Objective:To explore the role of lactate in Superoxide dismutase 2 (SOD2) lactylation in cerebral ischemia-reperfusion injury in mice.Methods:Male C57BL/6 mice were randomLy (random number) divided into 4 groups: sham operation group (Sham group), Middle Cerebral Artery Occlusion/Reperfusion group (MCAO/R group), Middle Cerebral Artery Occlusion/Reperfusion+2-Deoxy-D-glucose group (MCAO/R+2-DG group), Middle Cerebral Artery Occlusion/Reperfusion+sodium lactate group (MCAO/R+Nala group). Cerebral ischemia reperfusion injury model was established in the mice of MCAO/R group using the thread occlusion. In the MCAO/R+2-DG group, mice received an intraperitoneal injection of 250 mg/kg of 2-DG 90 min before ischemia. Mice in the MCAO/R+ Nala group was given an intraventricular injection of 2 μL of 100 mmol/L Nala 24 h before ischemia. Commercial kits was used to detect lactate levels, Hematoxylin & Eosin Staining (HE) was employed to observe cell morphology, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess cell apoptosis, and immunofluorescence was utilized to detect reactive oxygen species (ROS). Western blot was conducted to measure SOD2, Superoxide Dismutase 2 Lysine 114 Lactylation(SOD2-K114la), Iron regulatory protein 2(IRP2) and transferrin receptor protein 1(TFR1) levels. The above indicators were analyzed and compared by one-way variance.Results:Compared with the Sham group, the MCAO/R group showed increased levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2 and TFR1[lactate: (0.608±0.064) vs. (0.376±0.030), P<0.005; SOD2-K114la: (2.311±0.146) vs. (1.009±0.073), P<0.0005; TUNEL positive rate: (35.420±2.832) vs. (0.294±0.147), P<0.0001; ROS: (3.415±0.229) vs. (1.166±0.155), P<0.0001; IRP2: (1.735±0.125) vs. (1.000±0.000), P<0.0001; TFR1: (1.611±0.058) vs. (1.000±0.000), P<0.0001], while SOD2 decreased[(0.545±0.062) vs. (1.082±0.088), P<0.0001]. HE staining indicated brain damage. Compared with the MCAO/R group, the MCAO/R+2-DG group showed reduced levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2, and TFR1[lactate: (0.453±0.047) vs. (0.608±0.064), P<0.05; SOD2-K114la: (1.764±0.188) vs. (2.311±0.146), P<0.05; TUNEL positive rate: (23.800±3.168) vs. (35.420±2.832), P<0.005; ROS: (2.640±0.213) vs. (3.415±0.229), P<0.005; IRP2: (1.463±0.055) vs. (1.735±0.125), P<0.05; TFR1: (1.252±0.081) vs. (1.611±0.058), P<0.005], with higher level of SOD2 [(0.727±0.026) vs. (0.545±0.062), P<0.05]. Meanwhile, HE staining indicated reduced damage. Compared with the MCAO/R group, the MCAO/R+Nala group showed increased levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2 and TFR1[lactate: (1.021±0.051) vs. (0.608±0.064), P<0.0001; SOD2-K114la: (3.479±0.275) vs. (2.311±0.146), P<0.0005; TUNEL positive rate: (53.430±3.551) vs. (35.420±2.832), P<0.0001; ROS: (4.687±0.253) vs. (3.415±0.229), P<0.0001; IRP2: (2.463±0.117) vs. (1.735±0.125), P<0.0001; TFR1: (2.209±0.094) vs. (1.611±0.058), P<0.0001], with decreased levels of SOD2 [(0.286±0.040) vs. (0.545±0.062), P<0.0001]. And HE staining revealed worsened braindamage. Conclusions:Increased lactate levels can enhance the lactylation of SOD2, exacerbating brain damage after Cerebral ischemia reperfusion injury(CIRI). Inhibiting lactate production may alleviate brain injury by regulating iron Metabolism.

