Corynebacterium pseudotuberculosis(C.pseudotuberculosis)is a group of intracellular Gram-positive bacteria that can cause zoonotic diseases.This study investigated the mechanisms of inflammatory mediator secretion and the phagocytic and bactericidal functions of mouse peritoneal macrophages following C.pseudotuberculosis infection.Initially,transcriptomic sequencing was em-ployed to identify genes critical for C.pseudotuberculosis infection in macrophages.Subsequently,gene knockout mice were utilized to assess the impact of these key genes on inflammatory media-tor secretion,activation of inflammatory signaling pathways,and the phagocytic and bactericidal functions of macrophages infected with C.pseudotuberculosis.Techniques such as ELISA,Western blot,and immunofluorescence were employed in this analysis.Further,transcriptomic sequencing was conducted to identify key downstream genes.Following C.pseudotuberculosis infection,GO enrichment analysis was performed,and TLR2 was identified as the focal point of the study.Perito-neal macrophages from C57BL/6J and TLR2 knockout(TLR2-/-)mice were infected with C.pseudotuberculosis.ELISA results revealed that the levels of TNF-α,IL-1β,and IL-10 were signifi-cantly downregulated in TLR2-/-macrophages compared to C57BL/6J macrophages post-infec-tion.Western blot demonstrated that the absence of TLR2 led to a marked decrease in M APK(p38 and ERK)signaling pathway phosphorylation following C.pseudotuberculosis infection.Immuno-fluorescence results indicated that the phagocytic rate of TLR2-/-macrophages was significantly higher than that of C57BL/6J macrophages after infection.Subsequently,transcriptomic analysis of C57BL/6J and TLR2-/-macrophages infected with C.pseudotuberculosis was performed,followed by GO enrichment analysis of differential genes.IL-36a,Cx3cr1,TLR1,and TLR2 were identified as key differential genes.TLR2 plays a crucial role in the inflammatory response induced by C.pseudotuberculosis infection in mice,influencing the progression of the inflammatory response and host outcomes through the secretion of inflammatory mediators,activation of signaling pathways,and modulation of phagocytic and bactericidal functions.IL-36a and Cx3cr1 were identified as key downstream factors in this process.

Corynebacterium pseudotuberculosis(C.pseudotuberculosis)is a group of intracellular Gram-positive bacteria that can cause zoonotic diseases.This study investigated the mechanisms of inflammatory mediator secretion and the phagocytic and bactericidal functions of mouse peritoneal macrophages following C.pseudotuberculosis infection.Initially,transcriptomic sequencing was em-ployed to identify genes critical for C.pseudotuberculosis infection in macrophages.Subsequently,gene knockout mice were utilized to assess the impact of these key genes on inflammatory media-tor secretion,activation of inflammatory signaling pathways,and the phagocytic and bactericidal functions of macrophages infected with C.pseudotuberculosis.Techniques such as ELISA,Western blot,and immunofluorescence were employed in this analysis.Further,transcriptomic sequencing was conducted to identify key downstream genes.Following C.pseudotuberculosis infection,GO enrichment analysis was performed,and TLR2 was identified as the focal point of the study.Perito-neal macrophages from C57BL/6J and TLR2 knockout(TLR2-/-)mice were infected with C.pseudotuberculosis.ELISA results revealed that the levels of TNF-α,IL-1β,and IL-10 were signifi-cantly downregulated in TLR2-/-macrophages compared to C57BL/6J macrophages post-infec-tion.Western blot demonstrated that the absence of TLR2 led to a marked decrease in M APK(p38 and ERK)signaling pathway phosphorylation following C.pseudotuberculosis infection.Immuno-fluorescence results indicated that the phagocytic rate of TLR2-/-macrophages was significantly higher than that of C57BL/6J macrophages after infection.Subsequently,transcriptomic analysis of C57BL/6J and TLR2-/-macrophages infected with C.pseudotuberculosis was performed,followed by GO enrichment analysis of differential genes.IL-36a,Cx3cr1,TLR1,and TLR2 were identified as key differential genes.TLR2 plays a crucial role in the inflammatory response induced by C.pseudotuberculosis infection in mice,influencing the progression of the inflammatory response and host outcomes through the secretion of inflammatory mediators,activation of signaling pathways,and modulation of phagocytic and bactericidal functions.IL-36a and Cx3cr1 were identified as key downstream factors in this process.

Objective To study the common etiological components and bedside diagnostic skills of isolated transient vertigo (ITV).Methods We performed a single-center observational study that had consecutively recruited 424 middle-aged and elderly patients presenting with vertigo episodes lasted less than three minutes in the Neurology Department of Shanghai Tongji Hospital from January 2018 to January 2020.The clinical characteristics were collected and analyzed according to the established format and vertigo causes were analyzed.Binary Logistic regression analysis was used to determine some clinical parameters to benign paroxysmal positional vertigo (BPPV) and vascular ITV (vITV).Results In all 424 patients,BPPV accounted for 65.09% (n=276),vITV for 20.51% (n=87).Fifty-two cases (18.84%) of BPPV were diagnosed as posterior circulation ischemia (PCI) and 12 cases (14.63%) of vITV were suspected as BPPV.Logistic Regression analysis showed that onset time of vertigo<30 s,vertigo provoked by lying down or turning over in the supine position and typical nystagmus by positional maneuver were the independent predicted factors of BPPV (P<0.001).The higher risk of vITV was found in association with intolerance of head movement,trunk balance disturbance,spontaneous nystagmus and intolerance by positional maneuver (P<0.001).There was no independent correlation between vascular risk factors and vITV (P>0.05).Conclusion BPPV and vITV are the most common etiologies of ITV in the middle-aged and elderly patients.The etiology of ITV can be quickly and effectively distinguished by some specific clinical features and target bedside examinations.

