This study established a droplet digital PCR(ddPCR)EvaGreen assay and probe methods for Schistosoma japonicum detection,and evaluated their application in detecting early infections in the S.japonicum host oncomelania and mice.Primers and corresponding probes for both ddPCR methods were designed and synthesized,and plasmids containing target sequences were constructed.The sensitivity of the two methods was tested through detection of the corresponding plasmids,and infectious and mixed oncomelania genomic DNA.Their specificity was evaluated by the detection of genomic DNA of negative oncomelania,Schistosoma mansoni,Clonorchis sinensis,Spirometra mansoni,and S.japonicum(as a positive control).The ddPCR probe method was evaluated by detection of early infection of oncomelania exposed tomiracidium with various ratios and incubation times,and the early migration and distribution of cercaria or schistosomula in mouse hosts infected with 200 cercaria via abdominal skin contact.According to standard curves constructed through the detection of plasmid serial dilutions,the regression equation for the EvaGreen assay was y=-0.839 9x+7.050 9,with a correlation coefficient R2=0.988 1,and the regression equation for the probe method was y=-1.047 5x+7.255 1,with a correlation coefficient R2=0.999 8.The lowest limit of plasmid detection with the probe method was between 38.94 cp/μL and 194.74 cp/μL.Both methods successfully detected positive reactions in the genomic DNA samples of infectious oncomelania at concentrations above 0.002 ng/μL and in the genomic DNA of each group of oncomelania mixtures.No significant differences between probe methods were observed in the detection values in the control group and the genomic DNA of negative oncomelania,S.mansoni,C.sinensis,and S.mansoni.However,the detection value of genomic DNA of negative oncomelania(291 ng/μL)with the EvaGreen assay was(20.3±4.39)cp/μL,a value significantly higher than the(1.5±0.1)cp/μL observed in the control group.For detection of early infection in oncomelania,the probe method detected Schistosoma japonicum DNA after 30 s incubation at room temperature with a≥5∶1 ratio of miracidium to oncomelania;the detection value peaked after a short time(5 min),and the peak value showed a fold increase similar to the increase in the miracidium to oncomelania ratio.Detection of early stage infection in mice with the probe method revealed that the schistosomula entered the lungs on day 2 and the liver on day 4,and continually migrated within the organs with abundant blood supply(spleen,kidney,and brain)in the first 9 days;moreover,a tendency toward ectopic parasitism was observed in the heart and pancreas on day 9,and a constantly negative control level was observed in the testes.The ddPCR probe method was more accurate and specific than the EvaGreen assay in the detection of plasmids,and infectious and mixed oncomelania,and the latter showed non-specific reactions in negative oncomelania detection.In a practical application,the probe method was demonstrated to be sensitive,to effectively reflect the early infection of oncomelania,and to reveal schistosomula migration and distribution in multiple organs of infected mice.

This study established a droplet digital PCR(ddPCR)EvaGreen assay and probe methods for Schistosoma japonicum detection,and evaluated their application in detecting early infections in the S.japonicum host oncomelania and mice.Primers and corresponding probes for both ddPCR methods were designed and synthesized,and plasmids containing target sequences were constructed.The sensitivity of the two methods was tested through detection of the corresponding plasmids,and infectious and mixed oncomelania genomic DNA.Their specificity was evaluated by the detection of genomic DNA of negative oncomelania,Schistosoma mansoni,Clonorchis sinensis,Spirometra mansoni,and S.japonicum(as a positive control).The ddPCR probe method was evaluated by detection of early infection of oncomelania exposed tomiracidium with various ratios and incubation times,and the early migration and distribution of cercaria or schistosomula in mouse hosts infected with 200 cercaria via abdominal skin contact.According to standard curves constructed through the detection of plasmid serial dilutions,the regression equation for the EvaGreen assay was y=-0.839 9x+7.050 9,with a correlation coefficient R2=0.988 1,and the regression equation for the probe method was y=-1.047 5x+7.255 1,with a correlation coefficient R2=0.999 8.The lowest limit of plasmid detection with the probe method was between 38.94 cp/μL and 194.74 cp/μL.Both methods successfully detected positive reactions in the genomic DNA samples of infectious oncomelania at concentrations above 0.002 ng/μL and in the genomic DNA of each group of oncomelania mixtures.No significant differences between probe methods were observed in the detection values in the control group and the genomic DNA of negative oncomelania,S.mansoni,C.sinensis,and S.mansoni.However,the detection value of genomic DNA of negative oncomelania(291 ng/μL)with the EvaGreen assay was(20.3±4.39)cp/μL,a value significantly higher than the(1.5±0.1)cp/μL observed in the control group.For detection of early infection in oncomelania,the probe method detected Schistosoma japonicum DNA after 30 s incubation at room temperature with a≥5∶1 ratio of miracidium to oncomelania;the detection value peaked after a short time(5 min),and the peak value showed a fold increase similar to the increase in the miracidium to oncomelania ratio.Detection of early stage infection in mice with the probe method revealed that the schistosomula entered the lungs on day 2 and the liver on day 4,and continually migrated within the organs with abundant blood supply(spleen,kidney,and brain)in the first 9 days;moreover,a tendency toward ectopic parasitism was observed in the heart and pancreas on day 9,and a constantly negative control level was observed in the testes.The ddPCR probe method was more accurate and specific than the EvaGreen assay in the detection of plasmids,and infectious and mixed oncomelania,and the latter showed non-specific reactions in negative oncomelania detection.In a practical application,the probe method was demonstrated to be sensitive,to effectively reflect the early infection of oncomelania,and to reveal schistosomula migration and distribution in multiple organs of infected mice.

