BHK-21 Research Articles

Synthesis of Up-Conversion CaTiO3: Er3+ Films on Titanium by Anodization and Hydrothermal Method for Biomedical Applications.

The present study investigates the effects of Er3+ doping content on the microstructure and up-conversion emission properties of CaTiO3: Er3+ phosphors as a potential material in biomedical applications. The CaTiO3: x%Er3+ (x = 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0%) films were synthesized on Ti substrates by a hydrothermal reaction at 200 °C for 24 h. The SEM image showed the formation of cubic nanorod CaTiO3: Er3+ films with a mean edge size value of (1-5) μm. When excited with 980 nm light, the CaTiO3: Er3+ films emitted a strong green band and a weak red band of Er3+ ions located at 543, 661, and 740 nm. The CaTiO3: Er3+ film exhibited excellent surface hydrophilicity with a contact angle of ~zero and good biocompatibility against baby hamster kidney (BHK) cells. CaTiO3: Er3+ films emerge as promising materials for different applications in the biomedical field.

Synthesis of Heterostructured TiO2 Nanopores/Nanotubes by Anodizing at High Voltages.

This paper reports on the coating of heterostructured TiO2 nanopores/nanotubes on Ti substrates by anodizing at high voltages to design surfaces for biomedical implants. As the anodized voltage from 50 V to 350 V was applied, the microstructure of the coating shifted from regular TiO2 nanotubes to heterostructured TiO2 nanopores/nanotubes. In addition, the dimension of the heterostructured TiO2 nanopores/nanotubes was a function of voltage. The electrochemical characteristics of TiO2 nanotubes and heterostructured TiO2 nanopores/nanotubes were evaluated in simulated body fluid (SBF) solution. The creation of heterostructured TiO2 nanopores/nanotubes on Ti substrates resulted in a significant increase in BHK cell attachment compared to that of the Ti substrates and the TiO2 nanotubes.

In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing.

Electrospun (ES) fibrous nanomaterials have been widely investigated as novel biomaterials. These biomaterials have to be safe and biocompatible; hence, they need to be tested for cytotoxicity before being administered to patients. The aim of this study was to develop a suitable and biorelevant in vitro cytotoxicity assay for ES biomaterials (e.g. wound dressings). We compared different in vitro cytotoxicity assays, and our model wound dressing was made from polycaprolactone and polyethylene oxide and contained chloramphenicol as the active pharmaceutical ingredient. Baby Hamster Kidney cells (BHK-21), human primary fibroblasts and MTS assays together with real-time cell analysis were selected. The extract exposure and direct contact safety evaluation setups were tested together with microscopic techniques. We found that while extract exposure assays are suitable for the initial testing, the biocompatibility of the biomaterial is revealed in in vitro direct contact assays where cell interactions with the ES wound dressing are evaluated. We observed significant differences in the experimental outcome, caused by the experimental set up modification such as cell line choice, cell medium and controls used, conducting the phosphate buffer washing step or not. A more detailed technical protocol for the in vitro cytotoxicity assessment of ES wound dressings was developed.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Structural investigations and antibacterial, antifungal and anticancer studies on zinc salicylaldimine complexes.

Aim: Zinc salicylaldimines may act as multidrug agents.Results: Three zinc salicylaldimines C1-C3 and respective ligands HL1-HL3 were examined for antimicrobial/anticancer drug action and C3 was structurally analyzed (tetrahedral, triclinic). Against two fungi, C1 inhibited Candida albicans with 12mm (21mm for amphotericin B). Among four bacteria, two ligands inhibited Staphylococcus aureus and Escherichia coli (9-10mm), but the complexes inhibited all bacteria with 10-14mm (21-26mm for ampicillin). The half-maximal inhibitory concentrations for the ligands, complexes and doxorubicin were 195.5-310.7, 22.18-70.05 and 9.66μM against cancerous MCF-7 cells and 186.4-199.9, 14.95-18.87 and 36.42μM against normal BHK cells.Conclusion: The complexation produced pronounced enhancement in the ligand antimicrobial/anticancer activities, despite these activities are moderate comparing with standards.

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

BackgroundFlavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear.MethodsHeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells.ResultsWe found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication.ConclusionsIn summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential

AbstractA2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.

