disease caused by Marburg virus (MARV) infection, and MARV has been identified as a priority pathogen for vaccine development by the WHO. The glycoprotein (GP) of MARV mediates viral adhesion and invasion of host cells and therefore can be used as an effective target for vaccine development. Moreover, DNA vaccines have unique advantages, such as simple construction processes, low production costs, and few adverse reactions, but their immunogenicity may decrease due to the poor absorption rate of plasmids. Lysosome-associated membrane protein 1 (LAMP1) can direct antigens to lysosomes and endosomes and has great potential for improving the immunogenicity of nucleic acid vaccines. Therefore, we constructed a DNA vaccine based on a codon-optimized MARV GP (ID MF939097.1) fused with LAMP1 and explored the effect of a LAMP targeting strategy on improving the immunogenicity of the MARV DNA vaccine. ELISA, ELISpot, and flow cytometry revealed that the introduction of LAMP1 into the MARV DNA candidate vaccine improved the humoral and cellular immune response, enhanced the secretion of cytokines, and established long-term immune protection. Transcriptome analysis revealed that the LAMP targeting strategy significantly enriched antigen processing and presentation-related pathways, especially the MHC class II-related pathway, in the candidate vaccine. Our study broadens the strategic vision for enhanced DNA vaccine design and provides a promising candidate vaccine for MHF prevention. Full article

This study aimed to isolate and screen canine-derived probiotics with excellent probiotic properties. Strain characterization was conducted using a combination of in vitro and in vivo probiotic characterization and safety assessments, as well as complete genome analysis. The results showed that Limosilactobacillus reuteri LRA7 exhibited excellent bacteriostatic and antioxidant activities. The survival rate at pH 2.5 was 79.98%, and the viable counts after exposure to gastrointestinal fluid and 0.5% bile salts were 7.77 log CFU/mL and 5.29 log CFU/mL, respectively. The bacterium also exhibited high hydrophobicity, self-coagulation, and high temperature tolerance, was negative for hemolysis, and was sensitive to clindamycin. In vivo studies in mice showed that the serum superoxide dismutase activity level was 53.69 U/mL higher in the MR group of mice compared to that of the control group, the malondialdehyde content was 0.53 nmol/mL lower in the HR group, and the highest jejunal V/C value was 4.11 ± 1.05 in the HR group (p < 0.05). The L. reuteri LRA7 gene is 2.021 megabases in size, contains one chromosome and one plasmid, and is annotated with 1978 functional genes. In conclusion, L. reuteri LRA7 has good probiotic potential and is safe. It can be used as an ideal probiotic candidate strain of canine origin. Full article

Titanium-Niobium (TiNb) alloys are commonly employed in a number of implantable devices, yet concerns exist regarding their use in implantology owing to the biomechanical mismatch between the implant and the host tissue. Therefore, to balance the mechanical performance of the load-bearing implant with bone, TiNb alloys with differing porosities were fabricated by powder metallurgy combined with spacer material. Microstructures and phase constituents were characterized with energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The mechanical properties were tested by uniaxial compression, and the corrosion performance was determined via a potentiodynamic polarization experiment. To evaluate a highly matched potential implant with the host, biocompatibilities such as cell viability and proliferation rate, fibronectin adsorption, plasmid-DNA interaction, and an SEM micrograph showing the cell morphology were examined in detail. The results showed that the alloys displayed open and closed pores with a uniform pore size and distribution, which allowed for cell adherence and other cellular activities. The alloys with low porosity displayed compressive strength between 618 MPa and 1295 MPa, while the alloys with high porosity showed significantly lower strength, ranging from 48 MPa to 331 MPa. The biological evaluation of the alloys demonstrated good cell attachment and proliferation rates. Full article

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development. Full article

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3′ terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals. Full article

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a ‘minigenome’ or ‘minireplicon’ assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a ‘first-generation’ plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene. Full article

