ObjectiveTo establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus (MHV), mouse parvovirus (MPV), and reovirus type 3 (Reo-3) in laboratory mice. MethodsRecombinant plasmid standards were constructed using conserved genomic regions of the three target viruses. Specific primers and probes were designed, and a triplex TaqMan quantitative PCR (qPCR) system was optimized through preliminary experiments. Sensitivity was evaluated using 10-fold serial dilutions (10¹-10⁷ copies/μL). Intra- and inter-assay repeatability were assessed via reproducibility experiments. ResultsA strong linear correlation (R ²>0.99) was observed between copy number and Ct value at template concentrations of 10²-10⁷ copies/μL. Intra-assay and inter-assay coefficients of variation (CV) were both below 5%. No cross-reactivity was observed with mouse cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), Sendai virus (SeV), or pneumonia virus of mice (PVM). ConclusionThe triplex TaqMan quantitative PCR method established in this study has high sensitivity, good repeatability, and strong specificity. It enables the rapid, specific, and sensitive detection of MHV, MPV, and Reo-3, allows the simultaneous detection of multiple pathogens in a single tube, and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations, thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.

ObjectiveTo analyze the composition of respiratory virus pathogen spectrum and epidemiological characteristics among adult influenza-like illness (ILI) cases in Putuo District of Shanghai from 2015 to 2024, to summarize the epidemic patterns of respiratory viruses across different time periods and populations, and to provide scientific evidence for the prevention and control of adult respiratory virus infections in Putuo District of Shanghai. MethodsSurveillance data of 2 273 adult ILI cases from sentinel hospitals under the comprehensive surveillance system for acute respiratory infections in Putuo District of Shanghai from 2015 to 2024 were retrospectively collected. Descriptive epidemiological methods were adopted to analyze the temporal and population distribution characteristics of respiratory viruses, including influenza virus (IFV), respiratory syncytial virus (RSV), adenovirus (ADV), and human metapneumovirus (HMPV). The mixed infection status among different age groups was compared, and the distribution of influenza virus subtypes was described. SPSS 22.0 and WPS Office software were used for statistical analyses. ResultsThe overall detection rate of respiratory viruses in 2 273 adult ILI cases was 43.51%. The detection rate in females (45.48%) was significantly higher than that in males (41.25%), with statistical significance (χ²=4.113, P=0.043). Individuals aged 60 years old and above had the highest detection rate (47.41%) (χ²=8.430, P=0.014). The detection rates in winter (50.41%) and summer (48.42%) were higher than those in other seasons (χ²=47.028, P<0.001). The overall viral detection rate decreased after 2020 (χ²=208.411, P<0.001). Detection rates of various viruses varied by year, with IFV and ADV showing high rates from 2015 to 2019 (all P<0.001), while human coronavirus (HCoV) and rhinovirus/enterovirus (HRV/EV) exhibited higher rates from 2020 to 2022 (all P<0.001). Co-infection with IFV and HCoV was the most common, followed by co-infection with IFV and ADV, together accounting for 50.00% of all mixed infections. During the surveillance period, the detection rate of IFV was significantly higher than that of other respiratory viruses. Influenza B/Victoria lineage predominated in 2021, followed by successive predominance of influenza A(H1N1), A(H3N2), and the co-circulation of the two subtypes from 2022 to 2023. Influenza A(H1N1) re-emerged as the predominant subtype in the first half of 2024. ConclusionFrom 2015 to 2024, respiratory virus infections in adults prevailed in winter and summer, and people aged 60 years old and above were the key high-risk population. The detection rates of respiratory viruses declined after 2020. IFV was the predominant pathogen detected, and different influenza virus subtypes circulated alternately each year.

