Molecular analyses of the tubby-like protein gene family and their response to salt and high temperature in the foxtail millet (Setaria italica).

Tubby-like proteins (TLPs) are a group of proteins found in both eukaryotes and prokaryotes. They are significant in various physiological and biochemical processes, especially in plants' response to abiotic stress. However, the role of TLP in foxtail millet (Setaria italica) remains unclear. The millet genome has 16 members of the TLP family with typical Tub domains, which can be sorted into five subgroups based on gene structure, motif, and protein domain distribution. SiTLPs were discovered to be predominantly located in the nucleus and also had extracellular distribution. The interspecific evolutionary analysis indicated that SiTLPs had a closer evolutionary relationship with monocots and were consistent with the morphological classification of foxtail millet. When subjected to salt stress, the abundance of SiTLP was affected, and qRT-PCR results showed that the expression levels of certain SiTLP members were induced by salt stress while others remained unresponsive. Except for SiTLP14, all other SiTLP genes were up-regulated in response to high-temperature stress, implying a potentially crucial role for SiTLP in mitigating high-temperature-induced damage. This study provides valuable insights into understanding the functional significance of the TLP gene family in foxtail millet.

Development and Application of a Comprehensive Nontargeted Screening Strategy for Aristolochic Acid Analogues.

Aristolochic acid analogs (AAAs) are naturally occurring carcinogenic and toxic compounds that pose a safety threat to pharmaceuticals and the environment. It is challenging to screen AAAs due to their lack of characteristic mass spectral fragmentation and their presence of structural diversity. A comprehensive nontargeted screening strategy was proposed by taking into account diverse factors and incorporating various self-developed techniques, and a Python3-based toolkit called AAAs_finder was developed for its implementation. The main procedures consist of virtual structure and ultraviolet and visible (UV) spectra database creation, exact mass and UV spectra-based suspect data extraction, tandem mass spectra (MS/MS) anthropomorphic interpretation, and multicondition retention time (RT) prediction-based candidate structures ranking. To initially assess screening feasibility, eight hypothetical unknown samples were subjected to nontargeted screening using the AAAs_finder toolkit and two other advanced tools. The results showed that the former successfully identified all, while the latter two only managed to identify two and three, respectively, indicating that our strategy was more feasible. After that, the strategy was carefully evaluated for false positives and false negatives, instrument dependence, reproducibility, and sensitivity. After the evaluation, the strategy was successfully applied to the screening of AAAs in real samples, such as herbal medicine, spiked soil, and water. Overall, this study proposed a nontargeted screening strategy and toolkit independent of characteristic mass spectral fragmentation and able to overcome challenges posed by structural diversity for the AAAs screening, which is also valuable for other classes of compounds.

Molecular epidemiology and vaccine compatibility analysis of seasonal influenza A viruses in the context of COVID-19 epidemic in Wuhan, China.

