BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Objective To quantitatively measure the liver fat content and liver fibrosis degree in patients with type 2 diabetes mellitus(T2DM)using liver transient elastography(FibroScan)and analyze the risk factors for metabolism-related fatty liver disease(MAFLD).Methods A retrospective analysis was conducted on the clinical data of 258 patients with T2DM admitted to The Department of Endocrinology,Harbin First Hospital from August 2017 to November 2020.The patients were divided into the simple T2DM group(n=41)and the MAFLD(n=217)group based on whether they had MAFLD.Both groups were assessed using the FibroScan 502 system to measure liver fat content(CAP)and liver fibrosis degree(LSM).General information,TC,TG,LDL-C,HDL-C,FPG,HbA1c,FC-P,FIns,and liver function(AST,ALT,GGT)were compared between the two groups.Results BMI,TG,FC-P,FIns,AST,CAP,and LSM were significantly higher in the MAFLD group than in the simple T2DM group(P<0.05).Pearson phase analysis showed that the DM duration,TG,FC-P and LSM were positively correlated with CAP(P<0.05),while TG,AST and CAP were positively correlated with LSM(P<0.05).Logistic regression analysis revealed that BMI,TG,FC-P,CAP,and LSM were risk factors for T2DM combined with MAFLD.Conclusions Elevated BMI,TG,FC-P,CAP,and LSM are risk factors for the onset of T2DM combined with MAFLD.Additionally,TG is a common factor for the increase in CAP and LSM.FibroScan measurement of CAP and LSM values can quantitatively reveal the liver lesion status in patients with T2DM.

ObjectiveTo investigate the mechanism of Forsythiae Fructus-Lonicerae Japonicae Flos（FF） in the treatment of acute lung injury（ALI） by investigating the effects of FF on serum metabolomics of rats with ALI. MethodsThirty male SD rats were acclimated for 1 week， and 6 rats were randomly selected as the blank group. The other 24 rats were injected with lipopolysaccharide（LPS） solution by tracheal drip to establish an ALI model. After successful model establishment， the rats were randomly divided into the model group， the FF low-dose group（3.0 g·kg^-1）， the FF high-dose group（6.0 g·kg^-1）， and the dexamethasone group（5 mg·kg^-1）， with six rats in each group. The FF low- and high-dose groups and the dexamethasone group were received daily oral administration of the corresponding drug solution， and the blank group and the model group were gavaged with an equal amount of saline， treatment was administered continuously for 3 d. The pathological conditions of rat lung tissues were evaluated by hematoxylin-eosin（HE） staining， wet/dry mass ratio（W/D） of the lung tissues， and protein concentration in rat bronchoalveolar lavage fluid（BALF）. Metabolomic analysis of rat serum was performed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， combined with multivariate statistical analysis， the potential biomarkers of FF in treating ALI were screened by variable importance in the projection（VIP） value>1， P<0.05 from t-test， and log₂fold change（FC）>1 or log₂FC<-1. Kyoto Encyclopedia of Genes and Genomes（KEGG） database combined with MetaboAnalyst were used for pathway analysis of the screened differential metabolites. The protein expression levels of sphingosine-1-phosphate（S1P）， phosphatidylinositol 3-kinase（PI3K）， protein kinase B1（Akt1）， and phosphorylated Akt1（p-Akt1） were examined by Western bolt. The expression levels of interleukin（IL）-6， IL-1β， and tumor necrosis factor（TNF）-α in BALF were detected by enzyme-linked immunosorbent assay（ELISA）. ResultsCompared with the blank group， rats in the model group showed ALI pathological features such as alveolar lumen dilatation， interstitial hemorrhage and massive inflammatory cell infiltration， and the protein concentration in BALF and W/D of the lung tissues were significantly elevated（P<0.01）. Compared with the model group， the low- and high-dose groups of FF as well as the dexamethasone group exhibited reduced pulmonary bronchial hemorrhage in rats， and the protein concentration in BALF and W/D were significantly decreased（P<0.05）， and the lung injury was significantly alleviated. Analysis of rat serum metabolomics revealed that FF downregulated 38 biomarkers. Pathway enrichment analysis showed that FF primarily exerted therapeutic effects through 7 key metabolic pathways， including arginine biosynthesis， sphingomyelin metabolism， alanine， aspartate and glutamate metabolism， taurine and hypotaurine metabolism， α-linolenic acid metabolism， niacin and nicotinamide metabolism， and retinol metabolism. The results of Western bolt and ELISA showed that， compared with the blank group， the model group exhibited significantly elevated expression levels of S1P， PI3K， Akt1 and p-Akt1 proteins in the lung tissues， as well as increased expression levels of IL-6， IL-1β and TNF-α in BALF（P<0.01）. Compared with the model group， the expression levels of the aforementioned indicators were significantly downregulated in the low- and high-dose FF groups as well as the dexamethasone group（P<0.05， P<0.01）. ConclusionFF may play a role in ALI by regulating amino acid metabolism and lipid metabolism， and its mechanism may be related to the inhibition of S1P/PI3K/Akt1 signaling pathway to attenuate the inflammatory response caused by ALI.

