[Investigation of Virulence Variation of Enterovirus 71 Strains during Thermal Adaptive Evolution].

Objective: To study the virulence variation of enterovirus 71 (EV71) during thermal adaptive evolution, providing references for the prevention and control of the EV71-related hand, foot and mouth disease. Methods: Parental strains and thermal-adapted strains originating from EV71 sibling strains (lineage #100 and #101) were used for plaque assay validation, CCK-8 cytotoxicity experiment, and host proteomics studies after Vero cell infection. Plaque morphology and cell inhibition rate of the viral strains were obtained. Mass spectrometry was used to examine and analyze the functions of proteins that were differential expressed in the host cells. Results: Plaque morphology variation was found only in the heat-adapted strain of lineage #101. Increase in cell inhibition rate was observed in all the thermal-adapted strains, but the amount of increase varied in different strains. According to the results of clustering analysis and principal component analysis, after infection of Vero cells, the host cell protein profile of the heat-adapted strains was similar to that of the parental strains and the host cell protein profile of cold-adapted strains was similar to that of cell-adapted strains. It showed that 500 kinds of proteins presented inter-group difference in their expression, with 239 kinds being up-regulated proteins and 261 being down-regulated. The function of the up-regulated proteins were related to post-translational protein modification, while the functions of the down-regulated proteins were related to SRP-dependent cotranslational protein translocation/targeting to membrane and retrograde protein transport. Conclusion: Virulence variations of enterovirus 71 may accompany thermal adaptive evolution, but its mechanism of action still awaits further investigation.

Foot-and-mouth disease virus non-structural protein 2B negatively regulates the RLR-mediated IFN-ß induction.

Foot-and-mouth disease virus (FMDV) is the causative agent of Foot-and-mouth disease (FMD), which is an acute and highly contagious disease affecting pigs, cattle and other cloven-hoofed animals. Several studies have shown that FMDV has evolved multiple strategies to evade the host innate immune response, but the underlying mechanisms for immune evasion are still not fully understood. In the current research, we have demonstrated that FMDV utilizes its non-structural protein 2B to sabotage the host immune response. Over-expression of the FMDV 2B inhibited Poly(I:C)-induced or SeV-triggered up-regulation of IFN-ß, IL-6 as well as ISG15. When HEK293T cells were transfected with FMDV 2B, the phosphorylation of TBK1 and IRF3 was inhibited. Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-I and MDA5. Moreover, FMDV 2B also inhibited the expression of the RIG-I and MDA5. Thus, FMDV 2B negatively regulates the RLR-mediated IFN-ß induction by targeting RIG-I and MDA5.

A cholesterol tag at the N terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo.

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG3) conjugated to the peptides' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG3-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide's insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.

A comprehensive study of the colloidal properties, biocompatibility, and synergistic antioxidant actions of Antarctic krill phospholipids.

Excipient selection is crucial to address the oxidation and solubility challenges of bioactive substances, impacting their safety and efficacy. AKPL, a novel ω-3 polyunsaturated fatty acids (PUFAs) esterified phospholipid derived from Antarctic krill, demonstrates unique antioxidant capabilities and synergistic effects. It exhibits pronounced surface activity and electronegativity at physiological pH, as evidenced by a critical micelle concentration (CMC) of 0.15 g/L and ζ-potential of -49.9 mV. In aqueous environments, AKPL self-assembles into liposomal structures, offering high biocompatibility and promoting cell proliferation. Its polyunsaturated bond-rich structure provides additional oxidation sites, imparting antioxidant properties superior to other phospholipids like DSPC and DOPC. Additionally, AKPL augments the efficacy of lipophilic antioxidants, such as alpha-tocopherol and curcumin, in aqueous media through both intermolecular and intramolecular interactions. In sum, AKPL emerges as an innovative unsaturated phospholipid, offering new strategies for encapsulating and delivering oxygen-sensitive agents.