The COVID-19 pandemic had a significant impact on the global influenza vaccination and the epidemics of seasonal influenza. To further explore the molecular epidemiology of influenza viruses and assess vaccine effectiveness, we collected influenza cases in Wuhan during the 2022-2023 influenza season. Among 1312 clinical samples, 312 samples tested positive for influenza viruses using reverse transcription polymerase chain reaction. These positive samples included 146A/H1N1 subtypes (46.8%), 164A/H3N2 subtypes (52.6%) and 2 influenza B virus types (0.6%). Based on the whole genome sequence information of hemagglutinin (HA) and neuraminidase (NA) from 27A/H1N1 influenza virus strains and 26A/H3N2 influenza virus strains obtained in this study, a phylogenetic analysis was conducted. The analysis revealed that all A/H1N1 strains belonged to the evolutionary branch 6B.1A.5a.2a, and they exhibited specific substitutions at positions K71Q, Q206E, E241A, and R276K. Similarly, all A/H3N2 strains were classified into the 3C.2a1b.2a.1a subclade and displayed amino acid substitutions at positions S172H, N175Y, I176T, K187N, and S214P. Notably, the A/H3N2 strains also acquired a new potential glycosylation site at position N174. Using an epitope model, the predicted vaccine effectiveness was assessed for the A/H1N1 and A/H3N2 strains. The predicted vaccine effectiveness against the Wuhan influenza epidemic strain was over 85% for the A/H1N1 vaccine strain. However, the effectiveness against the A/H3N2 vaccine strain was only 48.7%. To further verify the protection of influenza vaccine against circulating influenza viruses in the region, we conducted in vivo and in vitro animal studies. The results of in vitro neutralization experiment showed that rabbit serum antibodies inoculated with quadrivalent isolated influenza vaccine had neutralization ability against all 24 isolated influenza viruses. In vivo experiments showed that vaccinated mice had fewer lung lesions when infected with the influenza strain circulating in Wuhan, suggesting that vaccination can effectively reduce the occurrence of severe lung damage. These findings emphasize the importance of accurately predicting seasonal influenza strains for effective influenza prevention and control, especially during the co-circulation of SARS-CoV-2 and influenza viruses. This study provides valuable information on the seasonal influenza virus in Wuhan during the COVID-19 pandemic and serves as a basis for vaccine prediction and updates.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

The Capsid Protein of Nervous Necrosis Virus Antagonizes Host Type I IFN Production by a Dual Strategy to Negatively Regulate Retinoic Acid-Inducible Gene-I-like Receptor Pathways.

Nervous necrosis virus (NNV), a highly pathogenic RNA virus, is a major pathogen in the global aquaculture industry. To efficiently infect fish, NNV must evade or subvert the host IFN for their replication; however, the precise mechanisms remain to be elucidated. In this study, we reported that capsid protein (CP) of red-spotted grouper NNV (RGNNV) suppressed the IFN antiviral response to promote RGNNV replication in Lateolabrax japonicus brain cells, which depended on the ARM, S, and P domains of CP. CP showed an indirect or direct association with the key components of retinoic acid-inducible gene-I-like receptors signaling, L. japonicus TNFR-associated factor 3 (LjTRAF3) and IFN regulatory factor (LjIRF3), respectively, and degraded LjTRAF3 and LjIRF3 through the ubiquitin-proteasome pathway in HEK293T cells. Furthermore, we found that CP potentiated LjTRAF3 K48 ubiquitination degradation in a L. japonicus ring finger protein 114-dependent manner. LjIRF3 interacted with CP through the S domain of CP and the transcriptional activation domain or regulatory domain of LjIRF3. CP promoted LjIRF3 K48 ubiquitination degradation, leading to the reduced phosphorylation level and nuclear translocation of LjIRF3. Taken together, we demonstrated that CP inhibited type I IFN response by a dual strategy to potentiate the ubiquitination degradation of LjTRAF3 and LjIRF3. This study reveals a novel mechanism of RGNNV evading host immune response via its CP protein that will provide insights into the complex pathogenesis of NNV.

Enhancing Electrospinnability of Chitosan Membranes in Low-Humidity Environments by Sodium Chloride Addition.

The electrospinning of pure chitosan nanofibers is highly sensitive to environmental humidity, which limits their production consistency and applicability. This study investigates the addition of sodium chloride (NaCl) to chitosan solutions to enhance spinnability and mitigate the effigurefects of low humidity. NaCl was incorporated into the electrospun chitosan solution, leading to increased conductivity and decreased viscosity. These modifications improved the electrospinning process. Comparative analyses between chitosan membranes (CM) and sodium-chloride-added chitosan membranes (SCM) revealed no significant differences in chemical structure, mechanical strength, or in vitro cell proliferation. This indicates that the addition of 1% (w/v) NaCl does not adversely affect the fundamental properties of the chitosan membranes. The findings demonstrate that NaCl addition is a viable strategy for producing electrospun chitosan nanofibers in low-humidity environments, maintaining their physicochemical properties while enhancing spinnability.

