Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.

Objective To establish a stable in vitro model of CD8+T cell exhaustion.Methods CD8+T cells were isolated and purified from the spleens of ovalbumin-specific CD8+T cell receptor(OT-I)transgenic mice and subjected to chronic antigen stimulation to induce exhaustion in vitro.Flow cytometry was employed to evaluate the expressions of exhaustion markers,secretion of effector cytokines,and transcription factor profiles in CD8+T cells.Exhausted and effector(non-exhausted)CD8+T cells were co-cultured with tumor cells before tumor cell viability was measured to assess the cytotoxic potential of CD8+T cells.Additionally,N-acetyl-L-cysteine(N-AC)was used as a positive control during exhaustion induction to validate the model.Results Chronic stimulation resulted in a significant upregulation of inhibitory receptors,including programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(TIM-3),T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motifdomain(TIGIT),and lymphocyte activation gene 3(LAG-3).Concurrently,the secretion of key effector cytokines such as tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)was markedly reduced.Exhausted CD8+T cells exhibited diminished cytotoxicity against tumor cells compared to effector CD8+T cells.Notably,treatment with N-AC effectively restored the function of exhausted CD8+T cells and enhanced their anti-tumor activity.Conclusion This study has established an effective in vitro model for CD8+T cell exhaustion.The use of N-AC demonstrates its potential to restore functionality in exhausted CD8+T cells,underscoring the reliability and utility of this model for investigating the anti-tumor potential of exhausted T cells.

Sustained inflammatory responses are closely related to various severe diseases,and inhibiting the excessive activation of inflammasomes and pyroptosis has significant implications for clinical treatment.Natural products have garnered considerable concern for the treatment of inflammation.Huanglian-Wumei decoction(HLWMD)is a classic prescription used for treating inflammatory diseases,but the necessity of their combination and the exact underlying anti-inflammatory mechanism have not yet been elucidated.Inspired by the supramolecular self-assembly strategy and natural drug compatibility theory,we successfully obtained berberine(BBR)-chlorogenic acid(CGA)supramolecular(BCS),which is an herbal pair from HLWMD.Using a series of characterization methods,we confirmed the self-assembly mechanism of BCS.BBR and CGA were self-assembled and stacked into amphiphilic spherical supra-molecules in a 2:1 molar ratio,driven by electrostatic interactions,hydrophobic interactions,and π-πstacking;the hydrophilic fragments of CGA were outside,and the hydrophobic fragments of BBR were inside.This stacking pattern significantly improved the anti-inflammatory performance of BCS compared with that of single free molecules.Compared with free molecules,BCS significantly attenuated the release of multiple inflammatory mediators and lipopolysaccharide(LPS)-induced pyroptosis.Its anti-inflammatory mechanism is closely related to the inhibition of intracellular nuclear factor-kappaB(NF-κB)p65 phosphorylation and the noncanonical pyroptosis signalling pathway mediated by caspase-11.

Sustained inflammatory responses are closely related to various severe diseases, and inhibiting the excessive activation of inflammasomes and pyroptosis has significant implications for clinical treatment. Natural products have garnered considerable concern for the treatment of inflammation. Huanglian-Wumei decoction (HLWMD) is a classic prescription used for treating inflammatory diseases, but the necessity of their combination and the exact underlying anti-inflammatory mechanism have not yet been elucidated. Inspired by the supramolecular self-assembly strategy and natural drug compatibility theory, we successfully obtained berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS), which is an herbal pair from HLWMD. Using a series of characterization methods, we confirmed the self-assembly mechanism of BCS. BBR and CGA were self-assembled and stacked into amphiphilic spherical supramolecules in a 2:1 molar ratio, driven by electrostatic interactions, hydrophobic interactions, and π-π stacking; the hydrophilic fragments of CGA were outside, and the hydrophobic fragments of BBR were inside. This stacking pattern significantly improved the anti-inflammatory performance of BCS compared with that of single free molecules. Compared with free molecules, BCS significantly attenuated the release of multiple inflammatory mediators and lipopolysaccharide (LPS)-induced pyroptosis. Its anti-inflammatory mechanism is closely related to the inhibition of intracellular nuclear factor-kappaB (NF-κB) p65 phosphorylation and the noncanonical pyroptosis signalling pathway mediated by caspase-11.