Human Umbilical Cord Mesenchymal Stem Cells and their Extracellular Vesicles Modulate Pro- and Anti-inflammatory Cytokines in Ligature-induced Periodontitis.

Background: Periodontitis is a chronic inflammatory condition that affects the tissues supporting the teeth, ultimately leading to tooth loss. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) play a crucial role in periodontitis by modulating the activities of gum cells and the immune system. Objective: To investigate the therapeutic potential of human umbilical cord mesenchymal stem cells (hUCSCs) and EVs in regulating the inflammatory response associated with periodontitis. Methods: hUCSCs were isolated, subjected to flow cytometry analysis of surface markers, and differentiated into adipocyte and osteocyte. hUCSC-EVs were isolated and characterized using flow cytometry and electron microscopy. A periodontitis animal model was established in 30 female C57Bl/6 mice. Experimental groups received hUCSCs or hUCSCs-EVs, or vehicles intravenously. Animals were monitored for 4 weeks, and the periodontal tissues were used to assess the effects of hUCSCs and hUCSCs-EVs on the expression of pro- (TNF-α, IFN-γ, and IL-17a) and anti-inflammatory cytokines (TGF-ß, IL-10, and IL-4). The secretion of these cytokines by splenocytes was also evaluated using ELISA. Results: The levels of IL-17a, IFN-γ, and TNFα significantly reduced, while TGF-ß and IL-10 significantly increased in the periodontal tissues of the hUCSC and hUCSCEVs-treated mice. The expression of TNF-α, IFN-γ, and IL-17a significantly decreased, while the production of IL-10 and TGF-ß significantly increased in splenocytes from the hUCSC and EVs-treated mice. Conclusion: hUCSCs and their EVs have the potential to attenuate the inflammatory response associated with periodontitis, possibly by downregulating pro-inflammatory cytokines and upregulating anti-inflammatory ones.

Anti-Inflammatory and Repairing Effects of Mesoporous Silica-Loaded Metronidazole Composite Hydrogel on Human Dental Pulp Cells.

In order to test an effective biopolymer scaffold in promoting the growth of human dental pulp stem cells (HDPSCs), mesoporous silica @ hydrogel (MSN@Gel) nanocomposites are invented as a new type of biopolymer scaffold for HDPSCs proliferation in this paper. The expression levels of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) are significantly increased in the MSN@Gel group so as to better repair damaged dentin. In order to inhibit the proliferation of bacteria in the dental pulp, metronidazole (MTR) is loaded into MSN. The study found that MSN could effectively prolong the half-life of MTR by 1.75 times, and the viability of HDPSCs could be better maintained in the MSN-MTR@Gel group so as to better promote its proliferation to repair pulpitis. However, with the increase of the MTR concentration, its proliferation effect on HDPSCs decreased gradually, and the proliferation effect is the best in 10 µmol/L. Therefore, the MSN-MTR@Gel scaffold is expected to become an effective method for pulpitis therapy in the future.

Molecular characterization of enterovirus 71 sibling strains for thermal adaption in Vero cells with adaptive laboratory evolution.

Enterovirus 71 is the main pathogen that causes severe and fatal hand-foot-mouth-disease (HFMD) cases. As the enterovirus virus mutation has implications for pathogenesis, vaccine development, antiviral therapy, and epidemiological disease management of the virus. In this study, we investigated the variations of enterovirus 71 in thermal adaption, using the method of adaptive laboratory evolution. The sibling virus strains were isolated from a 2-year-old severe case of HFMD (#100) and her symptomless close contact (#101). Both strains were cultured in Vero cells by serial passage of 36 generations at the temperatures of 28.0 °C, 33.0 °C and 39.5 °C to construct adaptive lineages. According to the comparative analysis of phenotypes between adapted strains and parental strains, differences in growth rate were observed in the sibling lineages and a larger plaque was found mainly in the hot adapted strains for lineage #101. Two sets of adaptive strains from six time points (parental, 12th 17th, 31st, 35th passage and endpoint) were sequenced and analyzed by both Sanger sequencing and Next Generation Sequencing. Several variations in most coding genes and one reverse mutation in 5'UTR was observed, along with the identity of 99.8% for complete genome for both lineages. Notably, thermal specific non-synonymous mutations were found in the gene of VP1\VP3\3A\2C\3C. Moreover, the concurrent mutations A292G, A434G and A355C/T of sibling lineages in VP1 showed quantificational trace with distinguishing patterns for different temperatures, which were suspected to be the thermo-sensitive mutation hotspots. These results highlight the possible rules of thermal adaption in enterovirus 71, produce a novel picture of genome evolution of the virus, and shed light on viral variation and evolution.

