Objective:To observe and analyze the correlation between the changes of macular microvascular structure and the level of intracocular fluid cytokines in patients with diabetic macular edema (DME).Methods:A prospective clinical study. From December 2022 to June 2024, 20 patients with 25 eyes of DME diagnosed by Department of Ophthalmology of Linyi People's Hospital were included in the study. Among them, 14 males had 17 eyes and 6 females had 8 eyes. Age was (55.08±10.34) years. Optical coherence tomography (OCT) and OCT angiography (OCTA) were used to scan the macular region at a range of 6 mm×6 mm. Central retinal thickness (CRT), blood flow density of superficial retinal capillary plexus (SCP) and area of fovea avascular zone (FAZ) were measured. The anterior aqueous humor was extracted before the first intravitreal injection of anti-vascular endothelial growth factor (VEGF), the concentrations of interleukin (IL-6), IL-8, VEGF, vascular cell adhesion molecule (VCAM), placental growth factor (PLGF) and monocyte chemotactic protein-1 (MCP-1) were detected. The correlation between macular microvascular structure and aqueous humor cytokines was analyzed by Spearman correlation analysis.Results:The CRT of the affected eyes was (617.40±167.64) μm, the SCP flow density was (39.56±1.55)%, and the FAZ area was (0.46±0.13) mm 2. The concentrations of IL-6, IL-8, VEGF, VCAM, PLGF and MCP-1 in aqueous humor were (301.36±690.52), (29.15±20.56), (71.37±29.32) and (5 621.22±7 241.06), (72.40±13.43), (464.07±163.26) pg/ml, respectively. Correlation analysis showed that there was a significant positive correlation between CRT and the concentrations of aqueous cytokines VEGF and PLGF ( r=0.460, 0.462, P<0.05). FAZ area was positively correlated with VEGF and MCP-1 concentrations ( r=0.414, 0.465; P<0.05). There was a significant negative correlation between SCP blood flow density and IL-6 ( r=0.401, P<0.05). Conclusion:There was a significant correlation between the morphological structure of macular area and the damage degree of microvessels around macular area in DME patients and the concentration of aqueous cytokines.

OBJECTIVE To explore the effect and underlying mechanism of leucine(Leu)on meta-bolic dysfunction-associated fatty liver disease induced by high fat diet(HFD)in mice.METHODS C57BL/6J male mice were randomly divided into chow diet(normal),chow diet+Leu(normal+Leu),HFD and HFD+Leu groups,with 10 mice in each group.The mice in the normal and normal+Leu groups received chow diet while those in the HFD and HFD+Leu groups received HFD.Drinking water for mice in the normal+Leu and HFD+Leu groups was supplemented with 1.5%Leu.The experiment lasted 24 weeks,total food and water intake of mice were recorded weekly to calculate energy and Leu intake respectively.Energy metabolism of mice was detected at week 20 by the Oxymas/CLAMS Animal Metabolic System heat production,CO2 exhalation,O2 consumption and respiratory exchange rate(RER).At the end of week 24,the mice were sacrificed and the livers were harvested,followed by the oil red O staining to reveal the fat content.Western blotting was performed to analyze the changes in the activity of the liver branched-chain α-keto acid dehydrogenase E1α(BCKDE1A),the activation of AMP-activated protein kinase alpha subunit(AMPKα),and the protein expressions of downstream effector molecules including silent information regulator 1(SIRT1)and peroxisome proliferator-activated receptor coactivator-1α(PGC-1α),fatty acid synthetase(FAS)and fatty acid binding protein 4(FABP4)in the liver of mice.RESULTS Total Leu intake of mice was significantly reduced in the HFD+Leu group,compared with the normal+Leu group.The mice fed with HFD significantly increased the energy intake body mass gain and liver mass,accompanied by fat accumulation in the liver,compared to the mice in the normal group.Simultaneously,the mice in the HFD group showed a decrease in CO2 exha-lation both by day and by night,and in the respiratory exchange ratio by day compared to the normal group.Compared with the HFD group,the body mass gain and liver mass obviously decreased in mice of the HFD+Leu group,and the liver fat accumulation was reduced.The mice in the HFD+Leu group exhib-ited higher heat production and O2 consumption,along with an increase in CO2 exhalation by day and by night.In addition,heat production,CO2 exhalation,and O2 consumption were significantly higher by night than by day(P<0.01).As for the respiratory exchange ratio,a night increase was seen in the mice from the normal group,normal+Leu group,and HFD group,but not in the HFD+Leu group.The results of Western blotting showed that compared with the normal group,the BCKDE1A phosphorylation inacti-vation was enhanced,AMPKα phosphorylation activation alleviated,the protein expressions of SIRT1 and PGC-1α downregulated(P<0.05),and the protein expressions of FAS and FABP4 increased in the livers of mice in the HFD group.Compared with the HFD group,the BCKDE1A phosphorylation inacti-vation was alleviated,AMPKα phosphorylation activation enhanced,the protein expressions of SIRT1 and PGC-1α increased,and the protein expressions of FAS and FABP4 downregulated in the livers of mice in the HFD+Leu group.CONCLUSION Leu can alleviate HFD-induced metabolic dysfunction-associated fatty liver disease in mice,which may be closely related to the promotion of energy metabo-lism and inhibition of fat synthesis.

