[Prediction of superantigen active sites and clonal expression of staphylococcal enterotoxin-like W].

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 ß-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel ß-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.

22-oxacalcitriol protects myocardial ischemia-reperfusion injury by suppressing NF-κB/TNF-α pathway.

OBJECTIVE: The aim of this study was to explore whether 22-oxacalcitriol could protect inflammatory response induced by ischemia-reperfusion injury (IRI) in rats, and to investigate its underlying mechanism. MATERIALS AND METHODS: 24 male Sprague Dawley rats were randomly assigned into the sham group, the IRI group and the 22-oxacalcitriol group, with 8 rats in each group. Serum and heart samples of each rat were collected 10 days after the animal procedure. The serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in each rat were detected by relative commercial kits. Pathological lesions in rat myocardium were observed by hematoxylin and eosin (HE) staining. Cardiomyocyte apoptosis in rat heart was accessed by TUNEL staining. Meanwhile, the serum levels of tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1ß), interleukin-6 (IL-6), and KC-GRO were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Also, the protein expression levels of NF-κB, TNF-α, VCAM-1, ICAM-1, and MCP-1 in rat myocardium were detected by Western blot and immunohistochemistry. RESULTS: The serum levels of CK-MB and LDH in rats of the IRI group were significantly higher than those of the sham group. 22-oxacalcitriol treatment remarkably decreased the serum levels of CK-MB and LDH when compared with the IRI group. However, cardiomyocyte apoptosis of the 22-oxacalcitriol group was markedly less than the IRI group. The activities of SOD, GSH, CAT and T-AOC in the cardiac homogenate of the 22-oxacalcitriol group were significantly elevated than those of the IRI group. Meanwhile, malondialdehyde (MDA) and reactive oxygen species (ROS) levels were remarkably decreased by 22-oxacalcitriol treatment. Furthermore, the serum levels of TNF-α, IL-1ß, IL-6 and KC-GRO were significantly downregulated in the 22-oxacalcitriol group. Western blot results showed that the protein expression levels of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 in the 22-oxacalcitriol group were significantly lower than those of the IRI group. CONCLUSIONS: 22-oxacalcitriol inhibits the inflammatory response in the myocardium by suppressing NF-kB/TNF-α pathway, thereby protecting myocardial ischemia-reperfusion injury in rats.

Discovery and identification of fatigue-related biomarkers in human saliva.

OBJECTIVE: To identify stable and specific biomarkers/biomarker combinations for fatigue assessment and establish a discriminant model. PATIENTS AND METHODS: Saliva was collected and electroencephalogram analysis was performed for 47 emergency physicians while awake and after continuoutas duty for 18-24 h. Physicians were divided into the fatigue and non-fatigue groups. Protein spectra of completely quantified saliva specimens were identified before and after long working hours using mass spectrometry. Data were analyzed through Proteome Discoverer software combined with SEQUEST to search protein databases. Proteins were characterized by collision-induced dissociation spectra. A global internal standard (GIS) was added to each group of samples and labeled by tandem mass tags m/z 131.1. All data were compared with GIS, and data between groups were further compared. Qualitative and quantitative data on proteins were exported for fatigue-related proteomic analysis, and a fatigue assessment model was established. RESULTS: We identified 767 salivary proteins in the fatigue group. The correct rates of the discriminant function of the non-fatigue and fatigue groups were 97.1% and 91.7%, respectively (the total correct rate was 95.7%). CONCLUSIONS: We identified 30 fatigue-related protein markers from saliva. We also established a fatigue assessment model for emergency physicians using salivary biomarkers.

Clinical and molecular features of adenovirus type 2, 3, and 7 infections in children in an outbreak in Taiwan, 2011.

OBJECTIVES: We studied paediatric patients with human adenovirus (HAdV) infection during the 2011 outbreak in northern Taiwan to define the clinical features of different HAdV genotypes in children. METHODS: Between January and December 2011, 637 patients <19 years of age exhibited culture-confirmed adenoviral infection in Chang Gung Memorial Hospital, and provided specimens available for genotyping by multiplex real-time PCR. Clinical data were collected retrospectively. RESULTS: Excluding five cases with multiple genotypes, 632 cases were included for analysis. Three genotypes were identified, including HAdV-3 (429/632; 67.6%), HAdV-7 (144/632; 22.6%) and HAdV-2 (59/632; 9.8%). Median age was 4.58 years (range 2 months to 18 years), with children infected with HAdV-3 significantly older (82.9% >3 years; p <0.001). Of the 621 inpatients, 98.2% had fevers and all exhibited respiratory symptoms, 75 patients (12.1%) had lower respiratory tract infections, 20 (3.2%) required intensive care (HAdV-2: 1; HAdV-3: 8; and HAdV-7: 11), and three died (all HAdV-7-infected). HAdV-3-infected patients were significantly more likely to have upper respiratory symptoms and a high serum C-reactive protein level >100 mg/L, whereas leucocytosis (white blood cell count >15 000/mm3) was more common in HAdV-2-infected patients (p 0.007). HAdV-7 infections were significantly associated with a longer duration of fever, leucopenia (white blood cell count <5000/mm3), thrombocytopenia (platelet count <150 000/mm3), lower respiratory tract infections, a longer length of hospital stay, and requiring intensive care (all p <0.001). CONCLUSION: Childhood HAdV-2, HAdV-3 and HAdV-7 infections may exhibit different clinical manifestations. Although HAdV-3 was the most prevalent genotype observed during the 2011 Taiwan outbreak, HAdV-7 caused more severe disease characteristics and outcomes.