Surface Functionalization of Electrospun Scaffolds by QK-AG73 Peptide for Enhanced Interaction with Vascular Endothelial Cells.

Rapid endothelialization still remains challenging for blood-contacting biomaterials, especially for long-term, functional, small-diameter vascular grafts. The vascular endothelial growth factor (VEGF)-mimicking QK peptide holds great promise in promoting vascular endothelial cellular activities such as adhesion, spreading, proliferation, and migration. Syndecans are transmembrane proteoglycans that are highly expressed on cell surfaces, including vascular endothelial cells, which can act as docking receptors to provide binding sites for a variety of cellular growth and signaling molecules. Herein, a novel peptide QK-AG73 that coupled the QK domain with the syndecan binding peptide AG73 was proposed, aiming to synergistically enhance the interaction with vascular endothelial cells. In addition, mechanically matched bioactive scaffolds based on poly(l-lactide-co-ε-caprolactone) were successfully prepared by surface functionalization of the covalently combined QK-AG73 peptide. The result showed that the adhesion of human umbilical vein endothelial cells (HUVECs) was increased by approximately 2-fold on QK-AG73-modified surface compared with those modified with a single QK or AG73 peptide. Moreover, surface functionalization of electrospun scaffolds by this QK-AG73 peptide was more efficient in specifically promoting the proliferation of HUVECs and allowing them to grow with an elongated cobblestone-like cell morphology. It was hypothesized that both VEGF receptors and transmembrane syndecan receptors were involved in cellular regulation by the QK-AG73 peptide, which resulted in synergistic improvement of the interactions with vascular endothelial cells and provided a promising strategy to promote endothelialization of small-diameter vascular grafts.

Methyltransferase-like 3 suppresses red spotted grouper nervous necrosis virus and viral hemorrhagic septicemia virus infection by enhancing type I interferon responses in sea perch.

Methylation at the N6 position of adenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, tightly associating with regulation of viral life circles and immune responses. Here, a methyltransferase-like 3 homolog gene from sea perch (Lateolabrax japonicus), designated LjMETTL3, was cloned and characterized, and its negative role in fish virus pathogenesis was uncovered. The cDNA of LjMETTL3 encoded a 601-amino acid protein with a MT-A70 domain, which shared the closest genetic relationship with Echeneis naucrates METTL3. Spatial expression analysis revealed that LjMETTL3 was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. LjMETTL3 expression was significantly upregulated at 12 and 24 h post RGNNV and VHSV infection in vitro. In addition, ectopic expression of LjMETTL3 inhibited RGNNV and VHSV infection in LJB cells at 12 and 24 h post infection, whereas knockdown of LjMETTL3 led to opposite effects. Furthermore, we found that LjMETTL3 may participate in boosting the type I interferon responses by interacting with TANK-binding kinase. Taken together, these results disclosed the antiviral role of fish METTL3 against RGNNV and VHSV and provided evidence for understanding the potential mechanisms of fish METTL3 in antiviral innate immunity.

A novel epitope tag from rabies virus has versatile in vitro applications.

Fusion tag technology is an important tool for rapid separation, purification, and characterization of proteins. Combined with monoclonal antibodies, tag epitope systems can be rapidly adapted to many assay systems. A monoclonal antibody that reacts with the matrix protein of the rabies virus CVS-11 strain was reported. The epitope (termed M) targeted by this antibody contains only six amino acids. We examine whether this specific sequence epitope can be applied as a protein tag. We show ectopic expression of M-tagged proteins has little impact on cell viability or major signaling pathways. The M tag system can be used for western blotting, immunoprecipitation, immunofluorescence staining, and flow cytometry assays. The results indicate the specificity, sensitivity, and versatility of this novel epitope tag system are comparable to the widely used FLAG tag system, providing researchers with an additional tool for molecular analysis. KEY POINTS: • A short peptide (Pro Pro Tyr Asp Asp Asp) can be applied as a new tag. • The new epitope-tagging fusion system has no effect on the main cellular signaling pathway. • The epitope-tagging fusion system can be widely used for western blotting, immunoprecipitation, immunofluorescence, flow cytometry, etc.