Inactivation of an Indonesian isolate of foot-and-mouth disease virus using formaldehyde.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that endangers livestock and the environment with significant economic consequences. This study aimed to validate the inactivation of the Indonesian isolate of foot-and-mouth disease virus (FMDV) with various formaldehyde concentration. The experiment started with FMDV being adapted on BHK-21 cells until cytopathic effects (CPE) appeared. The biological titer of the virus was determined using the 50% tissue culture infectious dose (TCID50) assay. The virus was inactivated by exposing the isolate to different formaldehyde (FA) concentrations (0.025%, 0.05%, 0.1%, and 0.2%) at 37°C for 24 h, and residual infectivity was assessed using CPE scoring of reinoculated BHK-21 cells. 72 h post-inoculation, the virulence of the FMDV isolate was indicated by complete CPE on BHK-21 monolayer cells, with a TCID50 value of 109/mL; CPE scoring did not signify significant differences (p < 0.05) among 0.025%, 0.05%, 0.1%, 0.2% FA, and the negative control. All treatment groups showed significant differences (p < 0.05) from the positive control (C+). FA concentrations inactivated the FMDV isolate under the given conditions. 0.025% and 0.05% FA continued to display CPE through the third passage, while 0.2% FA did not significantly differ from 0.1% FA (p > 0.05). 0.1% FA is the optimal concentration for safely and effectively inactivating FMDV. All of the formaldehyde concentrations can completely inactivate the FMDV isolate, with the most optimal and safe concentration being 0.1%.

Effect of current density on the morphology and electrochemical properties of nanotubular TiO2 for implant applications

This study focuses on investigating the influence of current density (i) (A/dm2) at values of 0.5 A dm−2, 1.0 A dm−2 1.5 A dm−2, and 2.0 A/dm2 on the surface structure of nanotubular titanium dioxide (TiO2) in an ethylene glycol solvent containing a certain amount of fluoride salt and water. The surface structure observed via FESEM images reveals that different current densities yield different nanotubular TiO2 structures, predominantly in the form of anatase TiO2 crystals. EIS and CV measurements indicate that at a current density of i = 1.5 A dm−2, the nanotubular TiO2 layer exhibits corrosion resistance performance up to 90.06% compared to the bare titanium (Ti) samples. Confocal laser scanning microscopy (CLSM) demonstrates enhanced attachment of BHK cells on anodized titanium surfaces compared to unmodified controls. These findings suggest that nanotubular TiO2 presents a biocompatible material with promising potential for biomedical implant applications.

Antimony complexes with SNN thiosemicarbazones: Synthesis, structural studies and cytotoxicity against bacterial and cancer cells

Addition of antimony(III) chloride to ligands {HL1 = 4-(4-nitrophenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide and HL2 = 4-(2-methoxyphenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide} produced the complexes [Sb(L1)Cl2] (C1) and [Sb(L2)Cl2] (C2), respectively. These complexes were formulated based on elemental, spectroscopic and solution conductivity data. X-ray crystallography elucidated the packing of C2 in the triclinic P-1 space group and the exhibition of pseudo octahedral geometry around the antimony cation. The complexes (20 mg/ml) showed the largest inhibition zones in Staphylococcus aureus (34 mm for C1 and 36 mm for C2) and Escherichia coli (33 mm for C1 and 36 mm for C2) strains. HL1 showed no inhibition in the bacterial strains and HL2 inhibited the strains of Staphylococcus aureus and E. coli at 11 and 10 mm, while ampicillin inhibited these strains at 21 and 25 mm, respectively. The ligands' cytotoxic effects were investigated on four human cancer cells and the greatest effect was on the breast MCF-7 cells. The compounds HL1, HL2, C1, C2 and doxorubicin revealed cytotoxicity with IC50 values of 52.4 ± 0.38, 153.7 ± 1.05, 37.5 ± 0.32, 21.8 ± 0.20 and 9.66±0.25 μM in the MCF-7 cancer cells and of 54.8 ± 0.85, 82.7 ± 0.44, 176.5 ± 0.70, 196.7 ± 1.16 and 36.42±0.08 μM in normal baby hamster kidney (BHK) cells.