Objective:To investigate the influence of prealbumin on cerebral infarction severity, hemorrhage transformation and 1-year prognosis.Methods:A retrospective study was conducted to select 752 patients with cerebral infarction who were treated in Beijing Tiantan Hospital,Capital Medical University from December 2018 to December 2019 as the study objects. Personal information and laboratory indicators of the patients were collected including prealbumin, hemoglobin, white blood cell count, etc.Patients were divided into group B1 (<238 mg/L) and group B2 (≥238 mg/L) based on median prealbumin. By inquiry patient's case, NIHSS score (<16 was classified as mild, ≥16 as moderate and severe)and cerebral infarction volume (<20 cm 3 as small infarct, >20 cm 3 as large infarct) were recorded to evaluate the severity of the disease, and whether hemorrhage transformation occurred during hospitalization was recorded. Patients were followed up 1 year after discharge, and prognostic information of patients was recorded, including neurological function recovery (mRS score <3 was classified as good recovery, ≥3 as poor recovery),all-cause case fatality rate, and recurrence of cardio-cerebrovascular events. Normally distributed measurement data were expressed as xˉ±s, non-normally distributed measurement data were expressed as median and quartiles[ M( Q1, Q3)], categorical variable were expressed as ratio and percent(%). Comparison between groups of measurement data were performed by independent sample t test and Mann-Whitney U test. Chi-square test were used on comparison between groups of categorical variable. Single-factor comparison, Spearman correlation analysis and multiple Logistic regression were used to analyze the correlation between prealbumin and other laboratory indicators, cerebral infarction severity, hemorrhage transformation and 1-year prognosis, respectively. Results:The NIHSS score and infarct volume of patients in group B1 were 5(2,10) and 3.18(0.72,18.00) cm 3, and those in group B2 were 3(2,7) and 2.0(0.5,10.0) cm 3, respectively, which were higher in group B1 than in group B2, the differences were statistically significant ( Z=3.85, P<0.001, Z=2.81, P=0.005). The proportion of mRS Score ≥3 in group B1 was 28.8%(107/371), and the all-cause case fatality rate was 7.5%(28/371), both higher than 20.5%(78/381) and 3.1%(12/381) in group B2, with statistical significance ( χ2=7.10, P=0.008, χ2=7.22, P=0.007). Hemorrhage transformation and recurrence of cardio-cerebrovascular events were 13.5%(50/371) and 11.6%(43/371) in group B1 and 9.2% (35/381) and 8.7%(33/381) in group B2, respectively, with no significant difference between the two groups ( χ2=3.45, P=0.063, χ2=1.78, P=0.183). Multivariate logistic regression analysis showed that, after adjusted for potential confounding factors, prealbumin was protective factor of NIHSS ( OR and 95% CI: 0.990(0.984-0.997), P=0.035), poor neurological recovery(mRS≥3) ( OR and 95% CI:0.992(0.988-0.997), P<0.001) and all-cause case fatality rate ( OR and 95% CI:0.991(0.983-0.999), while prealbumin had no significant influence on cardiocerebrovascular recurrence events ( OR and 95% CI: 0.999(0.993-1.005), P=0.729). Conclusion:Prealbumin is significantly associated with the severity of cerebral infarction and poor prognosis 1 year after discharge, and low prealbumin was an independent risk factor for NIHSS score(≥16), poor neurological recovery (mRS≥3) and all-cause case fatality rate.