Bioinspired polydopamine nanoparticles as efficient antioxidative and anti-inflammatory enhancers against UV-induced skin damage.

Excessive and prolonged ultraviolet radiation (UVR) exposure causes photodamage, photoaging, and photocarcinogenesis in human skin. Therefore, safe and effective sun protection is one of the most fundamental requirements. Living organisms tend to evolve various natural photoprotective mechanisms to avoid photodamage. Among them, melanin is the main functional component of the photoprotective system of human skin. Polydopamine (PDA) is synthesized as a mimic of natural melanin, however, its photoprotective efficiency and mechanism in protecting against skin damage and photoaging remain unclear. In this study, the novel sunscreen products based on melanin-inspired PDA nanoparticles (NPs) are rationally designed and prepared. We validate that PDA NPs sunscreen exhibits superior effects on photoprotection, which is achieved by the obstruction of epidermal hyperplasia, protection of the skin barrier, and resolution of inflammation. In addition, we find that PDA NPs are efficiently intake by keratinocytes, exhibiting robust ROS scavenging and DNA protection ability with minimal cytotoxicity. Intriguingly, PDA sunscreen has an influence on maintaining homeostasis of the dermis, displaying an anti-photoaging property. Taken together, the biocompatibility and full photoprotective properties of PDA sunscreen display superior performance to those of commercial sunscreen. This work provides new insights into the development of a melanin-mimicking material for sunscreens.

[Responses of blood parameters and hemoglobin subtypes in plateau zokors and plateau pikas to different altitude habitats].

The plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae) are native species unique to the Qinghai-Tibetan Plateau with successful adaptation to the hypoxic environment. In this study, the number of red blood cells, hemoglobin concentration, mean hematocrit and mean volume of red blood cells were measured in plateau zokors and plateau pikas at different altitudes. Hemoglobin subtypes of two plateau animals were identified by mass spectrometry sequencing. The forward selection sites in two animals' hemoglobin subunits were analyzed by PAML4.8 program. Homologous modeling was used to analyze the effect of forward selection sites on the affinity of hemoglobin to oxygen. The adapting strategies of plateau zokors and plateau pikas to hypoxia at different altitudes were analyzed through comparing blood parameters between the two species. The results indicated that, with increasing altitudes, plateau zokors responded to hypoxia by increasing red blood cell count and decreasing red blood cell volume, while plateau pikas took the opposite strategies to plateau zokors. In erythrocytes of plateau pikas, both adult α2ß2 and fetal α2ε2 hemoglobins were identified, while erythrocytes of plateau zokors only had adult α2ß2 hemoglobin, however the affinities and the allosteric effects of the hemoglobin of plateau zokors were significantly higher than those of plateau pikas. Mechanistically, in the α and ß subunits of hemoglobin of plateau zokors and pikas, the numbers and the sites of the positively selected amino acids as well as the side chain groups polarities and orientations of the amino acids differed significantly, which may result in the difference of the affinities to oxygen of hemoglobin between plateau zokors and pikas. In conclusion, the adaptive mechanisms to respond to hypoxia in blood properties of plateau zokors and plateau pikas are species-specific.

3-Alkyl-2-pyridyl Directing Group-Enabled C2 Selective C-H Silylation of Indoles and Pyrroles via an Iridium Catalyst.

An iridium-catalyzed, directing group-enabled site selective intra- and intermolecular silylation of indoles and pyrroles with hydrosilanes has been developed under ligand-free conditions. Fine-tuning of the removable 3-alkyl-2-pyridyl directing group was found to be crucial for achieving high yields for C2-silylated indole and pyrrole products. Moreover, the scalability was demonstrated, and further transformations of the silylation products were achieved.

