In vitro activities of thiazolidione derivatives combined with daptomycin against clinical Enterococcus faecium strains

Background Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG’ protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni – NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM. Results The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01). Conclusion These two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Neuroinflammation

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesized a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-a (TNF-a), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO’s anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb this phenotype was no longer observed, indicating Ngb is needed for CO’s modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated neuroinflammation in microglia.

Exploration of an Efficient Electroporation System for Heterologous Gene Expression in the Genome of Methanotroph

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.

Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Cell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

The Antibacterial and Anti-biofilm Activities of Two Thiazolidione Derivatives (H2-60 and H2-81) Against Clinical Enterococcus Faecium Strains

Background: Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase (HK) YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the YycG recombinant proteins containing HisKA and HATPase_c domain of E. faecium DO were in vitro expressed in E. coli competent cell BL21 (DE3) and then purified for the autophosphorylation test, indicating these two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium.Results: The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium was in the range from 3.125mg/L to 25mg/L. Moreover, either H2-60 or H2-81showed the excellent bactericidal activity against E. faecium with the single dose or its combination with daptomycin (4 × MIC) by time-killing assay. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by two thiazolidione derivatives (H2-60 and H2-81) within 1/4×MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with its monotherapy (78.17% and 74.48%vs.41.59%, respectively, P< 0.01). Conclusion: Two thiazolidione derivatives (H2-60 and H2-81) exhibit the robust antibacterial and anti-biofilm activity against E. faecium by targeting the histidine kinase (HK) YycG.

Rotavirus NSP1 contributes to intestinal viral replication, pathogenesis, and transmission

Rotavirus (RV)-encoded non-structural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development.

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

Engineering Bacillus subtilis as a Versatile and Stable Platform for Production of Nanobodies

ABSTRACT There is a growing need for a highly stable system to allow the production of biologics for diagnoses and therapeutic interventions on demand that could be used in extreme environments. Among the variety of biologics, nanobodies (Nbs) derived from single-chain variable antibody fragments from camelids have attracted great attention in recent years due to their small size and great stability with translational potentials in whole-body imaging and the development of new drugs. Intracellular expression using the bacterium Escherichia coli has been the predominant system to produce Nbs, and this requires lengthy steps for releasing intracellular proteins for purification as well as removal of endotoxins. Lyophilized, translationally competent cell extracts have also been explored as offering portability and long shelf life, but such extracts may be difficult to scale up and suffer from batch-to-batch variability. To address these problems, we present a new system to do the following: (i) engineer the spore-forming bacterium Bacillus subtilis to secrete Nbs that can target small molecules or protein antigens on mammalian cells, (ii) immobilize Nbs containing a cellulose-binding domain on a cellulose matrix for long-term storage and small-molecule capturing, (iii) directly use Nb-containing bacterial supernatant fluid to perform protein detection on cell surfaces, and (iv) convert engineered B. subtilis to spores that are resistant to most environmental extremes. In summary, our work may open a new paradigm for using B. subtilis as an extremely stable microbial factory to produce Nbs with applications in extreme environments on demand. IMPORTANCE It is highly desirable to produce biologics for diagnoses and therapeutic interventions on demand that could be used in a variety of settings. Among the many biologics, Nbs have attracted attention due to their small size, thermal stability, and broad utility in diagnoses, therapies, and fundamental research. Nbs originate from antibodies found in camelids, and >10 companies have invested in Nbs as potential drugs. Here, we present a system using cells of the bacterium Bacillus subtilis as a versatile platform for production of Nbs and then antigen detection via customized affinity columns. Importantly, B. subtilis carrying engineered genes for Nbs can form spores, which survive for years in a desiccated state. However, upon rehydration and exposure to nutrients, spores rapidly transition to growing cells which secrete encoded Nbs, thus allowing their manufacture and purification.

Sequential and Asynchronous Strengthening of the Influence of Temperature on the Endo- and Exocytosis of Insulin in the Isolated Vertebrata Hepatocytes: Summing up Previous Studies

Insulin internalization and processing of the Insulin Receptor Complex (IRC) inside the cell are important components of the intracellular Mechanism of Insulin Action (MIA). They define the continuation of intracellular signaling of IRC and allow utilization of the parts of the complex after ligand dissociation. Traditionally, changes in the insulin regulatory system associated with the vertebrate phylogenesis have been evaluated by changes of its two elements: the hormone and its receptor. A hormone-competent cell was considered as an evolutionarily completed element of insulin regulatory system. However, previous studies of the isolated hepatocytes of four classes of vertebrates (lamprey, frog, chicken, and rat) revealed significant differences in the state of internalization of 125I-insulin and intracellular IRC processing. Radical differences were noted in the regulation of 125I-insulin internalization and the intracellular fate of the IRC. Here, cytosolic efficient insulin degradation and a complete lack of 125I-insulin exocytosis were observed in the cyclostome cells, whereas in amphibians the hormone underwent lysosomal degradation and showed low levels of exocytosis, while birds and mammals were characterized by high volumes of the excreted 125Iinsulin containing proteolytic 125I-insulin fragments. Despite the established recognition of the importance of the temperature factor, a complete understanding of the molecular mechanisms underlying the temperature effects on MIA is still missing. This poorly studied problem of the MIA temperature dependence can be behind the differences in the effect of temperature on the intracellular action of insulin and IGF-I. In fact, at different phylogenetic stages, successive changes were reported for the temperature dependence of the 125Iinsulin internalization and exocytosis. The following regularities were reported for the effect of temperature on the 125I-insulin internalization in isolated hepatocytes of different origin: complete lack of receptibility of the process to temperature in lampreys, receptibility of the process in a narrow range of low temperatures (0-5°C) in amphibians, and flexible regulation of 125I-insulin internalization in a wide temperature range (6- 37°C) in the cells from endothermic organisms. Reported data make it possible to observe three stages in the alteration of temperature regulation of 125I-insulin internalization (in cells of cyclostomes, amphibians, and endothermic organisms) and two stages of temperature regulation of 125I-insulin exocytosis in cells of amphibians, birds, and mammals. The data presented in this study reflect the specificity of the developmental reorganization of the intracellular MIA regulation and hormone utilization, and emphasize the central role of temperature in selective MIA formation during vertebrate phylogenesis.