Objective:To investigate the neuroprotective efficacy of scutellarin(Scu)against cerebral ischemia-reperfusion(CIRI)injury in rats,with a focus on its regulatory mechanisms involving the CX3CL1/CX3CR1-PI3K/AKT signaling axis.Methods:The rat CIRI model was established using Longa's intraluminal suture method.The experimental design comprised four groups(n=15 per group):Sham operation group,CIRI model group,Scu treat-ment(40 mg/kg/d Scu gavage)group,and Scu+LY294002(Scu combined with PI3K inhibitor LY294002)group.Following 4 weeks of intervention,neurological function,pathological changes,and pathway protein expression were evaluated using Longa scoring,TTC staining,HE staining,transmission electron microscopy,immunofluorescence,or Western blot analysis.Results:Compared to the CIRI model group,Scu treatment significantly improved neurological function,reduced cerebral infarct volume and attenuated neuronal damage(P<0.05).Furthermore,Scu downregulat-ed CX3CL1/CX3CR1 expression,increased p-PI3K/PI3K and p-AKT/AKT ratios,elevated Bcl-2/Bax ratio,and de-creased IL-17A expression(P<0.05).These protective effects were partially reversed by LY294002.Conclusion:Scu exerts neuroprotective effects in cerebral CIRI injury by downregulating CX3CL1/CX3CR1 expression and activating the PI3K/AKT pathway,thereby attenuating inflammation and apoptosis.

Objective:To investigate, for multi-sensitized allergic rhinitis (AR) patients allergic to dust mites combined with other allergens (pollen, mold, animal dander, etc.), whether the single dust mite subcutaneous immunotherapy (SCIT) can improve the specific symptoms caused by other allergens in the patients, and to analyze the relationship between the effectiveness of symptom improvement in these patients and the type, quantity and severity of the allergens.Methods:A multicenter retrospective study was conducted to collect mul-sensitized AR patients from allergy or respiratory departments of 5 hospitals who received house dust mite allergen preparation SCIT for 12 to 36 months and met other inclusion and exclusion criteria from February to July 2024. General clinical data were collected and the perennial or seasonal symptoms before and after treatment were evaluated with visual analogue scale (VAS) to assess whether there was an perennial or allergen-specific symptom improvement (VAS score decrease ≥30%), by which the patients were divided into effective group and ineffective. R software was used to analyze the differences between groups by using Fisher′s exact test and Mann-Whitney U test. Results:A total of 62 patients were enrolled, and the treatment were effective in 39 of them, with an effective rate of 62.9%. For allergen-specific symptoms, the median age of the effective group was higher than that of the ineffective group (12 years old vs. 8 years old, P=0.039), and the effective rate in dust mite specific immunoglobin E (sIgE) grade ≤5 group was higher than that in sIgE grade >5 group (81.6% vs. 45.5%, P=0.008), and the effective rate of mold sIgE grade ≤2 group was higher than that of sIgE grade >2 group (83.3% vs. 28.6%, P=0.045), and there was no statistically significant correlation between the other allergen grades and the effective rate ( P>0.05). For perennial symptoms, the effective rate in the mold grade ≤2 group was higher than that in the sIgE grade >2 group (91.3% vs. 28.6%, P=0.010), and there was no statistically significant correlation between the other allergen grades and the effective rate ( P>0.05). There was no significant correlation between the treatment effectiveness of perennial or allergen-specific symptoms and the number of combined allergens, the grade of skin test, and the difference between the grade of combined allergens and that of dust mites ( P>0.05). Conclusion:Among the patients with multi-sensitized AR allergic to dust mites included in this study, single dust mite SCIT is effective in some of them, and for allergen-specific symptoms, the effective group was elder, and dust mite sIgE grade 6 and mold sIgE grade ≥2 was related to the low effective rate of SCIT. The present results are insufficient for selecting single or multiple AIT in any type of multi-sensitized patients.