Objectives:To explore the effects of infarct volume, infarct type, inflammation, and coagulation indicators on hemorrhagic transformation in patients with acute cerebral infarction.Methods:711 patients with cerebral infarction admitted to Beijing Tiantan Hospital were retrospectively included as the study objects from December 2018 to December 2019 [535 males and 176 females, age 22-95 years, mean age (59.6±12.1) years]. Clinical data, laboratory indicators such as inflammation and coagulation function of patients were collected, and information such as location, volume and type of infarction were recorded. The patients were divided into hemorrhage transformation group and non-hemorrhage transformation group according to whether hemorrhage transformation occurred during hospitalization. Normally distributed measurement data were expressed as xˉ± s, non-normally distributed measurement data were expressed as median and quartiles [ M( Q1, Q3)], categorical variable were expressed as ratio and percent (%). Comparison between groups of measurement data were performed by independent sample t test and Mann-Whitney U test. χ2 test were used on comparison between groups of categorical variable. Univariate comparison and multivariate Logistic regression were used to analyze the correlation between hemorrhage transformation and infarct volume, infarction type and laboratory indicators, respectively, to explore the risk factors of hemorrhage transformation. ROC curve analysis was used to evaluate the diagnostic value of indicators. Results:The rates of coronary heart disease and atrial fibrillation history in the hemorrhage transformation group were 23.5% (20/85) and 22.4% (19/85), respectively, which were significantly higher than those in the non-hemorrhage transformation group (13.9% (87/626) and 5.8% (36/626), respectively), and the difference between the two groups was statistically significant ( χ2=5.43, χ2=28.90, P=0.020, P<0.001, respectively). The NIHSS score [10(4,17) points] and infarct volume [46.50 (14.21,118.42) mL] in the hemorrhage transformation group were significantly higher than those in the non-hemorrhage transformation group [4(2,7) points, 2.00(0.51,8.94) mL]. The difference between the two groups was statistically significant ( Z values were 6.69 and 10.69, respectively, P<0.001). The results of multivariate Logistic regression analysis showed that atrial fibrillation (OR=2.604, 95% CI: 1.186-5.716, P=0.107), infarct volume (OR=1.009, 95% CI: 1.004-1.015, P=0.001), infarct type of Chinese ischemic stroke subclassfication (OR=1.371, 95% CI: 1.085-1.731, P=0.008) and neutrophil/lymphocyte ratio (OR=1.047, 95% CI: 1.006-1.090, P=0.023) were independent risk factors for hemorrhage transformation. ROC curve analysis showed that the area under curve (AUC) of infarct volume and neutrophil/lymphocyte ratio were 0.861 (0.821-0.901) and 0.684 (0.626-0.741), respectively, which were effective in predicting hemorrhage transformation after cerebral infarction. The prediction of infarct volume was more efficient. Conclusion:History of atrial fibrillation, classification of cardioembolic stroke, infarct volume, and neutrophil/lymphocyte ratio are all risk factors for hemorrhagic transformation after acute cerebral infarction.

IgA nephropathy(IgAN)is one of the most prevalent forms of primary glomerulonephritis globally and a leading cause of end-stage renal disease(ESRD).The exact pathogenesis and progression mechanisms of IgAN remain unclear.Its clinical manifestations are diverse,with varying degrees of kidney involvement reflected in both clinical presentations and pathological changes.Consequently,responses to treatment and prognoses differ significantly among patients.Currently,renal needle biopsy serves as the gold standard for diagnosing IgAN;how-ever,this invasive procedure is often not well-accepted by patients,limiting its widespread clinical application and complicating disease diagnosis.Therefore,non-invasive biomarkers are crucial for assisting in the diagnosis of IgAN and evaluating the risk of disease progression.Below,we will focus on various serum and urinary biomarkers associated with IgAN at both protein and nucleic acid levels.