Dengue virus poses a significant global health challenge, particularly in tropical and subtropical regions. Despite the urgent demand for vaccines in the control of the disease, the two approved vaccines, Dengvaxia and TV003/TV005, there are current questions regarding their effectiveness due to an increased risk of antibody-dependent enhancement (ADE) and reduced protection. These challenges have underscored the need for further development of improved vaccines for Dengue Virus. This study presents a new design using an in silico approach to generate a more effective dengue vaccine. Initially, our design process began with the collection of Dengue polyprotein sequences from 10 representative countries worldwide. And then conserved fragments of viral proteins were retrieved as the bases for epitope screening. The selection of epitopes was then carried out with criteria such as antigenicity, immunogenicity, and binding affinity with MHC molecules, while the exclusion criteria were according to their allergenicity, toxicity, and potential for antibody-dependent enhancement. We then constructed a core antigen with the selected epitopes and linked the outcomes with distinct adjuvant proteins, resulting in three candidate vaccines: PSDV-1, PSDV-2, and PSDV-3. Among these, PSDV-2 was selected for further validation due to its superior physicochemical and structural properties. Extensive simulations demonstrated that PSDV-2 exhibited strong binding to pattern recognition receptors, high stability, and robust immune induction, confirming its potential as a high-quality vaccine candidate. For its recombinant expression, a plasmid was subsequently designed. Our new vaccine design offers a promising additional option for Dengue virus protection. Further experimental validations will be conducted to confirm its protective efficacy and safety. Full article

Polar habitats offer excellent sites to isolate unique bacterial strains due to their diverse physical, geochemical, and biological factors. We hypothesize that the unique environmental conditions of polar regions select for distinct strains of lactic acid bacteria (LAB) with novel biochemical properties. In this study, we characterized ten strains of psychrotrophic LAB isolated from hitherto poorly described sources—High Arctic and maritime Antarctic soils and soil-like materials, including ornithogenic soils, cryoconites, elephant seal colonies, and postglacial moraines. We evaluated the physiological and biochemical properties of the isolates. Based on 16S rRNA and housekeeping genes, the four LAB strains were assigned to three Carnobacterium species: C. alterfunditum, C. maltaromaticum, and C. jeotgali. The remaining strains may represent three new species of the Carnobacterium genus. All isolates were neutrophilic and halophilic psychrotrophs capable of fermenting various carbohydrates, organic acids, and alcohols. The identified metabolic properties of the isolated Carnobacterium strains suggest possible syntrophic interactions with other microorganisms in polar habitats. Some showed antimicrobial activity against food pathogens such as Listeria monocytogenes and human pathogens like Staphylococcus spp. Several isolates exhibited unique metabolic traits with potential biotechnological applications that could be more effectively exploited under less stringent technological conditions compared to thermophilic LAB strains, such as lower temperatures and reduced nutrient concentrations. Analysis of extrachromosomal genetic elements revealed 13 plasmids ranging from 4.5 to 79.5 kb in five isolates, featuring unique genetic structures and high levels of previously uncharacterized genes. This work is the first comprehensive study of the biochemical properties of both known and new Carnobacterium species and enhances our understanding of bacterial communities in harsh and highly selective polar soil ecosystems. Full article

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the CXCR4 locus. In this study, we cloned variable domains of this antibody and generated a recombinant chimeric antibody (chAb) by combining it with the constant regions of the humanized antibody Trastuzumab. The new chAb displayed a high specificity and two-fold higher level of affinity than the parental mouse monoclonal antibody. In addition, chAb mediated up to 27–43% of the antibody-dependent cellular cytotoxicity towards cells expressing MT-C34 on their surface. The anti-MT-C34 chAb can be easily generated using plasmids available for the research community and can serve as a valuable tool for the detection, purification, and even subsequent elimination of HIV-1-resistant CD4 cells or CAR cells engineered to fight HIV-1 infection. Full article

Ectoine is a compatible solute naturally produced in some halophilic bacteria as a protective agent for survival in salty environments. It has gained special interest as a therapeutic agent in the pharmaceutical and healthcare sectors for the treatment of different diseases. Ectoine mainly produced by bacterial milking, chemical, and fed-batch fermentation methods under a high-salt medium. Unfortunately, the ectoine yield through these methods is still too low to meet high industrial demand, causing salinity issues. The biobrick method was potentially utilized for efficient ectoine biosynthesis under a low-salt medium with different conditions in E. coli BL21(DE3) harboring the pET-22bNS-EctA-EctB-EctC plasmid. Firstly, three genes, L-2,4-diamino-butyric acid acetyltransferase (ectA), L-2,4-diaminobutyric acid transaminase (ectB), and ectoine synthase (ectC) from Bacillus pseudofirmus OF4, were precisely assembled and expressed into E. coli BL21(DE3). After optimizing the reaction conditions in a whole-cell catalytic reaction [50 mM of the sodium phosphate buffer (pH~7.5) containing 300 mM L-aspartic acid, 100 mM glycerol, 1/20 g/mL cell pellets], the amount of ectoine in the plasmid pET-22bNS-A_LacB_TacC_Tac reached the maximum level of 167.2 mg/mL/d (6.97 mg/mL/h). Moreover, Western blot analysis revealed that high expression levels of EctA and EctC had a significant effect on ectoine biosynthesis, indicating that both proteins might be the key enzymes in ectoine production. We conclude that a high amount of ectoine achieved through the biobrick method and efficiently used for different industrial applications. Full article

