EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

Canagliflozin Inhibited the Activity of Hemolysin and Reduced the Inflammatory Response Caused by Streptococcus suis.

Highly virulent Streptococcus suis (S. suis) infections can cause Streptococcal toxic shock-like syndrome (STSLS) in pigs and humans, in which an excessive inflammatory response causes severe damage. Hemolysin (SLY) is a major virulence factor of S. suis serotype 2 that produces pores in the target cell membrane, leading to cytoplasmic K+ efflux and activation of the NLRP3 inflammasome, ultimately causing STSLS. The critical aspect of hemolysin in the pathogenesis of S. suis type 2 makes it an attractive target for the development of innovative anti-virulence drugs. Here, we use the S. suis toxin protein (SLY) as a target for virtual screening. A compound called canagliflozin, a hypoglycemic agent, was identified through screening. Canagliflozin significantly inhibits the hemolytic activity of hemolysin. The results combined with molecular dynamics simulation, surface plasmon resonance, and nano differential scanning fluorimetry show that canagliflozin inhibits the hemolytic activity of SLY by binding to SLY. In addition, canagliflozin markedly reduced the release of SC19-induced inflammatory factors at the cellular level and in mice. Importantly, the combination of canagliflozin and ampicillin had a 90% success rate in mice, significantly greater than the therapeutic effect of ampicillin. The findings suggest that canagliflozin may be a promising new drug candidate for S. suis infections.

ACE-Inhibitory Peptides Identified from Quinoa Bran Glutelin-2 Hydrolysates: In Silico Screening and Characterization, Inhibition Mechanisms of ACE, Coordination with Zinc Ions, and Stability.

To obtain Angiotensin-I-Converting Enzyme (ACE) inhibition peptides with Zn-chelating capacity, quinoa bran glutelin-2 hydrolysates (QBGH) by Flavourzyme and Papain were subjected to Sephadex G-15 gel chromatography, reverse phase-high liquid performance chromatography and UPLC-ESI-MS/MS analysis. Four oligopeptides including GGGSGH, EAGAE, AGGGAGGG and AVPKPS were identified. Of these, only the hexapeptide AVPKPS had both ACE-inhibitory activity (IC50: 123.13 µmol/L) and Zn-chelating ability (17.36 mg/g). Molecular docking showed AVPKPS could bind with active residues Glu384 and Ala354 (both belong to the central S1 pocket of ACE including) through short hydrogen bond and hydrophobic interactions, respectively. Inhibition kinetics verified that AVPKPS was a competitive inhibitor of ACE. Moreover, AVPKPS can affect the zinc tetrahedral coordination in ACE through binding with residues His387 and His383. Fourier-transform infrared spectroscopy analysis demonstrated that the amino and carboxyl groups of AVPKPS were the main chelating sites for zinc ions. Under the gastrointestinal digestion, the ACE inhibition capacity of AVPKPS was relatively stable, and the zinc solubility of AVPKPS-zinc complexes was more stable than zinc sulfate (p < 0.05). These results suggest that quinoa peptides have potential applications as ingredients for antihypertension or zinc fortification.

UBE4A catalyzes NRF1 ubiquitination and facilitates DDI2-mediated NRF1 cleavage.

The transcription factor nuclear factor erythroid 2 like 1 (NFE2L1 or NRF1) regulates constitutive and inducible expression of proteasome subunits and assembly chaperones. The precursor of NRF1 is integrated into the endoplasmic reticulum (ER) and can be retrotranslocated from the ER to the cytosol where it is processed by ubiquitin-directed endoprotease DDI2. DDI2 cleaves and activates NRF1 only when NRF1 is highly polyubiquitinated. It remains unclear how retrotranslocated NRF1 is primed with large amount of ubiquitin and/or very long polyubiquitin chain for subsequent processing. Here, we report that E3 ligase UBE4A catalyzes ubiquitination of retrotranslocated NRF1 and promotes its cleavage. Depletion of UBE4A reduces the amount of ubiquitin modified on NRF1, shortens the average length of polyubiquitin chain, decreases NRF1 cleavage efficiency and causes accumulation of non-cleaved, inactivated NRF1. Expression of a UBE4A mutant lacking ligase activity impairs the cleavage, likely due to a dominant negative effect. UBE4A interacts with NRF1 and the recombinant UBE4A can promote ubiquitination of retrotranslocated NRF1 in vitro. In addition, knocking out UBE4A reduces transcription of proteasomal subunits in cells. Our results indicate that UBE4A primes NRF1 for DDI2-mediated activation to facilitate expression of proteasomal genes.

