Objective:To evaluate the application of metagenomic next-generation sequencing（mNGS）in the identification of non-tuberculous mycobacteria（NTM）.Methods:A retrospective analysis was conducted on mNGS results of 358 bronchoalveolar lavage fluid（BALF）samples positive for NTM collected at the First Affiliated Hospital of Zhejiang University School of Medicine from February 2021 to January 2024. The analysis included the distribution of NTM species，the detection of mixed pathogens，and the performance of conventional mycobacterial detection methods.Results:The results showed that 362 strains of 15 NTM species were identified from 350 specimens，8 specimens were not precise to the species level. The most frequently detected species were Mycobacterium intracellulare（37.3%，135/362）， Mycobacterium abscessus（26.8%，97/362），followed by Mycobacterium avium（11.0%，40/362）， Mycobacterium kansasii（8.0%，29/362）and Mycobacterium chelonae（7.7%，28/362）. Single NTM species were detected in 339 specimens，while two or three NTM species were simultaneously detected in 11 specimens（3.1%，11/358）. Non-NTM microorganisms co-infected were detected in 53.4%（191/358）of NTM-positive BALF samples，including common pathogens such as Pseudomonas aeruginosa， Staphylococcus aureus，and Aspergillus fumigatus；and difficult-to-identify pathogens such as Legionella pneumophila and Talaromyces marneffei. In NTM-positive patients detected by mNGS，the results supported the diagnosis of NTM infection in 298 cases（298/358，83.2%）and 105 cases（105/358，29.3%）initiated anti-NTM treatment accordingly；while in 60 cases（60/358，16.8%）the positive results were considered as colonization or unrelated to clinical infection. For samples tested with acid-fast staining，mycobacterial liquid culture，and DNA microarray，the positivity rates for NTM were 31.5%（73/232），48.7%（57/117），and 43.0%（46/107），respectively. Conclusions:mNGS demonstrates advantages in identification of NTM. However，the test may detect multiple microorganisms，in that case，the interpretation with clinical and radiological results is requried to determine the main pathogens.

Objective:To evaluate the application of metagenomic next-generation sequencing（mNGS）in the identification of non-tuberculous mycobacteria（NTM）.Methods:A retrospective analysis was conducted on mNGS results of 358 bronchoalveolar lavage fluid（BALF）samples positive for NTM collected at the First Affiliated Hospital of Zhejiang University School of Medicine from February 2021 to January 2024. The analysis included the distribution of NTM species，the detection of mixed pathogens，and the performance of conventional mycobacterial detection methods.Results:The results showed that 362 strains of 15 NTM species were identified from 350 specimens，8 specimens were not precise to the species level. The most frequently detected species were Mycobacterium intracellulare（37.3%，135/362）， Mycobacterium abscessus（26.8%，97/362），followed by Mycobacterium avium（11.0%，40/362）， Mycobacterium kansasii（8.0%，29/362）and Mycobacterium chelonae（7.7%，28/362）. Single NTM species were detected in 339 specimens，while two or three NTM species were simultaneously detected in 11 specimens（3.1%，11/358）. Non-NTM microorganisms co-infected were detected in 53.4%（191/358）of NTM-positive BALF samples，including common pathogens such as Pseudomonas aeruginosa， Staphylococcus aureus，and Aspergillus fumigatus；and difficult-to-identify pathogens such as Legionella pneumophila and Talaromyces marneffei. In NTM-positive patients detected by mNGS，the results supported the diagnosis of NTM infection in 298 cases（298/358，83.2%）and 105 cases（105/358，29.3%）initiated anti-NTM treatment accordingly；while in 60 cases（60/358，16.8%）the positive results were considered as colonization or unrelated to clinical infection. For samples tested with acid-fast staining，mycobacterial liquid culture，and DNA microarray，the positivity rates for NTM were 31.5%（73/232），48.7%（57/117），and 43.0%（46/107），respectively. Conclusions:mNGS demonstrates advantages in identification of NTM. However，the test may detect multiple microorganisms，in that case，the interpretation with clinical and radiological results is requried to determine the main pathogens.

Objective To explore the expression of glial cell line-derived neurotrophic factor (GDNF) in rats with spinal cord injury (SCI) after epidermal neural crest stem cells (EPI-NCSCs) transplantation.Method EPI-NCSCs were isolated from GFP transgenic rats for transplantation.The rat SCI model was made by NYU-II impactor (10 g 25 mm) at T10 level.Then 30 SD rats were randomly divided into blank injury group (group A),DMEM transplantation group (group B),and experimental group (group C).The EPI-NCSCs were transplanted into the injured region one week after SCI.In DMEM group,the DMEM/F12 was used to substitute for the EPI-NCSCs.No treatment was done in blank injury group.The locomotor function was appraised by BBB score every week after transplantation.At sixth week after transplantation,GDNF mRNA and protein expression was detected.Result The BBB score in experimental group was significantly higher than the other two groups from two weeks after transplantation (P＜0.05).The expression of GDNF mRNA and protein in experimental group was significantly higher than the other two groups (P＜0.05).There was no significant difference between blank injury group and DMEM transplantation group (P ＞ 0.05).Conclusion The expression of GDNF can be up-regulated by EPI-NCSCs transplantation,which may be one of the mechanisms for EPI-NCSCs repairing SCI.

