Objective To investigate the mechanism by which fatty acid-binding protein 4(Fabp4)regulates neuroinflamma-tion after traumatic brain injury(TBI).Methods Adult wild-type C57 male mice were randomly divided into sham group and TBI group,with 6 mice in each group.Fabp4 knockout(Fabp4-/-)male mice were randomly divided into sham group and TBI group,with 6 mice in each group.Western blot was used to detect the expression levels of Fabp4,IL-1β,TNF-α,NLRP3,Caspase-1,and ASC proteins in brain tissue from each group.Brain water content was assessed using the wet-to-dry weight method,and neuronal damage was evaluated by Nissl staining.Immunofluorescence double staining was performed to detect the expression of microglial M1 polarization markers(CD86 and iNOS),M2 polarization marker(CD206),and astrocytic polarization marker(C3).Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of pro-inflammatory factors IL-1βand TNF-α in the culture supernatant of BV2 cells.MitoSOX red fluorescence staining was used to assess mitochondrial ROS levels in BV2 cells,and ELISA was employed to determine the levels of malondialdehyde(MDA)and 8-hydroxy-2'-deox-yguanosine(8-OHdG)in brain tissue after TBI.Immunofluorescence was performed to detect NLRP3 protein expression in BV2 cells.Results In wild-type mice,the expression of Fabp4 protein in the injured brain region was significantly increased at 12 hours post-TBI compared to the sham group(P＜0.01)and peaked at 3 days post-injury(P＜0.01).Compared to the wild-type TBI group,protein expression levels of IL-1β and TNF-α were reduced(P＜0.01),and brain water content was decreased(P＜0.05)in Fabp4-/-mice from TBI group.Nissl staining revealed that the volume of brain damage following TBI was reduced af-ter Fabp4 deletion(P＜0.05).Immunofluorescence experiments showed that M1 polarization of microglia was inhibited after Fabp4 deletion(P＜0.01),and M2 polarization was promoted(P＜0.01).Fabp4 deletion had no significant effect on astrocytic polarization.BV2 cell experiments demonstrated that the levels of IL-1β and TNF-α in the culture supernatant were increased(P＜0.01)after Fabp4 overexpression,and mitochondrial ROS production was elevated(P＜0.01).After LPS stimulation,Fabp4 protein expression in BV2 cells was increased(P＜0.01),the levels of IL-1β and TNF-α(P＜0.05)as well as mitochondrial ROS(P＜0.01)were reduced after BMS309403 intervention.In the mouse model,the levels of MDA and 8-OHdG in the brain tissue of Fabp4-/-TBI mice were significantly lower than those of wild-type TBI mice(P＜0.01).The levels of NLRP3,ASC,and Caspase-1 proteins in BV2 cells were increased after Fabp4 overexpression(P＜0.01),while ROS scavenger treatment reversed this effect(P＜0.01,P＜0.05,P＜0.01).In the mouse model,the levels of NLRP3,ASC,and Caspase-1 proteins in Fabp4-/-TBI mice were significantly lower than those in wild-type TBI mice(P＜0.01,P＜0.05,P＜0.01).Conclusion Fabp4 pro-motes microglia-induced neuroinflammation after TBI by regulating mitochondrial ROS production and NLRP3 inflammasome formation.These findings suggest that Fabp4 may serve as a potential therapeutic target for TBI.