OBJECTIVE T o verify and compare the values of the different risk assessment scores in prediction of in-fections in the ST elevation myocardial infarction(STEMI)patients undergoing percutaneous coronary interven-tion(PCI)therapy.METHODS A total of 226 STEMI patients who received PCI in The Second Medical Center of Chinese PLA General Hospital from Aug.2019 to Jul.2024 were recruited as the research subjects.The efficien-cies of the six types of risk assessment scores,including age,serum creatinine,or glomerular filtration rate/ejec-tion fraction(ACEF/AGEF)score,Canadian acute coronary syndrome(C ACS)score,embolism risk score 2(CHADS2)score,global register of acute coronary events(GRACE)score and for contrast induced nephropathy(Mehran)scorein prediction of infections and major adverse clinical events(M ACE)were analyzed.RESULTS All of the risk assessment scores showed remarkable discriminating capability in prediction of infections(AUC:0 746 to 0 791)except CHADS2 score[the area under the curve(AUC)=0.682;95％CI=0.652 to 0.712)].All of the risk assessment scores showed the excellent performance in calibration of infections except CACS risk assess-ment score(calibration slope=0.77;95％CI=0.18 to 1.35).The risk assessment scores also showed tremen-dous capability in discriminating MACE during the hospital stay except CHADS2 score,with the AUC ranging be-tween 0.700 and 0.786.All of the six types of risk assessment scores showed the most excellent performance in calibration of MACE during the hospital stay.CONCLUSION ACEF,AGEF,CACS,GRACE and Mehran scores show remarkable discriminating capability and calibration in prediction of infections and MACE.

Objective To compare the periodontally accelerated osteogenic orthodontics(PAOO)effects of corticotomy with full mu-coperiosteal flap(flapped corticotomy)and corticotomy-only on accelerating orthodontic tooth movement.Methods A total of 60 healthy male SD rats(weighing 180-200 g)were selected and randomly divided into three groups.There were 20 rats each in the flapped corticotomy group,the corticotomy-only group,and the control group.After applying orthodontic instruments,5 rats each in the surgical group and the control group were killed by excessive anesthesia on 0,1,3,and 7 days after tooth movement.The tooth move-ment distances of the rats in the control group and the experimental group were counted,and immunohistochemical staining was per-formed to observe the corresponding molecular biological changes.Results There was no significant difference in accelerating ortho-dontic tooth movement between the flapped corticotomy group and the corticotomy-only group.Compared with traditional orthodontic tooth movement,both the flapped corticotomygroup and the corticotomy-only group could bring an increase in the expression of RANKL on the pressed periodontal side while there was no significant difference between the two experimental groups.Both the flapped corticot-omy and non-flapped corticotomy could enlarge the area of tissue formation in the periodontal tension zone,and still there was no signif-icant difference between the two surgical methods.Compared with traditional orthodontic tooth movement,both the flapped corticotomy group and the corticotomy-only group could lead to an increase in ALP,OCN and OPN expression on the periodontal tension zone.Conclusion Both flapped corticotomy and corticotomy play a significant role in accelerating orthodontic tooth movement and promoting alveolar bone formation in the early stage.Both surgical methods of PAOO can provide more efficient and stable biological support for orthodontic treatment.

OBJECTIVE T o verify and compare the values of the different risk assessment scores in prediction of in-fections in the ST elevation myocardial infarction(STEMI)patients undergoing percutaneous coronary interven-tion(PCI)therapy.METHODS A total of 226 STEMI patients who received PCI in The Second Medical Center of Chinese PLA General Hospital from Aug.2019 to Jul.2024 were recruited as the research subjects.The efficien-cies of the six types of risk assessment scores,including age,serum creatinine,or glomerular filtration rate/ejec-tion fraction(ACEF/AGEF)score,Canadian acute coronary syndrome(C ACS)score,embolism risk score 2(CHADS2)score,global register of acute coronary events(GRACE)score and for contrast induced nephropathy(Mehran)scorein prediction of infections and major adverse clinical events(M ACE)were analyzed.RESULTS All of the risk assessment scores showed remarkable discriminating capability in prediction of infections(AUC:0 746 to 0 791)except CHADS2 score[the area under the curve(AUC)=0.682;95％CI=0.652 to 0.712)].All of the risk assessment scores showed the excellent performance in calibration of infections except CACS risk assess-ment score(calibration slope=0.77;95％CI=0.18 to 1.35).The risk assessment scores also showed tremen-dous capability in discriminating MACE during the hospital stay except CHADS2 score,with the AUC ranging be-tween 0.700 and 0.786.All of the six types of risk assessment scores showed the most excellent performance in calibration of MACE during the hospital stay.CONCLUSION ACEF,AGEF,CACS,GRACE and Mehran scores show remarkable discriminating capability and calibration in prediction of infections and MACE.