Objective To establish a novel real-time quality control method for rapidly identifying the random error of sodium ion con-centration in serum using an artificial intelligence voting algorithm,and evaluate the relevant effectiveness of the model established on this basis.Methods A total of 144 754 test results of serum sodium ion rom the inpatients measured by Beckman AU5400 biochemis-try analyzer from January to May 2021 were obtained retrospectively from laboratory information system of the Department of Clinical La-boratory,Nanjing Drum Tower Hospital,and all the data were used as unbiased data for the current study.The random errors were arti-ficially introduced to generate the corresponding biased data set.Subsequently,the voting algorithm-based internal quality control model(ViQC)was established using the principles of the voting algorithm.The ViQC model and five classical PBRTQC(patient-based real-time quality control)algorithms were performed direct to each biased data.The analytical performance of the ViQC model was evaluated by using classification model criteria.The trimmed average number of patient samples until error detection(tANPed)was used to com-pare the clinical detection efficacy of the ViQC model with those of the five classical algorithms,and the error detection curves were plotted.Results Compare with all the classical algorithms,the ViQC model showed a false positive rate below 0.002 and achieved ac-curacy above 0.951 in detecting all the deviations.When the error factors were 1.5,2.5,and 3.0,the false positive rate of the ViQC model was zero.When the error factor was 2.5,its accuracy reached 0.979.Compared to the five classical PBRTQC algorithms,the ViQC model reduced the overall average tANPed by up to 34％and showed higher sensitivity for error detection.In addition,the ViQC model demonstrated the area under the ROC curve was as high as 0.989 at TEa on the test set,but the value of tANPed wasonly five.Conclusion We successfully established a real-time quality control model for the data of patients based on artificial intelligence algo-rithms,and its efficacy of clinical detection was superior to the traditional PBRTQC algorithms.

Objective:To investigate the protective effect of REV-ERBα agonist SR9009 on hippocampal working memory in rats with acute rapid eye movement (REM) sleep deprivation after exploratory laparotomy and its possible mechanism.Methods:Ninety SD rats were randomly divided into control group, sleep deprivation group, exploratory laparotomy group, sleep deprivation+exploratory laparotomy group, and sleep deprivation+exploratory laparotomy+SR9009 group ( n=18). Rats in the sleep deprivation group, exploratory laparotomy group, and sleep deprivation+exploratory laparotomy group were given REM sleep deprivation for 96 h or (and) exploratory laparotomy, respectively. Rats in the sleep deprivation+exploratory laparotomy+SR9009 group accepted exploratory laparotomy after REM sleep deprivation for 96 h, and accepted intraperitoneal injection of 100 mg/kg SR9009 daily from the day after surgery to the 6 th d of surgery. The reversall escape latency of rats was recorded by contrapuntal space exploration training one-5 d after surgery. On the 5 th d of surgery, reversal space exploration experiment was conducted to record the number of times of rats crossing the original platform. Western blotting was used to detect the protein expressions of REV-ERBα and BMAL1 in the hippocampus of rats. The levels of interleukin (IL)-1β and IL-6 in the hippocampus of rats were detected by enzyme-linked immunosorbent assay. Immunofluorescent staining was used to detect the expressions of neuronal nucleoprotein (NeuN) and glial fibrillary acidic protein (GFAP). Results:(1) The escape latency in the sleep deprivation group, exploratory laparotomy group, and sleep deprivation+exploratory laparotomy group was significantly longer than that in the control group on the first, 2 nd, 3 rd, 4 th, 5 th d of surgery ( P<0.05); while the escape latency in the sleep deprivation group and sleep deprivation+exploratory laparotomy group was significantly longer than that in the exploratory laparotomy group ( P<0.05); on the 2 nd, 3 rd, 4 th, 5 th d of surgery, the reversal escape latency in the sleep deprivation+exploratory laparotomy+SR9009 group was statistically shorter than that in the sleep deprivation+exploratory laparotomy group ( P<0.05). The number of times of rats crossing the original platform in the sleep deprivation group, exploratory laparotomy group, and sleep deprivation+exploratory laparotomy group was significantly smaller than that of the control group ( P<0.05); that of rats in the sleep deprivation+exploratory laparotomy group was significantly smaller than that of the exploratory laparotomy group, and that of rats in the sleep deprivation+exploratory laparotomy+SR9009 group was significantly larger than that of the sleep deprivation+exploratory laparotomy group ( P<0.05). (2) As compared with the control group, the exploratory laparotomy group, sleep deprivation group and sleep deprivation+exploratory laparotomy group had significantly decreased expressions of REV-ERBα and BMAL1, and statistically increased IL-1β and IL-6 levels in the hippocampal tissues ( P<0.05); as compared with the sleep deprivation+exploratory laparotomy group, the sleep deprivation+exploratory laparotomy+SR9009 group had significantly increased expressions of REV-ERBα and BMAL1, and statistically decreased IL-1β and IL-6 levels ( P<0.05). (3) As compared with the control group, the exploratory laparotomy group, sleep deprivation group and sleep deprivation+exploratory laparotomy group had decreased amount of neurons in the hippocampal CA3 area and increased amount of activated astrocytes; as compared with the sleep deprivation+exploratory laparotomy group, the sleep deprivation+exploratory laparotomy+SR9009 group had increased amount of neurons in the hippocampal CA3 area and decreased amount of activated astrocytes. Conclusion:Acute REM sleep deprivation can lead to work memory impairment in rats accepted exploratory laparotomy, which might be associated with neuroinflammation and REV-ERBα/BMAL1 pathway, and SR9009 could alleviate the damage.