Sea perch (Lateolabrax japonicus) autophagy related gene 5 promotes RGNNV infection via inhibiting RLRs-interferon signaling pathway.

Autophagy-related gene 5 (Atg5), an essential component of autophagy machinery, is associated with innate immune responses. Here, the Atg5 of sea perch (Lateolabrax japonicus) (LjAtg5) was cloned and its role in regulating autophagy and interferon (IFN) response during red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. The LjAtg5 cDNA encoded a polypeptide of 275 amino acids with an APG5 domain, and had the closet genetic relationship with Micropterus salmoides Atg5. Autophagic detection showed LjAtg5 was conserved in inducing cell autophagy. Spatial expression analysis revealed LjAtg5 had a higher expression level in liver, brain, and kidney tissues of RGNNV-infected sea perch compared with the control group. In RGNNV-infected LJB cells, overexpression of LjAtg5 significantly increased the mRNA and protein levels of capsid protein, whereas knockdown of LjAtg5 led to the opposite effect, indicating LjAtg5 played a pro-viral role during RGNNV infection. Furthermore, dual luciferase reporter assay revealed LjAtg5 significantly suppressed the activation of sea perch type I IFN promoter in vitro, and overexpression of LjAtg5 strongly weaken the expression of genes related to the RIG-I-like receptors (RLRs) signaling pathway and IFN stimulated genes. These results suggested LjAtg5 promoted RGNNV infection by negatively regulating RLRs-IFN signaling pathway.

A cluster-randomized trial of workplace ergonomics and neck-specific exercise versus ergonomics and health promotion for office workers to manage neck pain - a secondary outcome analysis.

BACKGROUND: Neck pain is prevalent among office workers. This study evaluated the impact of an ergonomic and exercise training (EET) intervention and an ergonomic and health promotion (EHP) intervention on neck pain intensity among the All Workers and a subgroup of Neck Pain cases at baseline. METHODS: A 12-month cluster-randomized trial was conducted in 14 public and private organisations. Office workers aged ≥18 years working ≥30 h per week (n = 740) received an individualised workstation ergonomic intervention, followed by 1:1 allocation to the EET group (neck-specific exercise training), or the EHP group (health promotion) for 12 weeks. Neck pain intensity (scale: 0-9) was recorded at baseline, 12 weeks, and 12 months. Participants with data at these three time points were included for analysis (n = 367). Intervention group differences were analysed using generalized estimating equation models on an intention-to-treat basis and adjusted for potential confounders. Subgroup analysis was performed on neck cases reporting pain ≥3 at baseline (n = 96). RESULTS: The EET group demonstrated significantly greater reductions in neck pain intensity at 12 weeks compared to the EHP group for All Workers (EET: ß = - 0.53 points 95% CI: - 0.84- - 0.22 [36%] and EHP: ß = - 0.17 points 95% CI: - 0.47-0.13 [10.5%], p-value = 0.02) and the Neck Cases (EET: ß = - 2.32 points 95% CI: - 3.09- - 1.56 [53%] and EHP: ß = - 1.75 points 95% CI: - 2.35- - 1.16 [36%], p = 0.04). Reductions in pain intensity were not maintained at 12 months with no between-group differences observed in All Workers (EET: ß = - 0.18, 95% CI: - 0.53-0.16 and EHP: ß = - 0.14 points 95% CI: - 0.49-0.21, p = 0.53) or Neck Cases, although in both groups an overall reduction was found (EET: ß = - 1.61 points 95% CI: - 2.36- - 0.89 and EHP: ß = - 1.9 points 95% CI: - 2.59- - 1.20, p = 0.26). CONCLUSION: EET was more effective than EHP in reducing neck pain intensity in All Workers and Neck Cases immediately following the intervention period (12 weeks) but not at 12 months, with changes at 12 weeks reaching clinically meaningful thresholds for the Neck Cases. Findings suggest the need for continuation of exercise to maintain benefits in the longer term. CLINICAL TRIAL REGISTRATION: hACTRN12612001154897 Date of Registration: 31/10/2012.