Analysis of the noncoding RNA regulatory networks of H37Rv- and H37Rv△1759c-infected macrophages.

Noncoding RNAs regulate the process of Mycobacterium tuberculosis (M. tb) infecting the host, but there is no simultaneous transcriptional information of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) and the global regulatory networks of non-coding RNA. Rv1759c, a virulence factor, is a member of protein family containing the proline-glutamic acid (PE) in M. tb, which can increase M. tb survival. To reveal the noncoding RNA regulatory networks and the effect of Rv1759c on non-coding RNA expression during M. tb infection, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We found 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv infection, 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv△1759c infection. We constructed lncRNA/circRNA-miRNA-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. We demonstrated the role of one of the hubs of the networks, hsa-miR-181b-3p, for H37Rv survival in macrophages. We discovered that the expression changes of 68 mRNAs, 92 lncRNAs, 26 circRNAs, and 3 miRNAs were only related to the deletion of Rv1759c by comparing the transcription profiles of H37Rv and H37Rv△1759c. Here, our study comprehensively characterizes the transcriptional profiles in THP1-derived-macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of noncoding RNA and PE/PPE family functions during the infection process.

miR-199a-5p Reduces Chondrocyte Hypertrophy and Attenuates Osteoarthritis Progression via the Indian Hedgehog Signal Pathway.

Osteoarthritis (OA), the most common type of arthritis, is an age-associated disease, characterized by the progressive degradation of articular cartilage, synovial inflammation, and degeneration of subchondral bone. Chondrocyte proliferation is regulated by the Indian hedgehog (IHH in humans, Ihh in animals) signaling molecule, which regulates hypertrophy and endochondral ossification in the development of the skeletal system. microRNAs (miRNAs, miRs) are a family of about 22-nucleotide endogenous non-coding RNAs, which negatively regulate gene expression. In this study, the expression level of IHH was upregulated in the damaged articular cartilage tissues among OA patients and OA cell cultures, while that of miR-199a-5p was the opposite. Further investigations demonstrated that miR-199a-5p could directly regulate IHH expression and reduce chondrocyte hypertrophy and matrix degradation via the IHH signal pathway in the primary human chondrocytes. The intra-articular injection of synthetic miR-199a-5p agomir attenuated OA symptoms in rats, including the alleviation of articular cartilage destruction, subchondral bone degradation, and synovial inflammation. The miR-199a-5p agomir could also inhibit the Ihh signaling pathway in vivo. This study might help in understanding the role of miR-199a-5p in the pathophysiology and molecular mechanisms of OA and indicate a potential novel therapeutic strategy for OA patients.

Millet bran protein hydrolysates derived peptides-zinc chelate: Structural characterization, security prediction in silico, zinc transport capacity and stability against different food processing conditions.