BACKGROUND:Skeletal muscle insulin resistance is the key pathological link of type 2 diabetes.The traditional Chinese medicine compound Roujishuncuiyin can effectively improve skeletal muscle insulin resistance,but its mechanism has not been clarified.OBJECTIVE:To explore the mechanism of Roujishuncuiyin on skeletal muscle insulin resistance in type 2 diabetes mice.METHODS:Forty db/db mice with type 2 diabetes mellitus were randomized into a model group,a low-dose Roujishuncuiyin group,a high-dose Roujishuncuiyin group,and a positive drug group,with 10 mice in each group.The latter three administration groups were given 157.5 mg/g and 630 mg/g Roujishuncuiyin and 200 mg/g metformin hydrochloride aqueous solution by gavage once a day,respectively.In addition,10 db/dm mice were selected as the blank control group.Mice in the model and blank control groups were given the same dose of 0.9％NaCl solution by gavage.After 12 weeks of intervention,fasting blood glucose was measured in each group of mice,and oral glucose tolerance test was performed to calculate the area under the blood glucose curve.ELISA was used to detect serum insulin level and calculate the resistance index.Mitochondrial structure of skeletal muscle tissue was observed under transmission electron microscopy.Western blot was used to detect the expression levels and phosphorylation levels of protein kinase B(AKT)and glycogen synthase kinase 3β(GSK-3β)proteins in skeletal muscle.RESULTS AND CONCLUSION:(1)Compared with the blank control group,fasting blood glucose,fasting insulin and insulin resistance index were significantly higher in the model group(P＜0.05),the area under the curve of the oral glucose tolerance test was significantly increased(P＜0.05),the expression of p-AKT and p-GSK3β proteins in tibialis anterior muscle was significantly decreased(P＜0.05),and there was a large amount of mitochondrial damage in tibialis anterior muscle and a large number of lipid droplets in the interstitium.(2)Compared with the model group,fasting blood glucose,fasting insulin,and insulin resistance index were significantly reduced in the low-and high-dose Roujishuncuiyin groups and the positive control group(P＜0.05),the area under the curve of the oral glucose tolerance test was reduced(P＜0.05),the expression of p-AKT and p-GSK3β proteins in the tibialis anterior muscle was significantly elevated(P＜0.05),and mitochondrial damage in the tibialis anterior muscle was significantly ameliorated,with decreased lipid droplets in the interstitium.(3)The above indexes were better in the high-dose Roujishuncuiyin group than the low-dose Roujishuncuiyin group(P＜0.05),while there was no significant difference between the high-dose Roujishuncuiyin group and positive control group(P＞0.05).To conclude,by upregulating the protein levels of p-AKT and p-GSK3β in skeletal muscle tissue,the traditional Chinese medicine compound Roujishuncuiyin can improve structural disorders and mitochondrial morphology in skeletal muscle tissue,reduce insulin resistance in the skeletal muscle and regulate glucose homeostasis in the body.

OBJECTIVE To explore the effect and underlying mechanism of leucine(Leu)on meta-bolic dysfunction-associated fatty liver disease induced by high fat diet(HFD)in mice.METHODS C57BL/6J male mice were randomly divided into chow diet(normal),chow diet+Leu(normal+Leu),HFD and HFD+Leu groups,with 10 mice in each group.The mice in the normal and normal+Leu groups received chow diet while those in the HFD and HFD+Leu groups received HFD.Drinking water for mice in the normal+Leu and HFD+Leu groups was supplemented with 1.5%Leu.The experiment lasted 24 weeks,total food and water intake of mice were recorded weekly to calculate energy and Leu intake respectively.Energy metabolism of mice was detected at week 20 by the Oxymas/CLAMS Animal Metabolic System heat production,CO2 exhalation,O2 consumption and respiratory exchange rate(RER).At the end of week 24,the mice were sacrificed and the livers were harvested,followed by the oil red O staining to reveal the fat content.Western blotting was performed to analyze the changes in the activity of the liver branched-chain α-keto acid dehydrogenase E1α(BCKDE1A),the activation of AMP-activated protein kinase alpha subunit(AMPKα),and the protein expressions of downstream effector molecules including silent information regulator 1(SIRT1)and peroxisome proliferator-activated receptor coactivator-1α(PGC-1α),fatty acid synthetase(FAS)and fatty acid binding protein 4(FABP4)in the liver of mice.RESULTS Total Leu intake of mice was significantly reduced in the HFD+Leu group,compared with the normal+Leu group.The mice fed with HFD significantly increased the energy intake body mass gain and liver mass,accompanied by fat accumulation in the liver,compared to the mice in the normal group.Simultaneously,the mice in the HFD group showed a decrease in CO2 exha-lation both by day and by night,and in the respiratory exchange ratio by day compared to the normal group.Compared with the HFD group,the body mass gain and liver mass obviously decreased in mice of the HFD+Leu group,and the liver fat accumulation was reduced.The mice in the HFD+Leu group exhib-ited higher heat