DCLK1 inhibition attenuates tumorigenesis and improves chemosensitivity in esophageal squamous cell carcinoma by inhibiting ß-catenin/c-Myc signaling.

Doublecortin-like kinase 1 (DCLK1) is involved in tumorigenesis, tumor growth and metastasis, and epithelial-to-mesenchymal transition in many digestive tract tumors. It is reportedly highly expressed in Barrett's esophagus and esophageal adenocarcinoma, but its effects on the occurrence and progression of esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, real-time PCR and western blot analysis confirmed significant upregulation of DCLK1 expression in human ESCC tissues and cell lines. CCK-8 assay showed that transfection with siRNA against DCLK1 (si-DCLK1) markedly inhibited cell proliferation and colony formation in the ESCC cell lines Eca109 and TE1. Transwell assay revealed that si-DCLK1 transfection inhibited the migratory and invasive capacities of Eca109 and TE1 cells. Moreover, si-DCLK1 increased the chemosensitivity of these cells to cisplatin, as indicated by inhibited cell viability and colony formation, and increased ROS and apoptosis in cisplatin-treated cells. Western blot assay revealed that expression of nuclear ß-catenin and c-Myc was significantly increased in ESCC tissues and that si-DCLK1 markedly downregulated nuclear ß-catenin and c-Myc in Eca109 cells. Treatment with lithium chloride, an activator of ß-catenin signaling, partially abolished the si-DCLK1-induced inhibition of proliferation, migration, invasion, and chemoresistance of ESCC cells. These findings suggest that knockdown of DCLK1 may inhibit the progression of ESCC by regulating proliferation, migration, invasion, and chemosensitivity via suppressing the ß-catenin/c-Myc pathway, supporting a promising therapeutic target against ESCC.

Process evaluation of a workplace-based health promotion and exercise cluster-randomised trial to increase productivity and reduce neck pain in office workers: a RE-AIM approach.

BACKGROUND: This study uses the RE-AIM framework to provide a process evaluation of a workplace-based cluster randomised trial comparing an ergonomic plus exercise intervention to an ergonomic plus health promotion intervention; and to highlight variations across organisations; and consider the implications of the findings for intervention translation. METHOD: This study applied the RE-AIM (reach, effectiveness, adoption, implementation, maintenance) methodology to examine the interventions' implementation and to explore the extent to which differences between participating organisations contributed to the variations in findings. Qualitative and quantitative data collected from individual participants, research team observations and organisations were interrogated to report on the five RE-AIM domains. RESULTS: Overall reach was 22.7% but varied across organisations (range 9 to 83%). Participants were generally representative of the recruitment pool though more females (n = 452 or 59%) were recruited than were in the pool (49%). Effectiveness measures (health-related productivity loss and neck pain) varied across all organisations, with no clear pattern emerging to indicate the source of the variation. Organisation-level adoption (66%) and staffing level adoption (91%) were high. The interventions were implemented with minimal protocol variations and high staffing consistency, but organisations varied in their provision of resources (e.g. training space, seniority of liaisons). Mean adherence of participants to the EET intervention was 56% during the intervention period, but varied from 41 to 71% across organisations. At 12 months, 15% of participants reported regular EET adherence. Overall mean (SD) adherence to EHP was 56% (29%) across organisations during the intervention period (range 28 to 77%), with 62% of participants reporting regular adherence at 12 months. No organisations continued the interventions after the follow-up period. CONCLUSION: Although the study protocol was implemented with high consistency and fidelity, variations in four domains (reach, effectiveness, adoption and implementation) arose between the 14 participating organisations. These variations may be the source of mixed effectiveness across organisations. Factors known to increase the success of workplace interventions, such as strong management support, a visible commitment to employee wellbeing and participant engagement in intervention design should be considered and adequately measured for future interventions. TRIAL REGISTRATION: ACTRN12612001154897; 29 October 2012.