Objective To investigate the expression pattern of IQGAP2 in renal cell carcinoma tissues and cell lines,and to analyze its effects on the proliferation and migration of renal cell carcinoma cells.Methods Firstly,GEO database was used to screen differentially expressed genes between renal cell carcinoma tissues,and GEPIA,TIMER2.0 were used to analyze the expression level of IQGAP2 in renal cell carcinoma tissue.Subse-quently,knockdown(siRNA)and overexpression plasmids of IQGAP2 were constructed and transfected into ACHN and 786-O cell lines to perform a series of functional experiments to evaluate the effect of IQGAP2 on the malignant biological behavior of renal carcinoma cells.qRT-PCR and Western Blot were used to detect the expres-sion of EMT(epithelial-mesenchymal transition)related proteins after knockdown and overexpression of IQGAP2.Results In renal cell carcinoma tissues,the relative expression of IQGAP2 was significantly lower than in adja-cent normal tissues(P<0.001).Transfection of si-IQGAP2 in ACHN and 786-O cells effectively downregulated the mRNA and protein expression levels of IQGAP2(P<0.01),while transfection with an overexpression plasmid significantly upregulated its mRNA and protein expression(P<0.001).Further studies revealed that overexpression of IQGAP2 significantly inhibited the proliferation(P<0.05)and migration(P<0.01)of ACHN and 786-O cells,whereas knockdown of IQGAP2 enhanced their proliferation(P<0.05,P<0.001)and migration(P<0.01).Through qRT-PCR and Western blot analyses of EMT-related proteins,it was found that reduced expression of IQGAP2 promoted the epithelial-mesenchymal transition(EMT)process in renal cancer cells.Conclusions The expression of IQGAP2 is low in renal cell carcinoma tissues and cells,and the decrease of the expression level can promote the EMT process of renal cell carcinoma cells,and then enhance the proliferation and migration of renal cell carcinoma cells.IQGAP2 plays an important tumor suppressor role in renal cell carcinoma.

Objective:To report the nationwide surveillance results of pathogenic profiles and antimicrobial resistance patterns of Gram-positive bloodstream infections in China in 2023.Methods:The clinical isolates of Gram-posttive bacteria from blood cultures were collected in member hospitals of National Bloodstream Infection Bacterial Resistant Investigation Collaborative System（BRICS）during January to December 2023. Antimicrobial susceptibility testing was performed using the dilution method recommended by the Clinical and Laboratory Standards Institute（CLSI）. Statistical analyses were conducted using WHONET 5.6 and SPSS 25.0 software.Results:A total of 4 385 Gram-positive bacterial isolates were obtained from 60 participating center. The top five pathogens were Staphylococcus aureus（ n=1 544，35.2%），coagulase-negative Staphylococci（ n=1 441，32.9%）， Enterococcus faecium（ n=574，13.1%）， Enterococcus faecalis（ n=385，8.8%），and α-hemolytic Streptococci（ n=187，4.3%）. The prevalence of methicillin-resistant Staphylococcus aureus（MRSA）and methicillin-resistant coagulase-negative Staphylococci（MRCNS）was 26.2%（405/1 544）and 69.8%（1 006/1 441），respectively. Notably，all Staphylococci remained susceptible to glycopeptide or daptomycin. Staphylococcus aureus demonstrated excellent susceptibility（>97.0%）to cephalobiol，rifampicin，trimethoprim-sulfamethoxazole，linezolid，minocycline，tigecycline，and eravacycline. No Enterococcus exhibiting resistance to linezolid were detected. Glycopeptide resistance was uncommon but more frequent in Enterococcus faecium（resistance to vancomycin and teicoplanin：both 1.7%）compared to Enterococcus faecalis（both 0.3%）. The detection rates of MRSA and MRCNS exhibited significant regional variations across the country（ χ2=17.674 and 148.650，respectively，both P<0.001）. No vancomycin-resistant Enterococci were detected in central China. Institutional comparison demonstrated higher prevalence of MRSA（ χ2=14.111， P<0.001）and MRCNS（ χ2=4.828， P=0.028）in provincial hospitals than that in municipal hospitals. Socioeconomic analysis identified elevated detection rates of both MRSA（ χ2=18.986， P<0.001）and MRCNS（ χ2=4.477， P=0.034）in less developed regions（per capita GDP