Development and biological characterization of an infectious cDNA clone of NADC34-like PRRSV.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) causes high abortion rates in gestating sows and stillbirths, as well as high piglet mortality, seriously jeopardizing the pig industry in China and worldwide. In this study, an infectious clone containing the full-length genome of NADC34-like PRRSV was constructed for the first time using reverse genetic techniques. The gene was amplified segmentally onto a plasmid, transfected into BHK-21 cells, and the transfected supernatant was harvested and transfected into PAM cells, which showed classical cytopathic effects (CPE). The virus rJS-KS/2021 was successfully rescued which could be demonstrated by Western Blot and indirect immunofluorescence assays. Its growth curve was similar to the original strain. Replace the 5'UTR and 3'UTR of rJS-KS/2021 with 5'UTR and 3'UTR of HP-PRRSV (strain SH1) also failed to propagate on MARC-145. In this study, an infectious clone of NADC34-like was constructed by reverse genetics, replacing the UTR and changing the cellular tropism of the virus. These findings provide a solid foundation for studying the recombination of different PRRSVs and the adaption of PRRSVs on MARC-145 in the future.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 in patients exposed to MERS-CoV and SARS-CoV-2 antigens.

This study evaluated the potential for antibody-dependent enhancement (ADE) in serum samples from patients exposed to Middle East respiratory syndrome coronavirus (MERS-CoV). Furthermore, we evaluated the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination on ADE in individuals with a MERS infection history. We performed ADE assay in sera from MERS recovered and SARS-CoV-2-vaccinated individuals using BHK cells expressing FcgRIIa, SARS-CoV-2, and MERS-CoV pseudoviruses (PVs). Further, we analyzed the association of ADE to serum IgG levels and neutralization. Out of 16 MERS patients, nine demonstrated ADE against SARS-CoV-2 PV, however, none of the samples demonstrated ADE against MERS-CoV PV. Furthermore, out of the seven patients exposed to SARS-CoV-2 vaccination after MERS-CoV infection, only one patient (acutely infected with MERS-CoV) showed ADE for SARS-CoV-2 PV. Further analysis indicated that IgG1, IgG2, and IgG3 against SARS-CoV-2 S1 and RBD subunits, IgG1 and IgG2 against the MERS-CoV S1 subunit, and serum neutralizing activity were low in ADE-positive samples. In summary, samples from MERS-CoV-infected patients exhibited ADE against SARS-CoV-2 and was significantly associated with low levels of neutralizing antibodies. Subsequent exposure to SARS-CoV-2 vaccination resulted in diminished ADE activity while the PV neutralization assay demonstrated a broadly reactive antibody response in some patient samples.

Антимікробна та противірусна активність нанокомпозитів на основі поліелектролітних комплексів з наночастинками срібла