A novel peptide Ser-Asp-Asp-Val-Leu (SDDVL) of excellent zinc-chelating capacity (13.77 mg/g) was identified in millet bran protein hydrolysates. In silico prediction demonstrated that SDDVL had no potential toxicity. The results of structural characterization demonstrated that both amino group and carboxyl group of SDDVL were the primary zinc-chelating sites. Moreover, SDDVL-zinc chelate showed higher stability (p < 0.05) than ZnSO4 and zinc gluconate under different processing conditions including most pasteurization conditions, heating at 100°C for 10-50 min, various pH values (8.0-10.0), treatment of glucose (4-8 g/100 g) or NaCl (1-4 g/100 g), and simulated gastrointestinal digestion. In addition, SDDVL-zinc chelate showed higher zinc transport capacity than ZnSO4 and zinc gluconate in Caco-2 cells (p < 0.05). These results suggested that millet bran peptide had a positive effect on the gastrointestinal stability and bioavailability of Zn, and SDDVL-zinc chelate could be used as ingredient of zinc supplements. PRACTICAL APPLICATION: The current study provided a practical method to identify peptides of excellent zinc-chelating capacity from millet bran protein hydrolysates. This study demonstrated that in silico prediction assisted with suitable database was a fast, practical, and economic way to evaluate the security and to analysis the physicochemical properties of novel peptides. Moreover, it provided an efficient method to assess the stability of peptide-zinc chelate under different food processing conditions, which was the theoretical basis for utilization of peptide as ingredient of zinc fortifications.

Selenium-sensitive histone deacetylase 2 is required for forkhead box O3A and regulates extracellular matrix metabolism in cartilage.

INTRODUCTION: Selenium (Se) as well as selenoproteins are vital for osteochondral system development. Se deficiency (SeD) has a definite impact on the expression and activity of histone deacetylases (HDACs). Abnormal expression of some HDACs affects cartilage development. This current study aims to explore the relationship between differentially expressed HDACs and cartilage development, especially extracellular matrix (ECM) homeostasis maintenance, under SeD conditions. MATERIALS AND METHODS: Dark Agouti rats and C28/I2 cell line under SeD states were used to detect the differently expressed HDAC by RT-qPCR, western blotting and IHC staining. Meanwhile, the biological roles of the above HDAC in cartilage development and homeostasis maintenance were confirmed by siRNA transfection, western blotting, RNA sequence and inhibitor treatment experiments. RESULTS: HDAC2 exhibited lower expression at protein level in both animals and chondrocytes during SeD condition. The results of cell-level experiments indicated that forkhead box O3A (FOXO3A), which was required to maintain metabolic homeostasis of cartilage matrix, was reduced by HDAC2 knockdown. Meanwhile, induced HDAC2 was positively associated with FOXO3A in rat SeD model. Meanwhile, knockdown of HDAC2 and FOXO3A led to an increase of intracellular ROS level, which activated NF-κB pathway. Se supplementary significantly inhibited the activation of NF-κB pathway with IL-1ß treatment. CONCLUSION: Our results suggested that low expression of HDAC2 under SeD condition increased ROS content by decreasing FOXO3A in chondrocytes, which led to the activation of NF-κB pathway and ECM homeostasis imbalance.

A Micron-Sized Laser Photothermal Effect Evaluation System and Method.

The photothermal effects of lasers have played an important role in both medical laser applications and the development of cochlear implants with optical stimulation. However, there are few methods to evaluate the thermal effect of micron-sized laser spots interacting with other tissues. Here, we present a multi-wavelength micro-scale laser thermal effect measuring system that has high temporal, spatial and temperature resolutions, and can quantitatively realize evaluations in real time. In this system, with accurate 3D positioning and flexible pulsed laser parameter adjustments, groups of temperature changes are systematically measured when the micron-sized laser spots from six kinds of wavelengths individually irradiate the Pd/Cr thermocouple junction area, and reference data of laser spot thermal effects are obtained. This work develops a stable, reliable and universal tool for quantitatively exploring the thermal effect of micron-sized lasers, and provides basic reference data for research on light-stimulated neuron excitement in the future.

Activity of Oritavancin and Its Synergy with Other Antibiotics against Mycobacterium abscessus Infection In Vitro and In Vivo.