OBJECTIVES: To investigate the role and mechanism of copper overload-mediated endoplasmic reticulum stress (ERS) in vascular endothelial injury in Kawasaki disease (KD). METHODS: Four-week-old male C57BL/6 mice were randomly divided into four groups: control, KD, KD plus copper chelator tetrathiomolybdate (TTM), and KD plus ERS inhibitor AMG PERK 44 (AMG) (n=20 per group). A KD mouse model was established using Candida albicans extract. Human umbilical vein endothelial cells (HUVECs) were divided into control (intervention with healthy children's serum), KD (intervention with KD patients' serum), and KD+TTM (intervention with KD patients' serum plus 20 µmol/L TTM). Copper deposition in mouse heart tissue was assessed using rubeanic acid staining. Vascular pathological changes were observed using hematoxylin-eosin staining and measurement of abdominal aortic diameter and area. ERS activation was detected by transmission electron microscopy and immunofluorescence. HUVEC viability, apoptosis, and functional changes were evaluated using CCK8, flow cytometry, cell scratch assay, and angiogenesis experiments. ERS marker protein expression levels were measured by Western blot. RESULTS: Compared to the KD group, the KD+TTM and KD+AMG groups showed reduced copper deposition in the vascular wall, decreased swelling of coronary endothelial cells and endoplasmic reticulum, reduced inflammatory cell infiltration, and less abdominal aortic lesion expansion. The abdominal aortic diameter and area, and the fluorescence intensity of ERS marker proteins (GRP78 and CHOP) were significantly lower (P<0.05). Compared to the KD group, the KD+TTM group exhibited increased cell viability, tube number, and scratch healing rate, along with decreased apoptosis rate and expression of ERS marker proteins (GRP78, CHOP, ATF6, and p-PERK) (P<0.05). CONCLUSIONS Copper overload aggravates vascular endothelial injury in KD by activating the ERS pathway. TTM can exert protective effects on the endothelium by regulating copper metabolism and inhibiting the ERS pathway.
Endoplasmic Reticulum Stress ; Copper/toxicity* ; Male ; Mucocutaneous Lymph Node Syndrome/metabolism* ; Animals ; Humans ; Endoplasmic Reticulum Chaperone BiP ; Mice, Inbred C57BL ; Mice ; Human Umbilical Vein Endothelial Cells ; Apoptosis ; Endothelium, Vascular/injuries*