Objective To detect the myelinating role of olfactory ensheathing cells (OECs in the contused spinal cord and their impact on remyelination.Methods The rats were subjected to spinal cord injury at T10(10 g ×25 mm) using a NYU-Ⅱ impactor.One week later,the rats were transplanted with green fluorescence protein (GFP)-OECs (OECs group) or an equal volume of Dulbecco' s modification of Eagle's medium (DMEM) (control group) at epicenter of the injury as well as its rostral and caudal sites.Six weeks after transplantation,the spinal cords were removed for frozen section.Myelin basic protein (MBP),protein zero (P0),and S100 protein (S100) were determined with qualitative and semi-quantitative immunocytochemical assay.Moreover,plastic embedded semithin and ultrathin sections were prepared for qualitative and semi-quantitative examination under light microscopy and electroscopic study of myelin sheath ultrastructure.Results In OECs group,the nerve fibers labeled with S100,MBP,and PO were extended from the normal tissues to the injured region and even grew through the region with space consuming of 12.3％,11.6％,and 9.3％ respectively.Moreover,there were no statistical differences regarding the number of fibers labeled by the three proteins,but all were significantly larger than that in control group (2.89％,P ＜ 0.01).Number of myelinated nerve fibers in injured regions on hemithin sections was increased significantly to 354.67 ± 59.00 in OECs group,with significant difference compared with 167.33 ± 42.16 in control group (P ＜ 0.01).The regenerated myelin sheaths in OECs group were smaller and thicker than those in control group.Conclusions OECs can accelerate regeneration of myelinated nerve fibers.Additionally,some OECs form myelin sheaths themselves,but the sheath structures are relatively thinner.

Neural crest stem cells originated from hair follicle (epidermal neural crest stem cell, EPI-NCSC) are easy to obtain and have potentials to differentiate into various tissues, which make them eminent seed cells for tissue engineering. EPI-NCSC is now used to repair nerve injury, especially, the spinal cord injury. To investigate their effects on repairing peripheral nerve injury, EPI-NCSC from a GFP-SD rat were primarily cultured on coated dishes and on a poly lactic acid coglycolic acid copolymer (PLGA) membrane. Methyl thiazolyl tetrazolium (MTT) assay showed that the initial adhesion rate of EPI-NCSC was 89.7% on PLGA membrane, and the relative growth rates were 89.3%, 87.6%, 85.6%, and 96.6% on the 1st, 3rd, 5th, 7th day respectively. Cell cycles and DNA ploidy analysis demonstrated that cell cycles and proliferation indexes of cultured EPI-NCSC had the same variation pattern on coated dishes and PLGA membrane. Then cultured EPI-NCSC were mixed with equal amount of extracellular matrix and injected into a PLGA conduit to connect a 10 mm surgery excision gap of rat sciatic nerve, Dulbecco's Modified Eagle's medium (DMEM) was used to substitute EPI-NCSC in the control group. After four weeks of transplantation, the defected sciatic nerve achieved a histological restoration, the sensory function of rat hind limb was partly recovered and the sciatic nerve index was also improved. The above results showed that a PLGA conduit filled with EPI-NCSC has a good repair effect on the peripheral nerve injury.
Animals ; Cells, Cultured ; Neural Crest ; cytology ; Neural Stem Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; Spinal Cord Injuries ; Stem Cell Transplantation ; Tissue Engineering

ObjectiveTo investigate the influence of olsalazine on tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in patients with chronic ulcerative colitis.MethodsSixty patients with chronic ulcerative colitis(observation group),including 22 chronic recurrent cases and 38 chronic persistent cases,were enrolled and treated with olsalazine.Meanwhile,60 healthy volunteers without disease history of ulcerative colitis were selected as control group.The concentrations of TNF- α and IL-10 in serum of the two groups were detected by ELISA and compared.ResultsBefore treatment,the concentration of TNF- α in serum of the observation group was significantly higher than that of control group [ (57.2 ± 10.1 )ng/L vs.(27.2 ± 6.9) ng/L],while IL-10 was significantly lower than that of control group[ (9.2 ± 2.1 ) ng/L vs.(17.3 ±2.9) ng/L] (P ＜0.05).Before treatment,the concentration of TNF-α in serum in chronic persistent patients and chronic recurrent patients[ (56.9 ± 9.9),(57.3 ± 9.7) ng/L ] were significantly higher than that in control group,and serum IL-10 in chronic persistent patients and chronic recurrent patients [ (9.1 ± 2.3 ),(8.4 ± 2.5 ) ng/L ] was significantly lower than that in control group (P＜ 0.05 ).The concentration of TNF- α in serum in observation group after treatment was obviously lower than that before treatment [(28.1 ±8.9) ng/L vs.(57.2 ± 10.1 ) ng/L],and IL-10 was obviously higher than that before treatment [(13.4 ± 10.7) ng/L vs.(9.2 ±2.1 )ng/L] (P ＜ 0.05).The concentration of serum TNF-αand IL-10 in chronic persistent and chronic recurrent patients before and after treatment had statistical significance (P＜0.05 ).ConclusionsOlsalazine can significantly decrease the concentration of TNF- α and increase the concentration of IL-10 in serum in patients with chronic ulcerative colitis.It is worthy of application in clinic.