Recently, nanocomposite materials containing nanoparticles of metals such as silver, copper and zinc oxide have attracted most attention due to their pronounced pharmacological properties, such as antimicrobial, antiviral, antiinflammatory, immunomodulatory ability and high stability in extreme conditions. Polyelectrolyte complexes based on polymers of natural origin, namely polysaccharides of chitosan and pectin, which can stabilize nanoparticles of a smaller size than individual polymers have significant potential for creation of silver-containing nanocomposites. The aim of this article is to study the antimicrobial and antiviral activity of silver-containing nanocomposites based on polyelectrolyte complexes. Methods. Peculiarities of the structural organization of silver-containing nanocomposites were investigated by the method of wide-angle scattering on an XRD-7000 diffractometer. The morphology of Ag nanoparticles in polymer matrixes were studied by transmission electron microscopy method (transmission electron microscope JEOL 100 CXII). The antimicrobial activity of silver-containing nanocomposites was determined by agar diffusion assays against opportunistic pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Cytotoxicity and antiviral activity were investigated using the MTT method and staining by gentian violet. Results. Analysis of wide-angle X-ray diffractograms of silver-containing nanocomposites based on polyelectrolyte complexes Na-CMC (pectin) – chitosan showed that at reduction of Ag+ ions to metallic silver, there are two low-intensity diffraction maxima at 2θm ~ 380 and 440 in the diffractograms. These maxima correspond to the crystallographic planes of the face-centered cubic lattice of silver, are characterized by indices (111) and (200), respectively, and confirm the presence of metallic silver in the polymer system. Analysis of micrographs of silver-containing nanocomposites based on Na-CMC and chitosan showed that larger nanoparticles are formed with increasing the molecular weight of chitosan. The dependence of the size of silver nanoparticles on the wavelength of ultraviolet radiation at reduction of silver ion in polyelectrolyte-metal complexes Na-CMC–Ag+–chitosan of low molecular weight was also revealed. In particular, smaller particles are formed under irradiation by light with a shorter wavelength (λ =254 nm) than at λ=365 nm. Silver-containing nanocomposites Na-СMC-Ag-chitosan and pectin citrus-Ag-chitosan, obtained by reduction of Ag+ ions under ultraviolet irradiation at a wavelength λ = 365 nm and λ = 254 nm, exhibit high antimicrobial activity against the test cultures of microorganisms S. aureus, E. coli, and P. aeruginosa C. albicans. No significant dependence of antimicrobial activity on the molecular weight of the studied samples was noted: the obtained data were within close limits and had close values. In addition, no dependence of antimicrobial activity on the type of investigated test cultures of microorganisms was observed either. Nanocomposites based on Na-CMC-chitosan (λ = 365 nm) inhibited infection titer HSV-1 by (3.72–5.45) lgTCID50/mL, whereas the decrease in titer during incubation with samples based on citrus pectin-chitosan was within (2.39–2.42) lgTCID50/mL. A dose-dependent relationship between molecular weight of chitosan and reduction of infection titer was observed. It was found that silver-containing nanocomposites formed by reduction of silver ions in polyelectrolyte-metal complexes under ultraviolet radiation of different wavelengths had no cytotoxic effect on cells of MDCK and BHK. Conclusions. The investigated silver-containing nanocomposites based on Na-CMC (pectin)-chitosan polyelectrolyte complexes show antimicrobial activity against test cultures of S. aureus, E. coli P. aeruginosa, and C. albicans along with antiviral activity against herpes simplex virus type 1 and influenza virus. It was established that the obtained nanocomposites did not show a cytotoxic effect on MDCK and BHK cells. The obtained data allow us to assert that investigated silver-containing nanocomposites are promising antimicrobial means for the development of new effective strategies against microorganisms and viruses and improvement of the population health.

Green Synthesized Chitosan Nanoparticles for Controlling Multidrug-Resistant mecA- and blaZ-Positive Staphylococcus aureus and aadA1-Positive Escherichia coli.

Antimicrobial resistance has recently been considered an emerging catastrophe globally. The public health and environmental threats were aggravated by the injudicious use of antibiotics in animal farming, aquaculture, and croup fields, etc. Consequently, failure of antibiotic therapies is common because of the emergence of multidrug-resistant (MDR) bacteria in the environment. Thus, the reduction in antibiotic spillage in the environment could be an important step for overcoming this situation. Bear in mind, this research was focused on the green synthesis of chitosan nanoparticles (ChiNPs) using Citrus lemon (Assam lemon) extract as a cross-linker and application in controlling MDR bacteria to reduce the antibiotic spillage in that sector. For evaluating antibacterial activity, Staphylococcus aureus and Escherichia coli were isolated from environmental specimens, and their multidrug-resistant pattern were identified both phenotypically by disk diffusion and genotypically by detecting methicillin- (mecA), penicillin- (blaZ), and streptomycin (aadA1)-resistance encoding genes. The inhibitory zone's diameter was employed as a parameter for determining the antibacterial effect against MDR bacteria revealing 30 ± 0.4 mm, 34 ± 0.2 mm, and 36 ± 0.8 mm zones of inhibition against methicillin- (mecA) and penicillin (blaZ)-resistant S. aureus, and streptomycin (aadA1)-resistant E. coli, respectively. The minimum inhibitory concentration at 0.31 mg/mL and minimum bactericidal concentration at 0.62 mg/mL of yielded ChiNPs were used as the broad-spectrum application against MDR bacteria. Finally, the biocompatibility of ChiNPs was confirmed by showing a negligible decrease in BHK-21 cell viability at doses less than 2 MIC, suggesting their potential for future application in antibiotic-free farming practices.

Synthesis, Photophysical Properties, Theoretical Studies, and Living Cancer Cell Imaging Applications of New 7-(Diethylamino)quinolone Chalcones.