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.
Humans ; Polo-Like Kinase 1 ; Cell Cycle Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Mitosis/physiology* ; HeLa Cells ; Adenosine/genetics* ; Methyltransferases/metabolism* ; RNA, Messenger/metabolism* ; RNA-Binding Proteins/metabolism*

BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Objective To quantitatively measure the liver fat content and liver fibrosis degree in patients with type 2 diabetes mellitus(T2DM)using liver transient elastography(FibroScan)and analyze the risk factors for metabolism-related fatty liver disease(MAFLD).Methods A retrospective analysis was conducted on the clinical data of 258 patients with T2DM admitted to The Department of Endocrinology,Harbin First Hospital from August 2017 to November 2020.The patients were divided into the simple T2DM group(n=41)and the MAFLD(n=217)group based on whether they had MAFLD.Both groups were assessed using the FibroScan 502 system to measure liver fat content(CAP)and liver fibrosis degree(LSM).General information,TC,TG,LDL-C,HDL-C,FPG,HbA1c,FC-P,FIns,and liver function(AST,ALT,GGT)were compared between the two groups.Results BMI,TG,FC-P,FIns,AST,CAP,and LSM were significantly higher in the MAFLD group than in the simple T2DM group(P<0.05).Pearson phase analysis showed that the DM duration,TG,FC-P and LSM were positively correlated with CAP(P<0.05),while TG,AST and CAP were positively correlated with LSM(P<0.05).Logistic regression analysis revealed that BMI,TG,FC-P,CAP,and LSM were risk factors for T2DM combined with MAFLD.Conclusions Elevated BMI,TG,FC-P,CAP,and LSM are risk factors for the onset of T2DM combined with MAFLD.Additionally,TG is a common factor for the increase in CAP and LSM.FibroScan measurement of CAP and LSM values can quantitatively reveal the liver lesion status in patients with T2DM.

