Objective To explore the relationship between circadian rhythm genes and the occurrence, development, prognosis, and tumor microenvironment (TME) of lung adenocarcinoma (LUAD). Methods The Cancer Genome Atlas data were used to evaluate the expression, copy number variation, and somatic mutation frequency of circadian gene sets in LUAD. Gene ontology, Kyoto encyclopedia of genes and genomes, and gene set enrichment analysis were used to explore the potential mechanisms by which circadian rhythm genes affected LUAD progression. Cox regression, least absolute shrinkage and selection operator regression, support vector machine recursive feature elimination, and random forest screened circadian genes and established prognostic models, and on this basis constructed nomogram to predict patients’ 1-, 3-, and 5-year survival rates. Kaplan-Meier survival curves, receiver operating characteristic (ROC) curves, and time-dependent ROC curves were drawn to evaluate the predictive ability of the model, and the external dataset of GEO further verified the prognostic value of the prediction model. In addition, we evaluated the association of the prognostic model with immune cells and immune checkpoint genes. Single cell RNA sequencing (scRNA-seq) analysis was used to explore the molecular characteristics between prognostically relevant circadian genes and different immune cell populations in TME. Results Differentially expressed circadian rhythm genes were mainly enriched in biological processes related to cGMP-PKG signaling pathway, lipid and atherosclerosis, and JAK-STAT signaling pathway. Seven circadian rhythm genes: LGR4, CDK1, KLF10, ARNTL2, RORA, NPAS2, PTGDS were screened out, and a RiskScore model was established. According to the median RiskScore, samples were divided into a high-risk group and a low-risk group. Compared with patients in the low-risk group, patients in the high-risk group showed a poorer prognosis (P<0.001). Immunological characterization analysis showed that there were differences in the infiltration of multiple immune cells between the low-risk group and high-risk group. Most immune checkpoint genes had higher expression levels in the high-risk group than those in the low-risk group, and RiskScore was positively correlated with the expression of CD276, TNFSF4, PDCD1LG2, CD274, and TNFRSF9, and negatively correlated with the expression of CD40LG and TNFSF15. The scRNA-seq analysis showed that RORA and KLF10 were mainly expressed in natural killer cells. Conclusion The prognostic model based on seven feature circadian rhythm genes has certain predictive value for predicting survival of LUAD patients. Dysregulated expression of circadian genes may regulate the occurrence, progression as well as prognosis of LUAD through affecting TME, which provides a possible direction for finding potential strategies for treating LUAD from the perspective of mechanism by which circadian disorder affects immune cells.

Objective To determine the prognostic biomarkers and new therapeutic targets of the lung adenocarcinoma (LUAD), based on which to establish a prediction model for the survival of LUAD patients. Methods An integrative analysis was conducted on gene expression and clinicopathologic data of LUAD, which were obtained from the UCSC database. Subsequently, various methods, including screening of differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA), were employed to analyze the data. Cox regression and least absolute shrinkage and selection operator (LASSO) regression were used to establish an assessment model. Based on this model, we constructed a nomogram to predict the probable survival of LUAD patients at different time points (1-year, 2-year, 3-year, 5-year, and 10-year). Finally, we evaluated the predictive ability of our model using Kaplan-Meier survival curves, receiver operating characteristic (ROC) curves, and time-dependent ROC curves. The validation group further verified the prognostic value of the model. Results The different-grade pathological subtypes' DEGs were mainly enriched in biological processes such as metabolism of xenobiotics by cytochrome P450, natural killer cell-mediated cytotoxicity, antigen processing and presentation, and regulation of enzyme activity, which were closely related to tumor development. Through Cox regression and LASSO regression, we constructed a reliable prediction model consisting of a five-gene panel (MELTF, MAGEA1, FGF19, DKK4, C14ORF105). The model demonstrated excellent specificity and sensitivity in ROC curves, with an area under the curve (AUC) of 0.675. The time-dependent ROC analysis revealed AUC values of 0.893, 0.713, and 0.632 for 1-year, 3-year, and 5-year survival, respectively. The advantage of the model was also verified in the validation group. Additionally, we developed a nomogram that accurately predicted survival, as demonstrated by calibration curves and C-index. Conclusion We have developed a prognostic prediction model for LUAD consisting of five genes. This novel approach offers clinical practitioners a personalized tool for making informed decisions regarding the prognosis of their patients.