BACKGROUND:Oriented polycaprolactone/silk fibroin blend electrospinning scaffolds can repair peripheral nerve damage and promote nerve regeneration,but the repair effect is limited,and it is necessary to further combine bioactive materials,growth factors,and other factors to prepare bionic neural scaffolds.Calcium titanate nanoparticles have the ability to promote adhesion between Schwann cells and have been widely used in the field of biomedical materials.OBJECTIVE:To prepare polycaprolactone/silk fibroin-calcium titanate functionalized scaffolds by electrospinning technology,and to further explore the effect of the scaffold on Schwann cell viability and adhesion.METHODS:Polycaprolactone/silk fibroin scaffolds were prepared by electrospinning technology.The optimal parameters were selected by adjusting the syringe flow rate,receiving device speed and spinning time during the preparation process for subsequent experiments.Under the optimal parameters,randomly oriented polycaprolactone/silk fibroin scaffolds(denoted as PCL/SF-R),oriented polycaprolactone/silk fibroin scaffolds(denoted as CTO-0),and oriented polycaprolactone/silk fibroin scaffolds containing 0.1,0.5,and 1.0 mg/mL polydopamine modified calcium titanate(denoted as CTO-1,CTO-5,and CTO-10,respectively)were prepared by electrospinning technology.The physical properties of the five groups of scaffolds were characterized.The extracts of the five groups of scaffolds were co-cultured with rat fibroblast cell line(L929).Cell proliferation was detected by MTT assay.Rat Schwann cell line(RSC96)was inoculated on the five groups of scaffolds.Cell viability,cytoskeleton staining,and cytoskeleton and cell adhesion related genes were detected.RESULTS AND CONCLUSION:(1)The optimal parameters for preparing electrospinning scaffolds were as follows:syringe flow rate 15 μL/min,receiving device speed 1 800 r/min,and spinning time 1 hour.(2)The PCL/SF-R group had the largest water contact angle and the smallest elastic modulus,while the CTO-0 group had the smallest water contact angle and the largest elastic modulus.The degradation rate of the scaffolds in the CTO-0 group was lower than that in the CTO-1 group,and the degradation rate of the scaffolds decreased with the increase of the mass concentration of calcium titanate in the oriented scaffolds.(3)The scaffolds in the five groups had no toxic effects on L929 cells and had good biocompatibility.CCK-8 assay results showed that CTO-10 could improve the viability of RSC96 cells.Cytoskeleton staining results showed that the aspect ratio and cell protrusion length of cells on the oriented scaffolds were higher than those on the randomly oriented scaffolds.qPCR test results showed that the mRNA expressions of YAP and Cntn2 in the CTO-5 and CTO-0 groups were higher than those in the PCL/SF-R group(P＜0.05).(4)The results indicate that the oriented polycaprolactone/silk fibroin-calcium titanate scaffold has good biocompatibility,mechanical properties,and hydrophilicity,which can enhance the viability and migration ability of Schwann cells and promote peripheral nerve regeneration.

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

Objective:To investigate, for multi-sensitized allergic rhinitis (AR) patients allergic to dust mites combined with other allergens (pollen, mold, animal dander, etc.), whether the single dust mite subcutaneous immunotherapy (SCIT) can improve the specific symptoms caused by other allergens in the patients, and to analyze the relationship between the effectiveness of symptom improvement in these patients and the type, quantity and severity of the allergens.Methods:A multicenter retrospective study was conducted to collect mul-sensitized AR patients from allergy or respiratory departments of 5 hospitals who received house dust mite allergen preparation SCIT for 12 to 36 months and met other inclusion and exclusion criteria from February to July 2024. General clinical data were collected and the perennial or seasonal symptoms before and after treatment were evaluated with visual analogue scale (VAS) to assess whether there was an perennial or allergen-specific symptom improvement (VAS score decrease ≥30%), by which the patients were divided into effective group and ineffective. R software was used to analyze the differences between groups by using Fisher′s exact test and Mann-Whitney U test. Results:A total of 62 patients were enrolled, and the treatment were effective in 39 of them, with an effective rate of 62.9%. For allergen-specific symptoms, the median age of the effective group was higher than that of the ineffective group (12 years old vs. 8 years old, P=0.039), and the effective rate in dust mite specific immunoglobin E (sIgE) grade ≤5 group was higher than that in sIgE grade >5 group (81.6% vs. 45.5%, P=0.008), and the effective rate of mold sIgE grade ≤2 group was higher than that of sIgE grade >2 group (83.3% vs. 28.6%, P=0.045), and there was no statistically significant correlation between the other allergen grades and the effective rate ( P>0.05). For perennial symptoms, the effective rate in the mold grade ≤2 group was higher than that in the sIgE grade >2 group (91.3% vs. 28.6%, P=0.010), and there was no statistically significant correlation between the other allergen grades and the effective rate ( P>0.05). There was no significant correlation between the treatment effectiveness of perennial or allergen-specific symptoms and the number of combined allergens, the grade of skin test, and the difference between the grade of combined allergens and that of dust mites ( P>0.05). Conclusion:Among the patients with multi-sensitized AR allergic to dust mites included in this study, single dust mite SCIT is effective in some of them, and for allergen-specific symptoms, the effective group was elder, and dust mite sIgE grade 6 and mold sIgE grade ≥2 was related to the low effective rate of SCIT. The present results are insufficient for selecting single or multiple AIT in any type of multi-sensitized patients.

