BACKGROUND: Myocardial ischemia-reperfusion (I/R) is a serious and irreversible injury. Bone marrow-derived mesenchymal stem cells (MSCs) is considered to be a potential therapy for I/R injury due to the paracrine effects. High-mobility group box 1 (HMGB1) is a novel mediator in MSC and regulates the response of inflammation injury. Signal Transduction and Transcription Activator 3 (STAT3) is a critical transcription factor and important for release of paracrine factors. However, the relationship between HMGB1 and STAT3 in paracrine effect of MSC remains unknown. METHODS: In vitro, hypoxia/reoxygenation injury model was established by AnaeroPack System and examined by Annexin V flow cytometry, CCK8 assay and morphology observation. Detection of apoptotic proteins and protein expression of HMGB1 and STAT3 by Western blot. RESULTS: The conditioned medium of MSCs with or without LPS pretreatment was cocultured with H9C2 cells for 24 h before hypoxia treatment and MSC showed obvious cardiomyocytes protect role, as evidence by decreased apoptosis rate and improved cells viability, and LPS pretreated MSC exhibited better protect role than untreated MSC. However, such effect was abolished in HMGB1 deficiency group, silencing HMGB1 decreased the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin growth factor (IGF), cell viability, and the expression of STAT3. Furthermore, STAT3 silence attenuated the protective effect of LPS in MSC. CONCLUSIONS These findings suggested that LPS improved MSC-mediated cardiomyocytes protection by HMGB1/STAT3 signaling.

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca²⁺ current. Using noise analysis of Ca²⁺ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca²⁺ channels along with Ca_V1.3 expression. All these changes could act synergistically to enhance the Ca²⁺ influx into IHCs. Inhibition of intracellular Ca²⁺ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.
Animals ; Calcium ; Exocytosis ; Hair Cells, Auditory, Inner ; Mice ; Sodium Salicylate/pharmacology* ; Tinnitus/chemically induced*

Objective:To establish a disease risk prediction model for the newborn screening system of inherited metabolic diseases by artificial intelligence technology.Methods:This was a retrospectively study. Newborn screening data ( n=5 907 547) from February 2010 to May 2019 from 31 hospitals in China and verified data ( n=3 028) from 34 hospitals of the same period were collected to establish the artificial intelligence model for the prediction of inherited metabolic diseases in neonates. The validity of the artificial intelligence disease risk prediction model was verified by 360 814 newborns ' screening data from January 2018 to September 2018 through a single-blind experiment. The effectiveness of the artificial intelligence disease risk prediction model was verified by comparing the detection rate of clinically confirmed cases, the positive rate of initial screening and the positive predictive value between the clinicians and the artificial intelligence prediction model of inherited metabolic diseases. Results:A total of 3 665 697 newborns ' screening data were collected including 3 019 cases ' positive data to establish the 16 artificial intelligence models for 32 inherited metabolic diseases. The single-blind experiment ( n=360 814) showed that 45 clinically diagnosed infants were detected by both artificial intelligence model and clinicians. A total of 2 684 cases were positive in tandem mass spectrometry screening and 1 694 cases were with high risk in artificial intelligence prediction model of inherited metabolic diseases, with the positive rates of tandem 0.74% (2 684/360 814)and 0.46% (1 694/360 814), respectively. Compared to clinicians, the positive rate of newborns was reduced by 36.89% (990/2 684) after the application of the artificial intelligence model, and the positive predictive values of clinicians and artificial intelligence prediction model of inherited metabolic diseases were 1.68% (45/2 684) and 2.66% (45/1 694) respectively. Conclusion:An accurate, fast, and the lower false positive rate auxiliary diagnosis system for neonatal inherited metabolic diseases by artificial intelligence technology has been established, which may have an important clinical value.

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

Buffaloes ; Cells, Cultured ; In Vitro Techniques ; Primary Cell Culture ; Stem Cells ; Tretinoin

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

【Objective】To investigate the effects of Tc17 cells on disease severity and elimination of HBV-DNA in patients with HBV related acute-on-chronic liver failure（HBV-ACLF）.【Methods】Forty-three patients with HBV- ACLF were enrolled. Twenty patients with chronic hepatitis B（CHB）and 12 healthy subjects（NC）were enrolled as controls. Flow cytometry was used to detect the expression of peripheral Tc17 cells. HBV-DNA loads were measured，and the MELD，MELD-Na，CLIF-C ACLF and AARC scores were used to evaluate the disease severity. The effects of Tc17 cells on disease severity and decline of HBV-DNA were analyzed.【Results】Compared with NC group and CHB group， the expression of Tc17 cells in HBV-ACLF patients was significantly increased（P < 0.001 and P = 0.017）. Correlation analysis showed that Tc17 cells were positively correlated with AARC score，MELD score and MELD-Na score（r = 0.504，P = 0.001；r = 0.417，P = 0.005 and r = 0.382，P = 0.012），and a correlated trend was found with CLIF-C ACLF score（r = 0.294，P = 0.055）. And as the disease progressed，the expression of Tc17 cells gradually increased.In addition，Tc17 cells were positively correlated with HBV-DNA levels（r = 0.339，P = 0.026）at baseline，and the HBV-DNA levels were significantly decreased in patients with higher expression of Tc17 cells than in lower group after 4 weeks of treatment（P < 0.001）. Furthermore，it was found that baseline Tc17 cells and AST levels were associated with the HBV-DNA decline by multivariate linear regression analysis.【Conclusion】Tc17 cells-mediated inflammatory response helps clear the HBV-DNA，but excessive inflammatory response may aggravate the disease severity.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
China ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; RUNX1 Translocation Partner 1 Protein ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; WT1 Proteins

Ginkgo diterpene lactone raw material, as a raw material for ginkgo diterpene lactone meglumine injection, is extracted and purified from ginkgo leaf. ¹H-NMR content determination method and fingerprint analysis method were respectively established for ginkgo diterpene lactone raw material. Content determination was conducted in 3 batches of samples by using ¹H-qNMR, and then the results were basically consistent with the results in HPLC method. Twenty-four proton peaks were identified as common fingerprint peaks, and the fingerprint peaks were identified by using the control product and NMR information. Furthermore, 10 batches of samples were analyzed by ¹H-NMR fingerprint. The similarities were all higher than 0.99 and the common peaks were identified with the reference standards. This method is easy, fast, with good precision, stability and repeatability and could provide basis and new ideas for quality evaluation of ginkgo diterpene lactone raw material and its preparations.