Objective To explore the changes in neural activity in patients with type 2 diabetes mellitus(T2DM)and their corre-lation with executive function,and to analyze the neural mechanisms underlying the decline in executive function in T2DM patients.Methods Thirty-one T2DM patients(T2DM group)and thirty-two healthy controls(HC)(HC group)matched for body mass index(BMI)underwent resting-state functional magnetic resonance imaging(rs-fMRI)scans and N-back task tests were included.Differ-ences in the amplitude of low-frequency fluctuation(ALFF),regional homogeneity(ReHo),and seed-based functional connectivity(FC)between the two groups were compared,and partial correlation analyses were performed between the difference results and N-back task performance.Results The T2DM group showed prolonged reaction time(RT)in the 1-back and 2-back tasks.T2DM patients exhibited increased ALFF in the bilateral caudate nucleus,left medial superior frontal gyrus,and right postcentral gyrus,as well as elevated ReHo in the right putamen.FC analysis revealed significant alterations in FC between the caudate nucleus,putamen,and multiple brain regions in T2DM patients,with some of these FC changes significantly correlated with RT and accuracy(ACC)in the N-back task.Conclusion The decline in executive function in T2DM patients may be associated with abnormal neural activity in brain regions such as the striatum,salience network,and frontoparietal control network.FC further decreases under increased cognitive load.These findings provide evidence for the study of the neural mechanisms of executive function impairment in T2DM patients.

Objective"Red butterfly sore","yin-yang toxin"and"Bi disease"are different Chinese medicine diagnoses of systemic lupus erythematosus(SLE).It is not clear whether there are biological differences between these three types of Chinese medicine diagnoses.The aim of this study was to compare the different TCM diagnoses of SLE patients from the perspective of lymphocyte subsets and cytokines.Methods Patients diagnosed with SLE in our hospital from June 1,2021 to December 1,2023 were retrospectively collected,and the differences of T cell subsets,NK cells,B lymphocytes and Th1,Th2 and Th17 cytokines among different groups were compared by one-way ANOVA or nonparametric test.As well as differences in laboratory test indicators such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),immunoglobulin,and complement,receiver operating characteristic curve(ROC curve)was used to analyze the value of these indexes in the differential diagnosis of different types of SLE.Results From June 1,2021 to December 1,2023,291 patients diagnosed with SLE in our hospital for the first time were collected,and 104 cases meeting the exclusion criteria of this study were included,including 31 cases of red butterfly sores,30 cases of yin-yang toxin and 43 cases of BI disease.The absolute number and percentage of CD8+T cells,interleukin-10(IL-10)content and tumor necrosis factor α(TNF-α)content were different among the three groups of SLE patients diagnosed by different Chinese medicine,and the absolute number of CD8+T cells in the red butterfly sore group was significantly higher than that in the yin-yang toxicities group(P=0.039)and the disease group(P=0.008).CD8+T cell percentage in red butterfly sore group was significantly higher than that in yin-yang toxin group(P=0.014)and disease group(P=0.004),IL-10 and TNF-α levels in red butterfly sore group were significantly lower than those in disease group(P=0.015,P=0.036),and ROC curve analysis showed that,the absolute number and percentage of CD8+T cells can effectively distinguish red butterfly sores from yin-yang toxins(AUC=0.65,AUC=0.61,P<0.05),and the absolute number and percentage of CD8+T cells,IL-10 and TNF-α can effectively distinguish red butterfly sores from diseases(AUC=0.68,AUC=0.66,P<0.01,AUC=0.67,AUC=0.64,P<0.05).Conclusion Immune lymphocyte subtypes,cytokines,especially the absolute number and percentage of CD8+T cells,IL-10 and TNF-α may play an important role in the identification of different TCM diagnosis of SLE.

A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

Objective To investigate the effects of Xiaoyukang capsule on neuroinflammation and neuronal apoptosis after Intracerebral hemorrhage(ICH)in rats by regulating JNK/c-JUN signaling pathway.Methods Adult male SD rats were intrastriatally injected with bacterial collagenase Ⅶ to induce an ICH model and they were randomly divided into blank control group,model control group,Xiaoyukang capsule small dose group,medium dose group,and large dose group.Neurobehavioral tests,body mass measurements,hematoma volume statistics,hematoxylin-eosin(HE)staining,immunofluorescence,deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining,enzyme-linked immunosorbent assay(ELISA)and Western Blotting were performed after 3 and 5 days,respectively.Results Compared with the blank control group,the rats in the model control group had severe neurobehavioral defects and weight loss(P＜0.05).The arrangement of neurons in brain tissue was disordered,and there was microglia/macrophages activation,neutrophil infiltration,neuronal apoptosis(P＜0.05).The levels of pro-inflammatory factors TNF-α,IL-1β and the expression of p-JNK,p-c-JUN,Bax,Caspase-3 and Cleaved Caspase-3 protein around hematoma were significantly increased(P＜0.05),while the anti-inflammatory factor IL-10 and anti-apoptotic protein Bcl-2 were decreased(P＜0.05).Compared with the model control group,Xiaoyukang capsule large dose group significantly improved the neurobehavioral function of rats,promoted weight recovery and hematoma absorption(P＜0.05).Reduce the pathological injury of brain tissue,inhibition of microglia/macrophages activation,neutrophil infiltration and neuronal apoptosis(P＜0.05).In addition,the levels of pro-inflammatory factors TNF-α,IL-1β and the expression of p-JNK,p-c-JUN,Bax,Caspase-3,Cleaved Caspase-3 protein around hematoma were significantly decreased(P＜0.05),and the anti-inflammatory factor IL-10 and anti-apoptotic protein Bcl-2 were increased(P＜0.05).Conclusion Xiaoyukang capsule can improve neurobehavioral defects in ICH rats,promote body mass recovery and hematoma absorption,reduce pathological damage of brain tissue,inhibit microglia/macrophage activation and neutrophil infiltration,its mechanism may be achieved by inhibiting JNK/c-JUN-mediated neuroinflammation and neuronal apoptosis.