production and O2 consumption,along with an increase in CO2 exhalation by day and by night.In addition,heat production,CO2 exhalation,and O2 consumption were significantly higher by night than by day(P<0.01).As for the respiratory exchange ratio,a night increase was seen in the mice from the normal group,normal+Leu group,and HFD group,but not in the HFD+Leu group.The results of Western blotting showed that compared with the normal group,the BCKDE1A phosphorylation inacti-vation was enhanced,AMPKα phosphorylation activation alleviated,the protein expressions of SIRT1 and PGC-1α downregulated(P<0.05),and the protein expressions of FAS and FABP4 increased in the livers of mice in the HFD group.Compared with the HFD group,the BCKDE1A phosphorylation inacti-vation was alleviated,AMPKα phosphorylation activation enhanced,the protein expressions of SIRT1 and PGC-1α increased,and the protein expressions of FAS and FABP4 downregulated in the livers of mice in the HFD+Leu group.CONCLUSION Leu can alleviate HFD-induced metabolic dysfunction-associated fatty liver disease in mice,which may be closely related to the promotion of energy metabo-lism and inhibition of fat synthesis.

IgA nephropathy(IgAN)is one of the most prevalent forms of primary glomerulonephritis globally and a leading cause of end-stage renal disease(ESRD).The exact pathogenesis and progression mechanisms of IgAN remain unclear.Its clinical manifestations are diverse,with varying degrees of kidney involvement reflected in both clinical presentations and pathological changes.Consequently,responses to treatment and prognoses differ significantly among patients.Currently,renal needle biopsy serves as the gold standard for diagnosing IgAN;how-ever,this invasive procedure is often not well-accepted by patients,limiting its widespread clinical application and complicating disease diagnosis.Therefore,non-invasive biomarkers are crucial for assisting in the diagnosis of IgAN and evaluating the risk of disease progression.Below,we will focus on various serum and urinary biomarkers associated with IgAN at both protein and nucleic acid levels.

BACKGROUND:Skeletal muscle insulin resistance is the key pathological link of type 2 diabetes.The traditional Chinese medicine compound Roujishuncuiyin can effectively improve skeletal muscle insulin resistance,but its mechanism has not been clarified.OBJECTIVE:To explore the mechanism of Roujishuncuiyin on skeletal muscle insulin resistance in type 2 diabetes mice.METHODS:Forty db/db mice with type 2 diabetes mellitus were randomized into a model group,a low-dose Roujishuncuiyin group,a high-dose Roujishuncuiyin group,and a positive drug group,with 10 mice in each group.The latter three administration groups were given 157.5 mg/g and 630 mg/g Roujishuncuiyin and 200 mg/g metformin hydrochloride aqueous solution by gavage once a day,respectively.In addition,10 db/dm mice were selected as the blank control group.Mice in the model and blank control groups were given the same dose of 0.9％NaCl solution by gavage.After 12 weeks of intervention,fasting blood glucose was measured in each group of mice,and oral glucose tolerance test was performed to calculate the area under the blood glucose curve.ELISA was used to detect serum insulin level and calculate the resistance index.Mitochondrial structure of skeletal muscle tissue was observed under transmission electron microscopy.Western blot was used to detect the expression levels and phosphorylation levels of protein kinase B(AKT)and glycogen synthase kinase 3β(GSK-3β)proteins in skeletal muscle.RESULTS AND CONCLUSION:(1)Compared with the blank control group,fasting blood glucose,fasting insulin and insulin resistance index were significantly higher in the model group(P＜0.05),the area under the curve of the oral glucose tolerance test was significantly increased(P＜0.05),the expression of p-AKT and p-GSK3β proteins in tibialis anterior muscle was significantly decreased(P＜0.05),and there was a large amount of mitochondrial damage in tibialis anterior muscle and a large number of lipid droplets in the interstitium.(2)Compared with the model group,fasting blood glucose,fasting insulin,and insulin resistance index were significantly reduced in the low-and high-dose Roujishuncuiyin groups and the positive control group(P＜0.05),the area under the curve of the oral glucose tolerance test was reduced(P＜0.05),the expression of p-AKT and p-GSK3β proteins in the tibialis anterior muscle was significantly elevated(P＜0.05),and mitochondrial damage in the tibialis anterior muscle was significantly ameliorated,with decreased lipid droplets in the interstitium.(3)The above indexes were better in the high-dose Roujishuncuiyin group than the low-dose Roujishuncuiyin group(P＜0.05),while there was no significant difference between the high-dose Roujishuncuiyin group and positive control group(P＞0.05).To conclude,by upregulating the protein levels of p-AKT and p-GSK3β in skeletal muscle tissue,the traditional Chinese medicine compound Roujishuncuiyin can improve structural disorders and mitochondrial morphology in skeletal muscle tissue,reduce insulin resistance in the skeletal muscle and regulate glucose homeostasis in the body.