Objective:To report the results of bacterial resistant investigation collaborative system（BRICS）on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2023，and provide reference for clinical tretment of bloodstream infections and prevention and control of bacterial resistance.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of BRICS were collected during January 2023 to December 2023. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 were used to analyze the data.Results:During the study period，11 492 strains of Gram-negative bacteria were collected from 60 hospitals，of which 10 098（87.9%）were Enterobacterales and 1 394（12.1%）were non-fermentative bacteria. The top 5 bacterial species were Escherichia coli（50.0%）， Klebsiella pneumoniae（26.1%）， Pseudomonas aeruginosa（5.1%）， Acinetobacter baumannii complex（5.0%）and Enterobacter cloacae complex（4.1%）. The ESBL-producing rates in Escherichia coli， Klebsiella pneumoniae and Proteus mirablilis were 46.8%（2 685/5 741），18.3%（549/2 999）and 44.0%（77/175），respectively. The prevalence of carbapenem-resistant Escherichia coli（CREC）and carbapenem-resistant Klebsiella pneumoniae（CRKP）were 1.3%（76/5 741）and 15.0%（450/2 999）；32.9%（25/76）and 78.0%（351/450）of CREC and CRKP were sensitive to ceftazidime/avibactam combination，respectively. 94.7%（72/76）and 90.2%（406/450）of CREC and CRKP were sensitive to aztreonam/avibactam combination. Furthermore，57.9%（44/76）and 79.1%（356/450）were sensitive to imipenem/relebactam combination. The prevalence of carbapenem-resistant Acinetobacter baumannii（CRAB）complex was 64.6%（370/573），while more than 80.0% of CRAB complex was sensitive to tigecycline，eravacycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa（CRPA）was 17.0%（99/581）. There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of important Gram-negative bacteria resistance among different regions in China，with statistically significant differences in the prevalence of CREC，CRKP，CRPA and CRAB complex（ χ2=10.6，28.6，10.8 and 19.3， P<0.05）. The prevalence of ESBL-producing Escherichia coli， CREC，CRAB complex and CRKP were higher in provincial hospitals than those in municipal hospitals（ χ2=12.5，9.8，12.7 and 57.8，all P<0.01）. Conclusions:Gram-negative bacteria are the main pathogens causing bloodstream infections in China，and Escherichia coli is ranked in the top，while the trend of Klebsiella pneumoniae increases continuously with time. CRKP infection shows a slow upward trend，CREC infecton maintains a low prevalence level，and CRAB complex infection continues to exhibit a high prevalence rate. The composition and resistance patterns of pathogens causing bloodstream infections vary to some extent across different regions and levels of hospitals in China.

Objective To investigate the effects of Qizhi Zhoufei Granules on endoplasmic reticulum stress in rats with chronic obstructive pulmonary disease(COPD);To explore its mechanism.Methods COPD rat model was induced by lipopolysaccharide tracheal instillation and smoking.Totally 60 Wistar rats were divided into control group,model group,Bufei Huoxue Capsules group and Qizhi Zhoufei Granules low-,medium-and high-dosage groups using random number table method,with 10 rats in each group.Drug gavage intervention was carried out for the treatment group since the 29th day of modeling,and normal saline was given to the control group and model group for 28 d.Lung function tests were performed,HE staining was used to detect morphology of lung tissue,TUNEL staining was used to detect the degree of apoptosis in lung tissue,RT-qPCR and Western blot were used to detect the endoplasmic reticulum stress and apoptosis related molecular mRNA and protein expression in lung tissue.Results Compared with the control group,the lung function indexes of peak inspiratory flow(PIF),peak expiratory flow(PEF)and minute volume(MV)significantly decreased,and frequency of breathing(F)significantly increased in the model group(P＜0.05);the structural damage of the lung tissue was obvious,the lung injury score and apoptosis rate significantly increased(P＜0.05),the expressions of glucose regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK),C/EBP-homologous protein(CHOP),Caspase-3 and Caspase-9 mRNA were increased(P＜0.05),the protein expressions of GRP78,p-PERK,activating transcription factor 4(ATF4),CHOP,Caspase-3 and Caspase-9 were significantly increased(P＜0.05).Compared with the model group,PIF,PEF and MV significantly increased in Qizhi Zhoufei Granules medium-and high-dosage groups and Bufei Huoxue Capsules group,and F significantly decreased(P＜0.05);the damage in lung tissue was improved,and the lung injury score and cell apoptosis rate significantly decreased(P＜0.05),the mRNA expressions of GRP78,PERK,CHOP,Caspase-3 and Caspase-9 in lung tissue decreased(P＜0.05),and the protein expressions of GRP78,p-PERK,ATF4,CHOP,Caspase-3 and Caspase-9 decreased(P＜0.05).Conclusion Qizhi Zhoufei Granules can prevent cell apoptosis and excessive damage by inhibiting the expression of endoplasmic reticulum stress related factors in COPD rats,thereby promoting unfolded protein response and improving endoplasmic reticulum folding ability,constraining endoplasmic reticulum stress state,and assisting in its regulation.