Objective To evaluate the association between cervical insufficiency(CI)and spontaneous preterm birth(SPB),and to investigate the predictive value of shear wave elastography(SWE),cervical length(CL),and anterior cervical angle(ACA)in identifying SPB resulting from CI.Methods This study recruited 786 pregnant women who attended prenatal outpatient clinics or were hospitalized for treatment at the Affiliated Hospital of Nantong University and the Maternity Hospital affiliated with Jiangnan University from June 2023 to January 2024.Among these participants,723 were full-term pregnancies and 63 were preterm pregnancies.Cervical SWEI,CL,and cervical ACA were measured using a Mindray Resona R9 Pro/Eagus R9s ultrasound device equipped with shear wave elasticity imaging software.Additionally,the participants were categorized into four gestational age groups:19-23+6 weeks,24-27+6 weeks,28-33+6 weeks,and 34-36+6 weeks,to evaluate the relationship between gestational age and ultrasound parameters.Results The mean CL in the preterm group was 25.4 mm,significantly shorter than the 29.7 mm observed in the full-term group(P<0.001).The ACA in the preterm group was 121°,which was significantly higher than the 99° in the full-term group(P<0.001).Additionally,SWE values were significantly lower in the preterm group compared to the full-term group at all measured cervical sites:anterior external os(AE)6.47 kPa versus 9.91 kPa(P<0.001),anterior internal os(AI)10.98 kPa versus 18.62 kPa(P<0.001),posterior internal os(PI)11.32 kPa versus 21.09 kPa(P<0.001),and posterior external os(PE)8.16 kPa versus 13.24 kPa(P<0.001).A significant negative correlation was found between CL and gestational age(r=-0.278,P=0.001).The combined predictive indicators demonstrated high accuracy and specificity for predicting preterm birth,with an area under the curve(AUC)of 0.952,sensitivity of 95%,and specificity of 86%.Conclusions The integration of cervical SWE,CL measurement,and assessment of the angle of ACA can substantially enhance the predictive accuracy for SPB due to CI.This multimodal approach offers clinicians a more robust and precise tool for identifying high-risk pregnancies,enabling timely interventions that can reduce the incidence of preterm birth and improve maternal and neonatal outcomes.

Objective To evaluate the association between cervical insufficiency(CI)and spontaneous preterm birth(SPB),and to investigate the predictive value of shear wave elastography(SWE),cervical length(CL),and anterior cervical angle(ACA)in identifying SPB resulting from CI.Methods This study recruited 786 pregnant women who attended prenatal outpatient clinics or were hospitalized for treatment at the Affiliated Hospital of Nantong University and the Maternity Hospital affiliated with Jiangnan University from June 2023 to January 2024.Among these participants,723 were full-term pregnancies and 63 were preterm pregnancies.Cervical SWEI,CL,and cervical ACA were measured using a Mindray Resona R9 Pro/Eagus R9s ultrasound device equipped with shear wave elasticity imaging software.Additionally,the participants were categorized into four gestational age groups:19-23+6 weeks,24-27+6 weeks,28-33+6 weeks,and 34-36+6 weeks,to evaluate the relationship between gestational age and ultrasound parameters.Results The mean CL in the preterm group was 25.4 mm,significantly shorter than the 29.7 mm observed in the full-term group(P<0.001).The ACA in the preterm group was 121°,which was significantly higher than the 99° in the full-term group(P<0.001).Additionally,SWE values were significantly lower in the preterm group compared to the full-term group at all measured cervical sites:anterior external os(AE)6.47 kPa versus 9.91 kPa(P<0.001),anterior internal os(AI)10.98 kPa versus 18.62 kPa(P<0.001),posterior internal os(PI)11.32 kPa versus 21.09 kPa(P<0.001),and posterior external os(PE)8.16 kPa versus 13.24 kPa(P<0.001).A significant negative correlation was found between CL and gestational age(r=-0.278,P=0.001).The combined predictive indicators demonstrated high accuracy and specificity for predicting preterm birth,with an area under the curve(AUC)of 0.952,sensitivity of 95%,and specificity of 86%.Conclusions The integration of cervical SWE,CL measurement,and assessment of the angle of ACA can substantially enhance the predictive accuracy for SPB due to CI.This multimodal approach offers clinicians a more robust and precise tool for identifying high-risk pregnancies,enabling timely interventions that can reduce the incidence of preterm birth and improve maternal and neonatal outcomes.