BACKGROUND: Pulmonary disease caused by Mycobacterium abscessus (M. abscessus) spreads around the world, and this disease is extremely difficult to treat due to intrinsic and acquired resistance of the pathogen to many approved antibiotics. M. abscessus is regarded as one of the most drug-resistant mycobacteria, with very limited therapeutic options. METHODS: Whole-cell growth inhibition assays was performed to screen and identify novel inhibitors. The IC50 of the target compounds were tested against THP-1 cells was determined to calculate the selectivity index, and then time-kill kinetics assay was performed against M. abscessus. Subsequently, the synergy of oritavancin with other antibiotics was evaluated by using checkerboard method. Finally, in vivo efficacy was determined in an immunosuppressive murine model simulating M. abscessus infection. RESULTS: We have identified oritavancin as a potential agent against M. abscessus. Oritavancin exhibited time-concentration dependent bactericidal activity against M. abscessus and it also displayed synergy with clarithromycin, tigecycline, cefoxitin, moxifloxacin, and meropenem in vitro. Additionally, oritavancin had bactericidal effect on intracellular M. abscessus. Oritavancin significantly reduced bacterial load in lung when it was used alone or in combination with cefoxitin and meropenem. CONCLUSIONS: Our in vitro and in vivo assay results indicated that oritavancin may be a viable treatment option against M. abscessus infection.

Identification of a cartilage specific novel miRNA which directly targets PRMT3 in rats.

Through experiments to testify a candidate novel miRNA previously discovered by us is a real miRNA and involved in cartilage development. DESIGN: The miR-novel and the newly hairpin miRNA transcribed sequence (pre-miR-novel) was verified as a genuinely existing miRNA by northern blotting. The predicted secondary structure, sequence alignment and targets of pre-miR-novel were performed by "RNAstructure 5.3" program, LASTN2.8.0+/miRbase22 program and RNA hybird program, respective. GO/KEGG pathway analysis also were performed. The miR-novel expression in cartilage tissue during development was detected by RT-qPCR and dot blotting. The chondrocyte differentiation model was established to examine whether miR-novel is involved in cartilage development. The regulation of PRMT3 expression by novel miRNA was determined with the luciferase reporter gene assay and Western blotting after novel miRNA mimic or inhibitor transfection. RESULTS: It's potential role in specifically regulating rodent cartilage development and associated cellular processes. Furthermore, the expression of protein arginine N-methyltransferase 3 (PRMT3), as a predicted target of the novel miRNA, was found consistently downregulated at rat cartilage during developmental stages and RCJ3.1C5.18 (C5.18) cells during the proliferating and hypertrophic phases of the cartilage development, where the miR-novel expression was significantly up-regulated. Both the dual-luciferase reporter gene assay and the up- or down-regulation of miR-novel suggest that the later can specifically bind with the Prmt3 3'-UTR. CONCLUSION: Overall, this study provides the first comprehensive evidence that a genuine cartilage-specific novel miRNA directly targets PRMT3 and may regulate multitudinous cellular processes and signal transduction during cartilage development.

Design, Synthesis, and Antifungal Activity of Novel Aryl-1,2,3-Triazole-ß-Carboline Hybrids.

The copper catalytic azide and terminal alkyne cycloaddition reaction, namely "click chemistry", gives a new and convenient way to create l,4-disubstitutd-l,2,3-triazoles. In this work, 2-pyrrolecarbaldiminato⁻Cu(II) complexes were established as efficient catalysts for the three-component 1,3-dipolar cycloaddition reaction of arylboronic acid and sodium azide (NaN3) with terminal alkynes in ethanol at room temperature to 50 °C, 1,4-disubstituted 1,2,3-triazoles were synthesized. Following the optimized protocol, two series of new aryl-1,2,3-triazole-ß-carboline hybrids have been designed and synthesized, and the chemical structures were characterized by ¹H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). All of the target compounds were evaluated in vitro for their antifungal activity against Rhizoctorzia solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot by mycelia growth inhibition assay at 50 µg/mL. The antifungal evaluation of the novel hybrids showed that, among the tested compounds, 5a, 5b, 5c, and 9b showed good antifungal activity against sunflower sclerotinia rot. Specifically, compound 9b also exhibited high broad-spectrum fungicidal against all the tested fungi with inhibition rates of 58.3%, 18.52%, 63.07%, 84.47%, and 81.23%. However, for F. oxysporum, all the target compounds showed no in vitro antifungal activities with an inhibition rate lower than 20%. These results provide an encouraging framework that could lead to the development of potent novel antifungal agents.