Objective This study aimed to analyze the current epidemic situation of cancer in Yunnan province in 2020,and to provide theoretical basis for the prevention and treatment of cancer in Yunnan province.Methods The data on the incidence and mortality of cancer were collected from 129 cancer registration areas in Yunnan province in 2020.A total of 89 counties(cities,dis-tricts)that met the quality control standards were included in this analysis.Among them,there were 16 in urban areas,covering a pop-ulation of 7,593,622(24.16%),and 73 in rural areas,covering a population of 23,838,542(75.84%).The number of malignant tumor cases,deaths,the crude incidence and China age-standardized incidence(China Standardized Incidence),the crude mortality and China age-standardized mortality(China Standardized Mortality),the cumulative incidence and mortality from 0 to 74 years old,as well as the order of cancer incidence and death were statistical analyzed.Results In 2020,66,719 new cases of cancer were re-ported from 89 tumor registration areas in Yunnan province,with a crude rate of 212.26/100,000 and the China age-standardized in-cidence of 150.33/100,000;The reported number of deaths from cancer was 39,251,with a crude mortality of 124.88/100,000 and the China age-standardized mortality of 82.45/100,000.The incidence and mortality of cancer were higher in men than those in fe-male,and higher in rural than those in urban areas.According to the crude incidence rate and crude mortality,the top 5 cancers with the highest crude incidence were lung cancer,female breast cancer,colo-rectum cancer,liver cancer and thyroid cancer,and the top 5 cancers with the highest crude mortality were lung cancer,liver cancer,colo-rectum cancer,stomach cancer and female breast cancer.Conclusion Lung cancer,liver cancer,colo-rectum cancer and female breast cancer are still the key cancers for prevention and con-trol in Yunnan province.The incidence of thyroid cancer is relatively high,and there are significant differences in the incidence and mortality of cancer between urban and rural areas and between sexes.The corresponding cancer prevention should be carried out ac-cording to the current epidemic situation of cancer in Yunnan province.

Objective This study aimed to analyze the incidence and mortality of cervical cancer in tumor registration areas of Yunnan province in 2020,as well as the trend of incidence and mortality from 2012 to 2020,in order to provide a basis for the pre-vention and treatment of cervical cancer in Yunnan province.Methods The data of cervical cancer incidence and mortality were col-lected and sorted out in tumor registration areas of Yunnan province from 2012 to 2020.The crude incidence,crude mortality,age-standardized incidence rate by World standard population(ASIRW)(referred to the World standard incidence),age-standardized mortality rate by World standard population(ASMRW)(referred to the World standard mortality)and other indicators of cervical canc-er in 2020 were statistically analyzed according to the urban and rural areas.The annual percent change(APC)was used to evaluate the changing trend of cervical cancer incidence and mortality from 2012 to 2020,and the GM(1,1)model was used to predict the crude incidence and ASIRW from 2021 to 2025.Results In 2020,there were 2426 new cases of cervical cancer in tumor registration area of Yunnan province,ranking fifth in the incidence of female malignant tumors.The crude incidence and ASIRW were 15.83/100,000 and 11.16/100,000,respectively.There were 831 deaths from cervical cancer,ranking sixth deaths of female malignant tumors.The crude mortality and ASMRW were 5.42/100,000 and 3.52/100,000,respectively.ASIRW was higher in rural areas(11.86/100,000)than that in urban areas(9.11/100,000).ASMRW was slightly higher in urban areas(3.63/100,000)than that in rural areas(3.48/100,000).The age-specific incidence of cervical cancer increased rapidly after the age of 20,and reached a peak in the 55-59 age group;The age-specific mortality of cervical cancer increased rapidly after the age of 35,reaching a peak in the 75-79 age group.ASIRW of cervical cancer in Yunnan province from 2012 to 2020 showed a downward trend with annual changes(APC=-7.54%,95%CI:-13.19%--1.53%),and the trend change was statistically significant(P<0.05).The prediction of the GM(1,1)model showed that the crude incidence and ASIRW of cervical cancer in Yunnan province would continue to decline from 2021 to 2025.Conclusion The incidence and mortality of cervical cancer are relatively low in Yunnan province,but it is still a common malignant tumor in women.The incidence of cervical cancer in Yunnan province is showing a trend of becoming younger,and rural women are the key population for prevention and treatment.