Objective:To analyze the expression level of protein arginine methyltransferase 5 (PRMT5) in pancreatic cancer tissues and its correlation with prognosis using bioinformatics methods, and to explore its potential mechanisms.Methods:Expression analysis of the PRMTs family members was performed based on the gene expression profiles of 178 pancreatic cancer tissues from the Gene Expression Profiling Interactive Analysis (GEPIA) database and 171 normal pancreatic tissues from the TCGA and GTEx databases. Using the median expression level of PRMTs family members in pancreatic cancer tissues as the cutoff, patients were divided into high-expression and low-expression groups, and Kaplan-Meier curves for overall survival (OS) and disease-free survival (DFS) were plotted. The correlation between PRMT5 expression and the expression of oncogenes such as KRAS, TP53, BRCA1, BRCA2, and SLC39A4 was analyzed. Functional enrichment analysis was conducted on genes similar to PRMT5, and a protein-protein interaction (PPI) network was constructed to analyze the interaction relationships among these similar genes. Pancreatic cancer PANC1 and AsPC-1 cells cultured in standard medium served as the control group, while PANC1 and AsPC-1 cells cultured in medium supplemented with the PRMT5 inhibitor EPZ015666 served as the intervention group. Cell viability was assessed using the CCK-8 assay, and cell cycle progression was analyzed by flow cytometry.Results:The expression level of PRMT5 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues and showed a significant negative correlation with both OS and DFS. Patients with high PRMT5 expression had a shorter median survival time compared to those with low expression (17.2 months vs 19.5 months). PRMT5 expression was significantly positively correlated with the expression of oncogenes KRAS, TP53, BRCA1, BRCA2, SLC39A4, and KLF5. Genes similar to PRMT5 were primarily enriched in cell cycle-related signaling pathways, and NOP58 identified as a hub gene in the PPI network. Compared to the control group, the proliferation activity of PANC1 and AsPC-1 cells in the intervention group was significantly reduced [(67.3±1.9)% vs (100±4.4)% for PANC1; (60.5±2.7)% vs (100.0±1.7)% for AsPC-1], and the proportion of cells in the G1 phase was significantly increased [(51.6±0.7)% vs (35.3±2.7)% for PANC1; (51.2±0.9)% vs (29.7±2.2)% for AsPC-1]. All these differences were statistically significant (all P values <0.05). Conclusions:High expression of PRMT5 was closely associated with poor prognosis in pancreatic cancer patients, and it may contribute to pancreatic cancer progression by regulating the cell cycle.

Objective To investigate the mechanism by which fatty acid-binding protein 4(Fabp4)regulates neuroinflamma-tion after traumatic brain injury(TBI).Methods Adult wild-type C57 male mice were randomly divided into sham group and TBI group,with 6 mice in each group.Fabp4 knockout(Fabp4-/-)male mice were randomly divided into sham group and TBI group,with 6 mice in each group.Western blot was used to detect the expression levels of Fabp4,IL-1β,TNF-α,NLRP3,Caspase-1,and ASC proteins in brain tissue from each group.Brain water content was assessed using the wet-to-dry weight method,and neuronal damage was evaluated by Nissl staining.Immunofluorescence double staining was performed to detect the expression of microglial M1 polarization markers(CD86 and iNOS),M2 polarization marker(CD206),and astrocytic polarization marker(C3).Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of pro-inflammatory factors IL-1βand TNF-α in the culture supernatant of BV2 cells.MitoSOX red fluorescence staining was used to assess mitochondrial ROS levels in BV2 cells,and ELISA was employed to determine the levels of malondialdehyde(MDA)and 8-hydroxy-2'-deox-yguanosine(8-OHdG)in brain tissue after TBI.Immunofluorescence was performed to detect NLRP3 protein expression in BV2 cells.Results In wild-type mice,the expression of Fabp4 protein in the injured brain region was significantly increased at 12 hours post-TBI compared to the sham group(P＜0.01)and peaked at 3 days post-injury(P＜0.01).Compared