Objective:To investigate the influence of prealbumin on cerebral infarction severity, hemorrhage transformation and 1-year prognosis.Methods:A retrospective study was conducted to select 752 patients with cerebral infarction who were treated in Beijing Tiantan Hospital,Capital Medical University from December 2018 to December 2019 as the study objects. Personal information and laboratory indicators of the patients were collected including prealbumin, hemoglobin, white blood cell count, etc.Patients were divided into group B1 (<238 mg/L) and group B2 (≥238 mg/L) based on median prealbumin. By inquiry patient's case, NIHSS score (<16 was classified as mild, ≥16 as moderate and severe)and cerebral infarction volume (<20 cm 3 as small infarct, >20 cm 3 as large infarct) were recorded to evaluate the severity of the disease, and whether hemorrhage transformation occurred during hospitalization was recorded. Patients were followed up 1 year after discharge, and prognostic information of patients was recorded, including neurological function recovery (mRS score <3 was classified as good recovery, ≥3 as poor recovery),all-cause case fatality rate, and recurrence of cardio-cerebrovascular events. Normally distributed measurement data were expressed as xˉ±s, non-normally distributed measurement data were expressed as median and quartiles[ M( Q1, Q3)], categorical variable were expressed as ratio and percent(%). Comparison between groups of measurement data were performed by independent sample t test and Mann-Whitney U test. Chi-square test were used on comparison between groups of categorical variable. Single-factor comparison, Spearman correlation analysis and multiple Logistic regression were used to analyze the correlation between prealbumin and other laboratory indicators, cerebral infarction severity, hemorrhage transformation and 1-year prognosis, respectively. Results:The NIHSS score and infarct volume of patients in group B1 were 5(2,10) and 3.18(0.72,18.00) cm 3, and those in group B2 were 3(2,7) and 2.0(0.5,10.0) cm 3, respectively, which were higher in group B1 than in group B2, the differences were statistically significant ( Z=3.85, P<0.001, Z=2.81, P=0.005). The proportion of mRS Score ≥3 in group B1 was 28.8%(107/371), and the all-cause case fatality rate was 7.5%(28/371), both higher than 20.5%(78/381) and 3.1%(12/381) in group B2, with statistical significance ( χ2=7.10, P=0.008, χ2=7.22, P=0.007). Hemorrhage transformation and recurrence of cardio-cerebrovascular events were 13.5%(50/371) and 11.6%(43/371) in group B1 and 9.2% (35/381) and 8.7%(33/381) in group B2, respectively, with no significant difference between the two groups ( χ2=3.45, P=0.063, χ2=1.78, P=0.183). Multivariate logistic regression analysis showed that, after adjusted for potential confounding factors, prealbumin was protective factor of NIHSS ( OR and 95% CI: 0.990(0.984-0.997), P=0.035), poor neurological recovery(mRS≥3) ( OR and 95% CI:0.992(0.988-0.997), P<0.001) and all-cause case fatality rate ( OR and 95% CI:0.991(0.983-0.999), while prealbumin had no significant influence on cardiocerebrovascular recurrence events ( OR and 95% CI: 0.999(0.993-1.005), P=0.729). Conclusion:Prealbumin is significantly associated with the severity of cerebral infarction and poor prognosis 1 year after discharge, and low prealbumin was an independent risk factor for NIHSS score(≥16), poor neurological recovery (mRS≥3) and all-cause case fatality rate.