In this article, three unsymmetrical 7-(diethylamino)quinolone chalcones with D-π-A-D and D-π-A-π-D type push-pull molecular arrangements were synthesized via a Claisen-Schmidt reaction. Using 7-(diethylamino)quinolone and vanillin as electron donor (D) moieties, these were linked together through the α,β-unsaturated carbonyl system acting as a linker and an electron acceptor (A). The photophysical properties were studied, revealing significant Stokes shifts and strong solvatofluorochromism caused by the ICT and TICT behavior produced by the push-pull effect. Moreover, quenching caused by the population of the TICT state in THF-H2O mixtures was observed, and the emission in the solid state evidenced a red shift compared to the emission in solution. These findings were corroborated by density functional theory (DFT) calculations employing the wb97xd/6-311G(d,p) method. The cytotoxic activity of the synthesized compounds was assessed on BHK-21, PC3, and LNCaP cell lines, revealing moderate activity across all compounds. Notably, compound 5b exhibited the highest activity against LNCaP cells, with an LC50 value of 10.89 μM. Furthermore, the compounds were evaluated for their potential as imaging agents in living prostate cells. The results demonstrated their favorable cell permeability and strong emission at 488 nm, positioning them as promising candidates for cancer cell imaging applications.

The topological model of NS4B and its TMD3 in duck TMUV proliferation

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Evolutionary engineering and characterization of Sendai virus mutants capable of persistent infection and autonomous production

Persistent virus infection involves modifying the host immune response and maintaining viral infection. Acute infection with Mononegavirales, such as Sendai viruses (SeVs), can give rise to viruses capable of persistent infection. SeVs establish persistent infection through generating copyback-type defective interfering (cbDI) genomes or acquiring temperature-sensitive mutations. Herein, we identify novel mutations associated with persistent infection and recombinant SeV mutants capable of persistent infection and autonomous production at physiological body temperature, independent of cbDI genomes or temperature-sensitive mutations. Diverse SeV populations were generated by passing the cDNA-recovered SeV Z strain 19 times through embryonated chicken eggs and subsequently infecting LLC-MK2 cells with the SeV populations to finally obtain SeV mutants capable of persistent infection and autonomous production in several types of cultured cells. Sequence analysis identified 4 or 5 mutations in the genome of the persistently infectious SeVs, distinguishing them from other existing strains with persistent infection. Recombinant SeVs carrying 4 or 5 mutations in the Z strain genome (designated SeV-Zpi or SeV-Zpi2, respectively) exhibited persistent infection and autonomous production in LLC-MK2, BHK-21, and Neuro2a cells at 37°C. SeV-Zpi and SeV-Zpi2 consistently produced viral particles even after long-term passages without cbDI particles or temperature-sensitive phenotypes. These results highlight the ability of acute infections of SeVs to spontaneously acquire mutations during replication, thereby endowing persistent infection and autonomous production at body temperature. The vectorization of SeV-Zpi and SeV-Zpi2 will contribute to both basic research and medical applications.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-β secretion

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-β (TGF-β) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1β (IL-1β), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-β levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-β secretion.

CDK5-mediated rearrangement of vimentin during Duck Tembusu virus infection inhibits viral replication

Duck Tembusu virus (DTMUV) is a newly emerging pathogen that causes massive economic losses to the poultry industry in China and neighbouring countries. Vimentin, an intermediate filament protein, has been demonstrated to be involved in viral replication during infection. However, the specific role of vimentin in DTMUV replication has not been determined. In this study, we found that overexpression of vimentin in BHK-21 cells can inhibit DTMUV replication. Moreover, DTMUV replication was enhanced after vimentin expression was reduced in BHK-21 cells via small interfering RNA (siRNA). Further research indicated that DTMUV infection had no effect on the transcription or expression of vimentin. However, we found that DTMUV infection induced vimentin rearrangement, and the rearrangement of vimentin was subsequently confirmed to negatively modulate viral replication through the use of a vimentin network disrupting agent. Vimentin rearrangement is closely associated with its phosphorylation. Our experiments revealed that the phosphorylation of vimentin at Ser56 was promoted in the early stage of DTMUV infection. In addition, by inhibiting the phosphorylation of vimentin at Ser56 with a CDK5 inhibitor, vimentin rearrangement was suppressed, and DTMUV replication was significantly enhanced. These results indicated that DTMUV infection induced vimentin phosphorylation and rearrangement through CDK5, resulting in the inhibition of DTMUV replication. In summary, our study reveals a role for vimentin as a negative factor in the process of DTMUV replication, which helps to elucidate the function of cellular proteins in regulating DTMUV replication.