to the wild-type TBI group,protein expression levels of IL-1β and TNF-α were reduced(P＜0.01),and brain water content was decreased(P＜0.05)in Fabp4-/-mice from TBI group.Nissl staining revealed that the volume of brain damage following TBI was reduced af-ter Fabp4 deletion(P＜0.05).Immunofluorescence experiments showed that M1 polarization of microglia was inhibited after Fabp4 deletion(P＜0.01),and M2 polarization was promoted(P＜0.01).Fabp4 deletion had no significant effect on astrocytic polarization.BV2 cell experiments demonstrated that the levels of IL-1β and TNF-α in the culture supernatant were increased(P＜0.01)after Fabp4 overexpression,and mitochondrial ROS production was elevated(P＜0.01).After LPS stimulation,Fabp4 protein expression in BV2 cells was increased(P＜0.01),the levels of IL-1β and TNF-α(P＜0.05)as well as mitochondrial ROS(P＜0.01)were reduced after BMS309403 intervention.In the mouse model,the levels of MDA and 8-OHdG in the brain tissue of Fabp4-/-TBI mice were significantly lower than those of wild-type TBI mice(P＜0.01).The levels of NLRP3,ASC,and Caspase-1 proteins in BV2 cells were increased after Fabp4 overexpression(P＜0.01),while ROS scavenger treatment reversed this effect(P＜0.01,P＜0.05,P＜0.01).In the mouse model,the levels of NLRP3,ASC,and Caspase-1 proteins in Fabp4-/-TBI mice were significantly lower than those in wild-type TBI mice(P＜0.01,P＜0.05,P＜0.01).Conclusion Fabp4 pro-motes microglia-induced neuroinflammation after TBI by regulating mitochondrial ROS production and NLRP3 inflammasome formation.These findings suggest that Fabp4 may serve as a potential therapeutic target for TBI.

Objective To investigate the effects of Qizhi Zhoufei Granules on endoplasmic reticulum stress in rats with chronic obstructive pulmonary disease(COPD);To explore its mechanism.Methods COPD rat model was induced by lipopolysaccharide tracheal instillation and smoking.Totally 60 Wistar rats were divided into control group,model group,Bufei Huoxue Capsules group and Qizhi Zhoufei Granules low-,medium-and high-dosage groups using random number table method,with 10 rats in each group.Drug gavage intervention was carried out for the treatment group since the 29th day of modeling,and normal saline was given to the control group and model group for 28 d.Lung function tests were performed,HE staining was used to detect morphology of lung tissue,TUNEL staining was used to detect the degree of apoptosis in lung tissue,RT-qPCR and Western blot were used to detect the endoplasmic reticulum stress and apoptosis related molecular mRNA and protein expression in lung tissue.Results Compared with the control group,the lung function indexes of peak inspiratory flow(PIF),peak expiratory flow(PEF)and minute volume(MV)significantly decreased,and frequency of breathing(F)significantly increased in the model group(P＜0.05);the structural damage of the lung tissue was obvious,the lung injury score and apoptosis rate significantly increased(P＜0.05),the expressions of glucose regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK),C/EBP-homologous protein(CHOP),Caspase-3 and Caspase-9 mRNA were increased(P＜0.05),the protein expressions of GRP78,p-PERK,activating transcription factor 4(ATF4),CHOP,Caspase-3 and Caspase-9 were significantly increased(P＜0.05).Compared with the model group,PIF,PEF and MV significantly increased in Qizhi Zhoufei Granules medium-and high-dosage groups and Bufei Huoxue Capsules group,and F significantly decreased(P＜0.05);the damage in lung tissue was improved,and the lung injury score and cell apoptosis rate significantly decreased(P＜0.05),the mRNA expressions of GRP78,PERK,CHOP,Caspase-3 and Caspase-9 in lung tissue decreased(P＜0.05),and the protein expressions of GRP78,p-PERK,ATF4,CHOP,Caspase-3 and Caspase-9 decreased(P＜0.05).Conclusion Qizhi Zhoufei Granules can prevent cell apoptosis and excessive damage by inhibiting the expression of endoplasmic reticulum stress related factors in COPD rats,thereby promoting unfolded protein response and improving endoplasmic reticulum folding ability,constraining endoplasmic reticulum stress state,and assisting in its regulation.