OBJECTIVETo examine the proportion and reasons of drop-out from antiretroviral therapy (ART) among 8 367 adult HIV-infected individuals in Dehong prefecture, Yunnan province.

METHODSAll adult HIV-infected patients receiving ART before September 30 of 2014 were examined for the situation of drop-out from ART.

RESULTSThe proportion of drop-out from ART among adult HIV-infected patients in Dehong prefecture was 14.4% (1 202/8 367). Results from the univariate logistic regression analyses indicated that drop-out from ART was significantly correlated with factors as: living area, gender, age, marital status, HIV transmission route, baseline CD4⁺ T cell counts and initial treatment regimen of the patients. After adjusted for potential confounding variables by multiple logistic regression model, drop-out from ART was significantly correlated with residential area, marital status, HIV transmission route, baseline CD4⁺ T cell count and initial treatment regimen of the patients. HIV-infected patients who were living in Mangshi city, Lianghe county or Yingjiang County, being married or living with partner, HIV infection through sexual contact, with baseline CD4⁺ T cell counts ≤ 200 cells/mm³, and ART included in the initial treatment regimen etc., were less likely to drop out from ART. The proportion of drop out from ART was significantly decreasing along with the increasing time of ART. Data from specific investigation revealed that among the 1 202 patients who dropped out from ART, 704 (58.6%) were lost to follow-up, 303 (25.2%) did not adhere to treatment, 74 (6.2%) moved out the region, 64 (5.3%) were Burmese that had returned to Burma, 29 (2.4%) stopped the treatment according to doctors' advice, 18 (1.5%) were incarcerated and 10 (0.8%) were under other reasons. Reasons for the drop-out varied, according to the situation of patients.

CONCLUSIONThe proportion of drop-out from ART varied significantly according to the characteristics of HIV-infected patients in Dehong prefecture that underscoring the needs for tailored responses to reduce drop-out of ART. Focus should be targeted on reducing the loss to follow-up and improving the treatment adherence.

Adult ; Anti-Retroviral Agents ; therapeutic use ; China ; HIV Infections ; drug therapy ; Humans ; Patient Dropouts ; statistics & numerical data

Objective To examine the proportion and reasons of drop-out from antiretroviral therapy(ART)among 8 367 adult HIV-infected individuals in Dehong prefecture,Yunnan province. Methods All adult HIV-infected patients receiving ART before September 30 of 2014 were examined for the situation of drop-out from ART. Results The proportion of drop-out from ART among adult HIV-infected patients in Dehong prefecture was 14.4%(1 202/8 367). Results from the univariate logistic regression analyses indicated that drop-out from ART was significantly correlated with factors as:living area,gender,age,marital status,HIV transmission route,baseline CD4+T cell counts and initial treatment regimen of the patients. After adjusted for potential confounding variables by multiple logistic regression model,drop-out from ART was significantly correlated with residential area,marital status,HIV transmission route,baseline CD4+T cell count and initial treatment regimen of the patients. HIV-infected patients who were living in Mangshi city,Lianghe county or Yingjiang county,being married or living with partner,HIV infection through sexual contact,with baseline CD4+T cell counts≤200 cells/mm3,and ART included in the initial treatment regimen etc.,were less likely to drop out from ART. The proportion of drop out from ART was significantly decreasing along with the increasing time of ART. Data from specific investigation revealed that among the 1 202 patients who dropped out from ART,704(58.6%)were lost to follow-up,303(25.2%) did not adhere to treatment,74(6.2%)moved out the region,64(5.3%)were Burmese that had returned to Burma,29 (2.4%) stopped the treatment according to doctors’advice,18(1.5%)were incarcerated and 10 (0.8%) were under other reasons. Reasons for the drop-out varied,according to the situation of patients. Conclusion The proportion of drop-out from ART varied significantly according to the characteristics of HIV-infected patients in Dehong prefecture that underscoring the needs for tailored responses to reduce drop-out of ART. Focus should be targeted on reducing the loss to follow-up and improving the treatment adherence.