Objective To compare the periodontally accelerated osteogenic orthodontics(PAOO)effects of corticotomy with full mu-coperiosteal flap(flapped corticotomy)and corticotomy-only on accelerating orthodontic tooth movement.Methods A total of 60 healthy male SD rats(weighing 180-200 g)were selected and randomly divided into three groups.There were 20 rats each in the flapped corticotomy group,the corticotomy-only group,and the control group.After applying orthodontic instruments,5 rats each in the surgical group and the control group were killed by excessive anesthesia on 0,1,3,and 7 days after tooth movement.The tooth move-ment distances of the rats in the control group and the experimental group were counted,and immunohistochemical staining was per-formed to observe the corresponding molecular biological changes.Results There was no significant difference in accelerating ortho-dontic tooth movement between the flapped corticotomy group and the corticotomy-only group.Compared with traditional orthodontic tooth movement,both the flapped corticotomygroup and the corticotomy-only group could bring an increase in the expression of RANKL on the pressed periodontal side while there was no significant difference between the two experimental groups.Both the flapped corticot-omy and non-flapped corticotomy could enlarge the area of tissue formation in the periodontal tension zone,and still there was no signif-icant difference between the two surgical methods.Compared with traditional orthodontic tooth movement,both the flapped corticotomy group and the corticotomy-only group could lead to an increase in ALP,OCN and OPN expression on the periodontal tension zone.Conclusion Both flapped corticotomy and corticotomy play a significant role in accelerating orthodontic tooth movement and promoting alveolar bone formation in the early stage.Both surgical methods of PAOO can provide more efficient and stable biological support for orthodontic treatment.

Background Grassroots medical and health personnel are an important component of China's public health system, and guaranteeing their physical and mental health will have a profound impact on the development of China's health service. Objective To identify potential influencing factors of occupational stress, anxiety, depression, and insomnia as well as their interactions. Methods In August 2021, a cross-sectional survey was conducted among all the staff (2675 medical and health personnel) at 7 secondary public hospitals and 8 health institutions in Xiong’an New Area of Hebei Province by cluster sampling. Occupational stress, anxiety, depression, and insomnia were evaluated by the Effort-Reward Imbalance Questionnaire (ERI), the Generalized Anxiety Disorder-7 (GAD-7), the Patient Health Questionnaire-9 (PHQ-9), and the Athens Insomnia Scale (AIS). The bnlearn and gmodels packages of R4.2.1 software were used to construct Bayesian networks on work and personal factors-occupational stress- health effects. The Bayesian network model was visualized by Netica32.0. Results Among the 3018 questionnaires distributed, a total of 2675 valid questionnaires were recovered, with an effective recovery rate of 88.63%. The positive rates of occupational stress, anxiety, depression, and insomnia were 51.48%, 62.13%, 62.50%, and 56.37%, respectively in the grassroots medical and health personnel. The positive rate of occupational stress among the medical and health workers varied by age, educational level, and organization type (P<0.05); the positive rate of anxiety varied by age, marital status, and daily working hours (P<0.05); and the positive rates of depression and insomnia varied by gender, educational level, and overcommitment (P<0.05). The final Bayesian network contained 14 nodes and 18 directed edges, and its prediction accuracy was 85.4%. .Job title, daily working hours, overcommitment and exercise directly associated with occupational stress, and other work and personal factors associated with occupational stress indirectly. Occupational stress associated with insomnia directly or indirectly via anxiety. Anxiety and insomnia associated with reporting depression. Conclusion Grassroots medical and health personnel of Xiong'an New Area, Hebei Province report high levels of occupational stress, anxiety, depression, and insomnia. Occupational stress directly or indirectly associates with anxiety, depression, and insomnia. Work and personal factors associate with occupational stress, and associate with anxiety, depression, and insomnia via occupational stress. Occupational stress may be an early warning factor of general work and personal factors affecting anxiety, depression, insomnia, and other mental health problems. Attention should be paid to the assessment and intervention of occupational stress in medical and health personnel to prevent mental health problems.

Objective Hepatitis c patients with HCV RNA loads and anti HCV and liver function index of correlation.Methods Retrospective analysis in January 2011 to December 2013 in our hospital outpatient,hospital,medical examination in patients with hepatitis c antibody positive,male 150,female 229,age 32-87,application of real-time fluorescent quantitative PCR detection of HCV RNA viruses,automatic biochemical analyzer test liver function 8 indicators,according to the number of HCV RNA viral load points HCV RNA negative groups,HCV RNA in the low level group,high levels of HCV RNA.Results Three groups of age distribution proportion by the statistical test has no obvious difference (P ＞ 0.05),but low in the proportion of women is slightly higher than that of males,Three groups of liver function and biochemical indicators is not identical results (P ＜ 0.05 or P ＜ 0.01),of which the HCV RNA in the low level group and high level of serum ALT,AST,GGT and ALP,TBIL compared with negative groups have difference (P ＜ 0.05 or P ＜ 0.01) ; TP,propagated,DBIL comparison,though no significant statistical differences between the three groups,but the low level and high level group results in a downward trend,in the low level and high level group comparison ALT,AST,TBIL also have obvious difference (P ＜ 0.05),viral load,the higher the ALT,AST results,after treatment with interferon with loads of decline index of liver enzymes.Conclusion No matter the discretion of the viral load to last as long as HCV RNA positive,can cause the damage of liver cells and liver function abnormalities,clinical should be antiviral treatment as soon as possible.Real-time PCR fluorescence probe technique is advanced,mature technology,stable results,to early diagnosis of HCV infection,help clinical early antiviral treatment,prevent the spread of HCV and development.