The inhibitory effect of SEMA3C/3D mutations on Neuro-2a cells migration and axonal growth in patients with Hirschsprung′s disease

Objective:To explore the effect and mechanism of SEMA3C/3D mutations on axonal growth of neurons and cell migration in Hirschsprung′s disease(HSCR)patients.Methods:HEK293 Tcells were transfected with wild-type and mutant SEMA3C/3D plasmids.The supernatants that contained SEMA3C/3D-AP fusion proteins were collected and added into the Neuro-2acells.The changes in the cell morphology were observed by immunofluorescence staining.The expression and phosphorylation levels of cofilin,ERM and CRMP2 were determined by western blotting.The cell migration rate was measured by transwell assay.Results:Compared with wild-type SEMA3D,SEMA3D-P615T mutant suppressed cofilin phosphorylation in Neuro-2a cells(P <0.05).The neural cells treated by five mutant SEMA3C/3D-AP fusion proteins presented different levels of axon atrophy,growth cone collapse,and sometimes,loss of normal structure.SEMA3C-S329 G,SEMA3C-V337M and SEMA3D-P615T mutants cells exhibited a significantly reduced migration rate as compared with wild-type SEMA3C/3D treated cells(P <0.01).Conclusion:SEMA3C and SEMA3 D mutations in HSCR patients could lead to the inhibition of Neuro-2a cells′migration and axonal growth.The mechanism of SEMA3 DP615T mutant might be related to down-regulation of the expression of p-cofilin,which consequently lead to cytoskeleton structure collapse and migrating ability decrease.Our study might provide important clues for the pathogenesis of HSCR.

More common RNAemia in the early stage of severe SARS-CoV-2 BF.7.14 infections in pediatric patients

The risk factors of severe infections in children during the severe acute respiratory syndrome coronavirus 2(SARS‐CoV‐2)outbreak in Beijing remain elusive.SARS‐CoV‐2‐positive children admitted to the intensive care unit(ICU)with collected plasma specimens were enrolled and screened for common pathogens using capillary electrophoresis‐based multiplex PCR from December 12,2022,to January 24,2023.The SARS‐CoV‐2 subvariants were identified using next‐generation sequencing.Plasma was positive for two(positive;P),one(suspicious;S),or no(negative;N)SARS‐CoV‐2 genes were classified as plasmatic RNA‐positive(RNAemia;P+S)or without RNAemia(N).Clinical and laboratory data of the enrolled cases were then collected and analyzed.The 34 enrolled children included 26 males and 24 younger than three years.All were negative for other respiratory pathogens.BF.7.14(18/29)was the predominant subvariant.Viral loads in respiratory specimens,hours from symptom onset to the first respiratory specimen collection(time‐variable),with comorbidities and BF.7.14 and BA.5.2 distributions were significantly different in P vs.N and RNAemia vs.without RNAemia group.Among most cases,the T lymphocyte ratios decreased,while the cytokine level and the B lymphocyte ratio increased.The time variables were 2.22±2.05 and 4.00±2.49 days in BF.7.14 and BA.5.2 infections,respectively.In conclusion,SARS‐CoV‐2 was more likely to cause severe infections among males aged≤3 years old with comorbidities during the SARS‐CoV‐2 outbreak in Beijing,while RNAemia is more common in children at the early stage of severe BF.7.14 infections,and most had high cytokine levels and B‐cell activation.

Surveillance of Mycoplasma pneumoniae infection among children in Beijing from 2007 to 2012

Background Mycoplasma pneumonia （M.pneumoniae） is one of the key pathogens of community-acquired pneumonia.A global pandemic of M.pneumoniae has occurred since 2010.The aim of this study was to survey the prevalence of M.pneumoniae in children in Beijing from 2007-2012.Methods A total of 3 073 clinical specimens were obtained from pediatric patients with respiratory tract infections from January 2007 to December 2012,and examined by nested polymerase chain reaction.PCR products were visualized by 2％ agarose gel electrophoresis,positive products sequenced,and compared with reference sequences in GenBank.Macrolide resistance-associated mutations were also detected for some positive samples.Results Of the 3 073 specimens,588 （19.13％） were positive for M.pneumoniae,12.4％ of which were accompanied by viral infections.Positive rates for M.pneumoniae were highest in 2007 and 2012,showing a significant difference when compared with other years.Infections tended to occur in autumn and winter and positive rates were significantly higher for children aged 3-16.The rate of macrolide resistance-associated mutations was 90.7％,and the predominant mutation was an A→G transition （89.92％） at position 2063 in domain V of the 23S rRNA gene.Conclusions M.pneumoniae outbreaks occurred in 2007 and 2012 in pediatric patients in Beijing,which is consistent with the global prevalence of M.pneumoniae.M.pneumoniae can cause multi-system infections in children,and may be accompanied with viral infections.We determined that school-age children are more susceptible to this disease,particularly in autumn and winter.Gene mutations associated with macrolide resistance were very common in M.pneumoniae-positive specimens during this period in Beijing.

A single tube modified allele-specific-PCR for rapid detection of erythromycin-resistant Mycoplasma pneumoniae in Beijing

Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.