Objective To examine the proportion and reasons of drop-out from antiretroviral therapy(ART)among 8 367 adult HIV-infected individuals in Dehong prefecture,Yunnan province. Methods All adult HIV-infected patients receiving ART before September 30 of 2014 were examined for the situation of drop-out from ART. Results The proportion of drop-out from ART among adult HIV-infected patients in Dehong prefecture was 14.4%(1 202/8 367). Results from the univariate logistic regression analyses indicated that drop-out from ART was significantly correlated with factors as:living area,gender,age,marital status,HIV transmission route,baseline CD4+T cell counts and initial treatment regimen of the patients. After adjusted for potential confounding variables by multiple logistic regression model,drop-out from ART was significantly correlated with residential area,marital status,HIV transmission route,baseline CD4+T cell count and initial treatment regimen of the patients. HIV-infected patients who were living in Mangshi city,Lianghe county or Yingjiang county,being married or living with partner,HIV infection through sexual contact,with baseline CD4+T cell counts≤200 cells/mm3,and ART included in the initial treatment regimen etc.,were less likely to drop out from ART. The proportion of drop out from ART was significantly decreasing along with the increasing time of ART. Data from specific investigation revealed that among the 1 202 patients who dropped out from ART,704(58.6%)were lost to follow-up,303(25.2%) did not adhere to treatment,74(6.2%)moved out the region,64(5.3%)were Burmese that had returned to Burma,29 (2.4%) stopped the treatment according to doctors’advice,18(1.5%)were incarcerated and 10 (0.8%) were under other reasons. Reasons for the drop-out varied,according to the situation of patients. Conclusion The proportion of drop-out from ART varied significantly according to the characteristics of HIV-infected patients in Dehong prefecture that underscoring the needs for tailored responses to reduce drop-out of ART. Focus should be targeted on reducing the loss to follow-up and improving the treatment adherence.

OBJECTIVETo establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.

METHODSFollowing bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.

RESULTSThe amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.

CONCLUSIONSYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.

Actins ; genetics ; Biopsy ; Calibration ; Carcinoma, Hepatocellular ; genetics ; pathology ; DNA Methylation ; Feasibility Studies ; Female ; Fluorescence ; Genes, p16 ; Humans ; Luminescent Measurements ; Male ; Middle Aged ; Nucleic Acid Denaturation ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Transition Temperature

Objective To study the preparation of hepatitis C viruses (HCV) genotyping oligochip and its application in the detection of 76 hepatitis C patients.Methods Oligonucleotide probes and primers were designed in the 5’noncoding region and core region of HCV. The HCV typing chip was prepared by spotting the modified probes onto nylon membrane. Products of the second PCR were labeled with Dig-dUTP. Furthermore, 6 PCR products were sequenced.Results Using the chip,15 subtypes in 11 types of HCV were analyzed.Results of hybridization indicates that 76 hepatitis C patients were all positive and 20 health people were negative.Among 76 patients, 64 cases were 1b type, 11 cases were 2a type and 1 case was 3a type. Mix infection was not found. The results obtained by sequencing 6 samples and chip arraying were the same.Conclusion The HCV genotyping chip could be used in detecting serum HCV RNA and analyzing its genotypes.

Objective To prepare a gene chip for detecting polymorphisms on coding region of genes in hepatocellular carcinoma tissuses. Methods Genes harboring in loss regions with high frequency of hepatocellular carcinoma (HCC) were selected, the related information was inquired and the cSNP sequences were obtained in the SNP database of National Center for Biotechnology Information(NCBI).Then the appropriate primers and oligonucleotide probes were designed according to the SNP sites and the gene chip for the detection of SNPs were constructed. This chip includes 48 cSNPs of 25 hepatocellular carcinoma-related genes. The PCR products labeled by Dig-dUTP were hybridized with cSNP chip. Results The sensitivity, influence of probes and reiteration of the chip were detected. The results indicated that the chip was sensitive about 6?10~(-3) ng/?l. The signal of hybridization was down with lower concentrition of probes. By the chip, 7 of polymorphisms of caspase9 ( rs2308941) C→T and DOK2(rs2242241)T→G, 6 of polymorphisms of EGFL3(rs947345)A →G,caspase9 ( rs2308938)C→G and PHGDH(rs1801955)T→A, 5 of polymorphisms of E2F2(rs3218170) G→A,4 of polymorphisms of MUTYH(rs1140507)T→C and BNIP3L(rs1055806)G→T, 1 of polymorphism of TNFRSF1B (rs1061622)T→G were detected in the tissues of 10 HCC. Samples of caspase9 ( rs2308941G) and ( rs2308941A) were verified by PCR-SSCP and sequencing. Conclusion We prepare the cSNP chip of hepatocellular carcinoma-related genes, which can accelerate the discovery of polymorphic markers on hepatocellular carcinoma.

Objective To detection the methylation of p16INK4A in primary hepatocellular carcinoma, a nested bisulfite sequencing and methylation-specific polymerase chain reaction (BS-MSP) protocol was designed and used.Methods Bisulfite-modified DNA were amplified to evaluate the quality of templates with a pair of bisulfite sequencing primers in the first round of PCR, then subjected to methylation assay with corresponding methylation or unmethylation specific PCR primers.Representative PCR products were sequenced to confirm its correctness.Results 3 of 40 cases (7.5%) were failed to assay due to poor quality of templates, and 29 of 37 cases (78%) were detected p16INK4A methylation.Sequencing results confirmed that templates were correctly amplified.Conclusion BS-MSP technique might be valuable for methylation study on carcinogenesis and clinical assay.