Objective To analyze the clinical manifestations and genetic mutations in 3 pedigrees with hereditary spastic paraplegia (HSP).Methods Three pedigrees diagnosed as having HSP in our hospital from January 2014 to November 2015,were chosen;the clinical manifestations,electrophysiology and imaging features of the patients in these three families were analyzed.Genomic DNA was extracted from peripheral venous blood,and the targeted gene capturing was employed to identify the disease-causing genes of these patients.Results The patients from the first family was familiar HSP,and the main clinical features were progressive lower limbs weakness and abnormal gait without cognitive impairment;the patients from the second family were familiar HSP and those from the third family were HSP without family history,and the main clinical features of the two pedigrees were slowly progressive spastic paraplegia and cognitive impairment.In addition,thin corpus callosum was visible in MR imaging of family three.Genetic testing showed the first family presented with a known mutation c.715C＞T ofA TL1 exon 7 and the loci co-segregated in the family.The second family presented with novel compound heterozygous mutations in the SPG11 gene:c.3099_3103delGTTTG mutation of exon 17and c3817 3818insTGA mutation of exon 22;novel compound heterozygous mutations in the SPG11 gene in the third family were detected as follows:c.6194C ＞G mutation of exon 32 and c.5121+1C＞T splicing mutation ofintro 29.Conclusions Four novel mutations in SPG11 gene and one known mutation in A TL1 gene are found,which enriches the known HSP mutation types.Targeted gene capture is an efficient and rapid tool for identifying the causation of some complex and genetically heterogeneous neurodegenerative diseases.

Objective To investigate the influence of one kind of defective gene NOTCH3 (R90C) of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in proliferation of oligodendrocyte lineage cells HS683 and their related molecular mechanism.Methods (1) A pCMV-Sport6.0 was chosen as the expression vector and site-directed mutation was used to construct the mutant NOTCH3 (p.R90C) expression vector;eukaryotic cell transfection technique was used to respectively transfect the pCMV-Sport6.0 empty vector,wild NOTCH3 vector (p.NOTCH3) and mutant NOTCH3 (p.R90C) expression vector to HS683 cells (blank control vector group,wild NOTCH3 vector group,and mutant NOTCH3 vector group);the protein expressions of NOTCH3,p53,phosphorylated p53 and p21 were detected by Westem blotting.(2) Wild NOTCH3 vector group,mutant NOTCH3 vector group and mutant NOTCH3 vector+pifithrin-α group were divided,and after wild NOTCH3 vector (p.NOTCH3) and mutant NOTCH3 (p.R90C) vector transfection,the latter two groups were added 0 or 1 μmol/L pifithrin-α,respectively;CCK-8 assay was employed to test the proliferation oftransfected HS683 cells 24,48 and 72,and 96 h after transfection.Results (1) As compared with wild NOTCH3 vector group,mutant NOTCH3 vector group had significantly lower absorbance value 24,48 and 72 h after transfection (P＜0.05);72 h after transfection,wild NOTCH3 vector group and mutant NOTCH3 vector group had significantly higher NOTCH3 protein expression as compared with blank control vector group (P＜0.05),and mutant NOTCH3 vector group had significantly higher p53,phosphorylated-p53 and p21 protein expressions as compared with wild NOTCH3 vector group and blank control vector group (P＜0.05).(2) The absorbance value in the mutant NOTCH3 vector+pifithrin-α group was significantly increased as compared with that in the mutant NOTCH3 vector group 48,72 and 96 h after transfection (P＜0.05).Conclusion Mutation of NOTCH3 (R90C) may inhibit the proliferation of oligodendrocyte cell lineage via p53 dependent way,which might play a direct role in demyelination pathology of CADASIL caused by NOTCH3(R90C).

Objective To understand the laboratory testing current situation of ABO hemolytic disease of the newborn(ABO-HDN)in Chizhou area,and to analyze the test results of serological three indexes tests in order to provide the basis for clinical diag-nosis.Methods The ABO blood group identification and serological three indexes tests(direct antiglobulin test,free antibody test, antibody release test)were performed by using microcolumn gel method.Results A,B,O and AB blood groups were 29.13%, 31.09%,37.82% and 1.96%;the total positive rate of ABO-HDN was 22.41%(80/357),the positive rates of ABO-HDN in A and B blood groups were 38.46% (40/104)and 36.04% (40/111 )respectively;the occurrence rate of ABO-HDN had no statistical difference between blood group A and B (P >0.05);the positive rates of the direct antiglobulin test,free antibody test and antibody release test were 1.96%(7/357),4.76%(17/357)and 22.41%(80/357)respectively.Conclusion The serological three indexes tests are the main basis for the diagnosis of ABO-HDN,the antibody release test shows the highest positive rate.If clinically consid-ering HDN,the newborns should conduct the ABO-HDN screening as early as possible for clarifying the diagnosis and performing the early treatment.