Objective:To explore the efficacy and safety of one-hole split endoscope (OSE) minimally invasive surgery for the treatment of single-segment thoracic ossification of the ligamentum flavum (TOLF).Methods:This retrospective non-randomized controlled study included 41 patients with single-segment TOLF who underwent surgery at Qilu Hospital of Shandong University between July 2019 and July 2023. Patients were divided into two groups: the OSE group (19 cases) treated with one-hole split endoscope minimally invasive surgery and the open group (22 cases) treated with traditional laminectomy and pedicle screw fixation. There were no significant differences between the two groups on gender, age, disease duration, affected segment, presence or absence of dural ossification, and residual cross-sectional vertebral canal area on CT ( P>0.05). Additionally, perioperative surgical time, estimated blood loss (EBL), incision length, hospital stay duration, hospitalization costs and follow-up duration were compared. The Japanese Orthopaedic Association (JOA) score and Oswestry Disability Index (ODI) were compared preoperatively and at the last follow-up. Complications were also recorded. Results:All patients successfully completed the surgery with no significant differences at the last follow-up ( P>0.05). Compared with the open group, the OSE group had a significantly shorter operative time (133.1±16.8 vs. 160.5±22.6 min), lower EBL (91.2±15.0 vs. 192.5±43.8 ml), shorter incision length (2.6±0.5 vs. 7.9±1.9 cm), reduced hospital stay (3.9±0.8 vs. 5.6±0.8 days), and lower hospitalization costs (34,874.9±4,568.6 vs. 53,162.3±9,815.6 yuan) (all P<0.05). AAt the final follow-up, JOA scores (8.5±0.8 vs. 8.6±1.2) and ODI values (16.7%±2.1% vs. 17.7%±4.4%) showed no significant differences between the OSE and open groups ( P>0.05). During the perioperative period and follow-up, complications occurred in 2 patients in the OSE group (1 cerebrospinal fluid leak, 1 poor wound healing) and in 8 patients in the open group (5 cerebrospinal fluid leaks, 1 neurological deterioration, 2 poor wound healing). Conclusion:OSE minimally invasive surgery is an effective treatment for single-segment thoracic ossification of the ligamentum flavum. Compared with open surgery, it provides advantages such as minimal invasiveness and fewer complications.

ObjectiveTo analyze the incidence and trend of stroke in Beilun District, so as to provide evidence for identifying influencing factors and reducing stroke incidence. MethodsStroke cases from 2012 to 2023 were extracted from the Ningbo Chronic Disease Collaborative Management System. Population information of Beilun District during the same period was also collected. The annual incidence and trends of stroke were analyzed. ResultsFrom 2012 to 2023, the age-standardized incidence rate of stroke in Beilun District, Ningbo City was 317.68/100 000, showing an increasing trend with an average annual percentage change (AAPC) of 2.267% (P=0.034). Among all subdistricts in Beilun District, two showed a downward trend in incidence, while the rest showed an upward trend. The crude incidence rate of stroke was significantly higher in males than that in females (P<0.001). The age-standardized incidence rate in males was 406.08/100 000, showing an increasing trend (AAPC=3.956%, P<0.001). The incidence of stroke also showed an increasing trend in the following age groups: 30‒<45 years (AAPC=6.340%, P=0.004), 45‒<60 years (AAPC=4.997%, P<0.001), and 60‒<75 years (AAPC=3.282%, P=0.042). Across all years, males had higher crude incidence rates in both ischemic and hemorrhagic stroke than females (P<0.05). The age-standardized incidence rate of ischemic stroke showed a rising trend in both males and the general population (male AAPC=4.905%, P<0.001; overall population AAPC=3.065%, P=0.001). ConclusionThe age-standardized incidence of stroke in Beilun District is on the rise, with higher crude incidence rate in males than that in females. The onset age of stroke is gradually declining. The age-standardized incidence rate of male ischemic stroke shows a clear upward trend.