Objective:To analyze the risk factors for 6-month prognosis of traumatic spinal cord injury (TSCI).Methods:A retrospective analysis was conducted of the 133 patients with TSCI who had been admitted to Department of Orthopaedics, The First Hospital Affiliated to Jinan University from January 2017 to August 2021. The patients with TSCI were categorized into an improved group ( n=82) and a non-improved group ( n=51) according to the improvements in the American Spinal Injury Association (ASIA) grading between admission and 6 months after injury. To identify the risk factors that might affect the prognosis of TSCI patients at 6 months after injury, univariate and logistic regression analyses were conducted of indicators such as gender, age, length of MRI spinal cord signal change, maximum canal compromise (MCC), maximum spinal cord compression (MSCC), brain and spinal cord injury center(BASIC)score, neutrophil-to-lymphocyte ratio (NLR) within 3 days after injury, ASIA grading within 3 days after injury, mean arterial pressure (MAP) at 3 days after operation, and presence of complications. Results:The univariate analysis showed significant differences between the improved and non-improved groups in length of MRI spinal cord signal change, MCC, MSCC, BASIC, NLR within 3 days after injury, ASIA grading within 3 days after injury, MAP at 3 days after operation, and presence of complications (all P<0.05). The logistic regression analysis showed that NLR ( OR=0.463, 95% CI: 0.287 to 0.748, P=0.002) and ASIA grading ( OR=11.684, 95% CI: 1.684 to 81.086, P=0.013) within 3 days after injury, as well as MAP at 3 days after operation ( OR=2.224, 95% CI: 1.306 to 3.787, P=0.003), were risk factors affecting the 6-month prognosis in TSCI patients. Conclusion:The NLR and ASIA grading within 3 days after injury, and MAP at 3 days after operation are risk factors that may affect the prognosis of TSCI patients at 6 months after injury.

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT- HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.
Animals ; Hematopoietic Stem Cells/cytology* ; Cyclin-Dependent Kinase Inhibitor p21/genetics* ; Mice ; Cell Self Renewal ; Repressor Proteins/genetics* ; Mice, Inbred C57BL ; Mice, Knockout ; Humans ; Gene Expression Regulation

Background Phenolic compounds may adversely affect human health, but the current relevant studies are mostly limited to the impact of single phenolic compound exposure on human health, and there is still a lack of studies on the population-based association between combined exposure to multiple common phenolic compounds and dyslipidemia. Objective To explore the association of phenolic compound combined exposure and dyslipidemia based on principal component analysis-random forest (PCA-RF) strategy. Methods The data were from the National Health and Nutrition Examination Survey (2013–2016). A total of 1301 adult residents aged ≥ 20 years with complete information on demographics and lifestyle, urine phenol concentrations (bisphenol A, bisphenol F, bisphenol S, triclocarban, benzophenone, and triclosan), and serum concentrations of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were included in this study. The concentrations of six urinary phenolic compounds were determined by solid phase extraction coupled with high performance liquid chromatography and tandem mass spectrometry, and the lipid indicators were determined by enzymatic methods. Principal component analysis combined with random forest model was used for model construction. First, principal component analysis was performed on 18 original variables including 6 phenolic compounds and 12 basic characteristic indicators, and then random forest model was established with dyslipidemia and its four evaluation indicators as dependent variables and the extracted principal components as independent variables, respectively. Results The PCA-RF analysis showed that bisphenol A, bisphenol F, and benzophenone may be important factors for dyslipidemia in the study subjects; bisphenol A, bisphenol F, and triclosan may be important factors for TC level in the study subjects; bisphenol A, bisphenol F, triclocarban, and benzophenone may be important factors for TG level in the study subjects; bisphenol A may be an important factor for LDL-C level in the study subjects; bisphenol F and benzophenone may be important factors for HDL-C level in the study subjects. Conclusion Phenolic compound exposure may be an important risk factor for the development of dyslipidemia. PCA-RF strategy can be effectively used to explore the association between phenolic compound exposure and dyslipidemia in the population.

Enterotoxigenic Escherichia coli（ETEC）infection can induce watery diarrhea，leading to dehydration，electrolyte disturbance，and even death in severe cases. Recombinant B subunit/inactivated whole-cell cholera（rBS/WC）vaccine is effective in preventing ETEC infectious diarrhea. On the basis of the latest evidence on etiology and epidemiology of ETEC，as well as the effectiveness，safety，and health economics of rBS/WC vaccine，National Clinical Research Center for Child Health（The Children’s Hospital，Zhejiang University School of Medicine）and Zhejiang Provincial Center for Disease Control and Prevention invited experts to develop expert consensus on rBS/WC vaccine in prevention of ETEC infectious diarrhea. It aims to provide the clinicians and vaccination professionals with guidelines on using rBS/WC vaccine to reduce the incidence of ETEC infectious diarrhea.