BACKGROUND:Oriented polycaprolactone/silk fibroin blend electrospinning scaffolds can repair peripheral nerve damage and promote nerve regeneration,but the repair effect is limited,and it is necessary to further combine bioactive materials,growth factors,and other factors to prepare bionic neural scaffolds.Calcium titanate nanoparticles have the ability to promote adhesion between Schwann cells and have been widely used in the field of biomedical materials.OBJECTIVE:To prepare polycaprolactone/silk fibroin-calcium titanate functionalized scaffolds by electrospinning technology,and to further explore the effect of the scaffold on Schwann cell viability and adhesion.METHODS:Polycaprolactone/silk fibroin scaffolds were prepared by electrospinning technology.The optimal parameters were selected by adjusting the syringe flow rate,receiving device speed and spinning time during the preparation process for subsequent experiments.Under the optimal parameters,randomly oriented polycaprolactone/silk fibroin scaffolds(denoted as PCL/SF-R),oriented polycaprolactone/silk fibroin scaffolds(denoted as CTO-0),and oriented polycaprolactone/silk fibroin scaffolds containing 0.1,0.5,and 1.0 mg/mL polydopamine modified calcium titanate(denoted as CTO-1,CTO-5,and CTO-10,respectively)were prepared by electrospinning technology.The physical properties of the five groups of scaffolds were characterized.The extracts of the five groups of scaffolds were co-cultured with rat fibroblast cell line(L929).Cell proliferation was detected by MTT assay.Rat Schwann cell line(RSC96)was inoculated on the five groups of scaffolds.Cell viability,cytoskeleton staining,and cytoskeleton and cell adhesion related genes were detected.RESULTS AND CONCLUSION:(1)The optimal parameters for preparing electrospinning scaffolds were as follows:syringe flow rate 15 μL/min,receiving device speed 1 800 r/min,and spinning time 1 hour.(2)The PCL/SF-R group had the largest water contact angle and the smallest elastic modulus,while the CTO-0 group had the smallest water contact angle and the largest elastic modulus.The degradation rate of the scaffolds in the CTO-0 group was lower than that in the CTO-1 group,and the degradation rate of the scaffolds decreased with the increase of the mass concentration of calcium titanate in the oriented scaffolds.(3)The scaffolds in the five groups had no toxic effects on L929 cells and had good biocompatibility.CCK-8 assay results showed that CTO-10 could improve the viability of RSC96 cells.Cytoskeleton staining results showed that the aspect ratio and cell protrusion length of cells on the oriented scaffolds were higher than those on the randomly oriented scaffolds.qPCR test results showed that the mRNA expressions of YAP and Cntn2 in the CTO-5 and CTO-0 groups were higher than those in the PCL/SF-R group(P＜0.05).(4)The results indicate that the oriented polycaprolactone/silk fibroin-calcium titanate scaffold has good biocompatibility,mechanical properties,and hydrophilicity,which can enhance the viability and migration ability of Schwann cells and promote peripheral nerve regeneration.