De novo synthesis of the complete genome of coxsackievirus A10 based on Golden Gate cloning combined with promoter reconstruction.

In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the -35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the -35 region of the tac promoter was introduced into the 3' end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering.

Altered frequency of Th17 and Treg cells in new-onset systemic lupus erythematosus patients.

BACKGROUND: T helper 17 (Th17) and regulatory T (Treg) cells play an important role in pathogenesis of systemic lupus erythematosus (SLE). Their imbalance was reported in treated SLE patients, while very little is known about the relationship between Th17 and Treg cells in new-onset untreated SLE patients. AIM: To assess the role of Th17/Treg cells in the pathogenesis of new-onset SLE. MATERIALS AND METHODS: Thirty-nine new-onset SLE patients and 33 age-matched healthy adults were enrolled. We analysed Th17 and Treg cells in different level, including their frequencies in peripheral blood mononuclear cell, the expression of interleukin-17 A (IL-17A) and forkhead box P3 (FoxP3) proteins, the expression of retinoid-related orphan nuclear receptor γt (RORγt) and FoxP3 genes and plasma level of IL-17A. RESULTS: The frequency of Th17 and Treg cells, the expression of IL-17A among Th17 cell, the plasma level of IL-17A, the expression of RORγt and FoxP3 genes were all significantly higher in SLE patients. Th17 cells were negatively correlated with Treg cells. We also found that plasma level of IL-17A was positively correlated with SLE disease activities index (SLEDAI) scores and an equation among the level of C3, IgA, IL-17A and SLEDAI scores. CONCLUSIONS: Results indicate that Th17 and Treg cells take roles in the pathogenesis of SLE. Th17 cells might suppress the differentiation of Treg cells, and feedback effects might exist between them during SLE pathogenesis. The measure of plasma level of IL-17A may be useful for evaluation of disease activity in new-onset SLE patients.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Angiotensin II aggravates lipopolysaccharide induced human pulmonary microvascular endothelial cells permeability in high glucose status.

Lung infection is one of the most common infections in diabetes mellitus and is characterized by increased pulmonary microvascular endothelial permeability. Local Angiotensin II (AngII) plays an important role in the pathogenesis of lung diseases. However, whether AngII can aggravate diabetic infectious lung injury is not clear. Therefore, we investigated the effects of AngII on the permeability of human pulmonary microvascular endothelial cells (HPMVECs) challenged by lipopolysaccharide (LPS) in high glucose states in vitro. HPMVECs were divided into five groups: a control group (CON), a high glucose group (HG), an LPS + high glucose group (LH), an LPS + high glucose + AngII group (LHA), and an LPS + high glucose + Losartan group (LHL). The HPMVECs permeability as well as the F-actin levels, cytoskeleton, apoptosis and TNF-α concentrations were evaluated. Compared to the CON group, the HG, LH and LHA groups had significantly higher cellular permeability, cellular apoptosis and TNF-α levels, with more extensive cytoskeletal damage and lower F-actin levels. Additionally, cells in the LHA group exhibited significantly elevated permeability, apoptosis and TNF-α concentrations, lower F-actin levels and more extensive cytoskeletal damage than either the LH or HG group. However, compared to the HG or LH group, the LHL group showed significantly lower cellular permeability, cell apoptosis, cytoskeletal damage and TNF-α concentrations and higher F-actin levels. This study suggests that in a diabetic infectious lung injury cellular model, AngII could aggravate the permeability of HPMVEC via F-actin dynamics and cell apoptosis. Furthermore, blocking the Angiotension II Type 1 Receptor could significantly alleviate the hyperpermeability of HPMVECs.