Rapid identification of Staphylococcus aureus, Vibrio parahaemolyticus and Shigella sonnei in foods by solid phase microextraction coupled with gas chromatography-mass spectrometry.

A novel approach for rapid identification of three foodborne pathogens including Staphylococcus aureus, Vibrio parahaemolyticus and Shigella sonnei in foods by solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was established. After cultivation 24, 18 and 20 h for Staphylococcus aureus, Vibrio parahaemolyticus and Shigella sonnei, respectively, the microbial volatile organic compounds (MVOCs) were extracted with a SPME device equipped with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fibers. The DB-1701P column was applied for separation of MVOCs. A total of 17, 13 and 14 volatile organic compounds were identified as characteristic MVOCs of Staphylococcus aureus, Vibrio parahaemolyticus and Shigella sonnei, respectively. Similarity of the MVOC chromatographic fingerprints for the bacteria were calculated and compared, and the results showed that the established method is stable, reproducible, accurate and has the potential to identify the three bacteria in food samples.

Preparation of a poly(styrene-co-DPHA-co-EDMA) monolith and its application for the separation of small molecules and biomacromolecules by HPLC.

A high performance liquid chromatography (HPLC) monolithic column was prepared by redox polymerization of styrene, dipentaerythritol hexaacrylate (DPHA) and ethylene glycol dimethacrylate (EDMA) in a porogen system of n-propanol/PEG400. The monolith was characterized by scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP) and the results indicated that the monolith had a stable and homogeneous structure. The porosity of the monolithic column was 75.86% and average pore diameter was 2.1µm. Several alkylbenzenes and anilines were used to evaluate the column performance in terms of hydrophobicity. Then the column was applied to separate small molecules including phytosterol and BSA tryptic digest. Finally, five standard proteins, egg white and plasma were separated respectively and high separation capacity of protein was obtained.

Fabrication of a novel hemin-based monolithic column and its application in separation of protein from complex bio-matrix.

A novel polymer-based monolithic column was prepared via redox initiation system within the confines of a stainless steel column with 4.6mm i.d. In the processes, hemin and lauryl methacrylate were used as co-monomers; ethylene dimethacrylate as crosslinking agent; n-butyl alcohol, ethanediol, and N, N-dimethylformamide as tri-porogens; benzoyl peroxide and N, N-dimethyl aniline as redox initiation system. The resulting polymer-based monolithic columns were characterized by scanning electron microscopy, nitrogen adsorption-desorption instrument, and mercury intrusion porosimeter, respectively. The results illustrated that the improved monolith had relative uniform porous structure, good permeability, and low back pressure. Aromatic compounds were used to test the chromatographic behavior of the monolith, resulting in highest column efficiency of 19 880 plates per meter with reversed-phase mechanism. Furthermore, the homemade monolith was used as the stationary phase of high performance liquid chromatography to separate proteins from complex bio-matrix, including human plasma, egg white, and snailase. The results showed that the monolithic column occupied good separation ability with these complex bio-samples. Excellent specific character of the homemade hemin-based monolith was that it could simultaneously remove high-abundance proteins (including human serum albumin, immunoglobulin G, and human fibrinogen) from human plasma and separate other proteins to different fractions.