Immunotherapy combined with chemicals and genetic engineering tools is emerging as a promising strategy to treat triple-negative breast cancer (TNBC), which is more aggressive with poorer progress than other breast cancer subtypes. In this study, lipid-based nanoparticles (LNPs) possessed an NK cell-like function that could deliver tumor-specific therapeutics and inhibit tumor growth. LNPs fused with an NK cell membrane protein system (NK-LNP) have three main features: (i) hydrophilic plasmid DNA can inhibit TNBC metastasis when encapsulated within LNPs and delivered to cells; (ii) the lipid composition of LNPs, including C18 ceramide, exhibits anticancer effects; (iii) NK cell membrane proteins are immobilized on the LNP surface, enabling targeted delivery to TNBC cells. These particles facilitate the targeted delivery of HIC1 plasmid DNA and the modulation of immune cell functions. Delivered therapeutic genes can inhibit metastasis of TNBC and then induce apoptotic cell death while targeting macrophages to promote cytokine release. The anticancer effect is expected to be applied in treating various difficult-to-treat cancers with LNP fused with NK cell plasma membrane proteins, which can simultaneously deliver therapeutic chemicals and genes. Full article

Bacteriocins produced by lactic acid bacteria are known to be useful tools for food biopreservation and fermentation control. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0 isolated from different samples of Mongolian traditional fermented milk, airag, had been reported to produce listericidal bacteriocin-like inhibitory substances with similar but slightly different properties. In this study, the antibacterial properties and the related gene sequences of both strains were compared, and then their bacteriocins were purified and identified. Strain 406 was superior to strain 213M0 in cell growth and antibacterial activity against many strains. However, the activity of 213M0 was stronger than that of 406 against a few strains. DNA sequencing revealed two and three plasmids in 406 and 213M0, respectively, and each one of them harbored an almost identical mesentericin Y105–B105 gene cluster. Removal of these plasmids resulted in a complete loss of activity, indicating that the antibacterial activity of both strains was generated by bacteriocins encoded on the plasmids. Mesentericins Y105 and B105 were purified from both cultures, and another novel bacteriocin, named mesentericin M, was identified from the 213M0 culture only. Its structural gene was coded on a 213M0 plasmid and, surprisingly, its C-terminal three amino acid residues were post-translationally cleaved. To our knowledge, this is the first report of a C-terminal truncated bacteriocin. In conclusion, the novel bacteriocin should be mainly responsible for the difference in antibacterial properties between the two strains. Full article

The aim of the present work was to genetically characterise cefotaxime-resistant enterobacteria isolated from community carriers in Bulgaria. In total, 717 faecal samples from children and adults in five medical centres in Sofia, Pleven and Burgas were examined. Antimicrobial susceptibility was evaluated by the disk diffusion method. bla_ESBL or plasmidic AmpC (pAmpC) genes were detected by PCR and sequencing. MLST and ERIC-PCR were used to detect clonal relatedness. Among the faecal samples, 140 cefotaxime-resistant enterobacteria were found. The most frequently detected species was Escherichia coli (77.9%, 109/140 samples), followed by Klebsiella pneumoniae (7.9%, 11/140). Among the isolates, bla_CTX-M-15 (37.1%) was predominant, followed by bla_CTX-M-3 (19.2%), bla_CTX-M-14 (10%), and bla_CTX-M-27 (4.3 %). Genes encoding pAmpC were observed in 11.4% (bla_DHA-1, 16/140) and in 1.4% (bla_CMY-2, 2/140). The frequency of ESBL and pAmpC producers among the subjects was 14.6% and 2.5%, respectively. No carbapenem-resistant isolates were found. Four main clonal complexes (CC131, CC10, CC38, and CC155) were detected among E. coli isolates. The most common type was ST131, phylogroup B2 (16.5%). The increased frequency of ESBL- and pAmpC-producing enterobacteria in the community is a prerequisite for treatment failures of the associated infections and a good background for further studies. Full article