Objective The aim of this study was to analyze the incidence and mortality of liver cancer among residents in cancer registration areas of Yunnan province in 2020,as well as the trend from 2012 to 2020,in order to provide reference for the pre-vention and control of liver cancer in Yunnan province.Methods The incidence and death data of all liver cancer patients included in the national cancer registration annual report from 2012 to 2020 in tumor registration areas of Yunnan province were collected.The crude incidence and crude mortality of liver cancer in cancer registration areas of Yunnan province in 2020 were analyzed using Excel 2010 and SAS 9.4 statistical software,and the change trend of age-standardized incidence rate by World standard population(ASIRW)and age-standardized mortality rate by World standard population(ASMRW)of liver cancer in cancer registration areas of Yunnan province from 2012 to 2020 was analyzed using Joinpoint 4.8.0.1 software.Results In 2020,the crude incidence of liver cancer in tumor registration areas of Yunnan province was 20.32/100,000(male 29.41/100,000 and female 10.78/100,000),the crude incidence in urban areas was 19.45/100,000,the crude incidence in rural areas was 20.60/100,000.The crude mortality of liver cancer in tumor registration areas of Yunnan province was 20.54/100,000(male 29.60/100,000 and female 11.02/100,000),17.70/100,000 for urban areas,and 21.44/100,000 for rural areas.In 2020,the crude incidence in tumor registration areas of liver cancer in Yunnan province was at a low level before the age of 34,but rapidly increased at the age of 35.The peak rate of the 80-84 age group was 81.37/100,000,and the crude mortality was at a low level before the age of 34.After the age of 35,the mortality rapid-ly increased,and the peak rate of the 75-79 age group was 92.14/100,000.From 2012 to 2020,the annual change percentage of ASIRW of liver cancer in tumor registration areas of Yunnan province showed an upward trend(APC=1.82%,95%CI:0.07%-3.61%,t=2.45,P<0.05),and the annual change percentage of ASMRW showed an upward trend(APC=2.88%,95%CI:1.52%-4.26%,t=5.04,P<0.05).Conclusion In 2020,the crude incidence rate and crude mortality of liver cancer in tumor registration areas of Yunnan Province were higher in men than in women,and higher in rural areas than in urban areas.From 2021-2020,ASIRW and ASMRW of liver cancer are on the rise.We should continue to strengthen the prevention and treatment of liver cancer in Yunnan Province,strengthen the early screening and diagnosis of liver cancer through the combination of medicine and prevention,improve the tumor registration report detection system,strengthen health education,and improve the overall health awareness and cancer prevention knowledge of residents.

Objective This study aimed to analyze the current epidemic situation of cancer in Yunnan province in 2020,and to provide theoretical basis for the prevention and treatment of cancer in Yunnan province.Methods The data on the incidence and mortality of cancer were collected from 129 cancer registration areas in Yunnan province in 2020.A total of 89 counties(cities,dis-tricts)that met the quality control standards were included in this analysis.Among them,there were 16 in urban areas,covering a pop-ulation of 7,593,622(24.16%),and 73 in rural areas,covering a population of 23,838,542(75.84%).The number of malignant tumor cases,deaths,the crude incidence and China age-standardized incidence(China Standardized Incidence),the crude mortality and China age-standardized mortality(China Standardized Mortality),the cumulative incidence and mortality from 0 to 74 years old,as well as the order of cancer incidence and death were statistical analyzed.Results In 2020,66,719 new cases of cancer were re-ported from 89 tumor registration areas in Yunnan province,with a crude rate of 212.26/100,000 and the China age-standardized in-cidence of 150.33/100,000;The reported number of deaths from cancer was 39,251,with a crude mortality of 124.88/100,000 and the China age-standardized mortality of 82.45/100,000.The incidence and mortality of cancer were higher in men than those in fe-male,and higher in rural than those in urban areas.According to the crude incidence rate and crude mortality,the top 5 cancers with the highest crude incidence were lung cancer,female breast cancer,colo-rectum cancer,liver cancer and thyroid cancer,and the top 5 cancers with the highest crude mortality were lung cancer,liver cancer,colo-rectum cancer,stomach cancer and female breast cancer.Conclusion Lung cancer,liver cancer,colo-rectum cancer and female breast cancer are still the key cancers for prevention and con-trol in Yunnan province.The incidence of thyroid cancer is relatively high,and there are significant differences in the incidence and mortality of cancer between urban and rural areas and between sexes.The corresponding cancer prevention should be carried out ac-cording to the current epidemic situation of cancer in Yunnan province.

Objective This study aimed to analyze the incidence and mortality of cervical cancer in tumor registration areas of Yunnan province in 2020,as well as the trend of incidence and mortality from 2012 to 2020,in order to provide a basis for the pre-vention and treatment of cervical cancer in Yunnan province.Methods The data of cervical cancer incidence and mortality were col-lected and sorted out in tumor registration areas of Yunnan province from 2012 to 2020.The crude incidence,crude mortality,age-standardized incidence rate by World standard population(ASIRW)(referred to the World standard incidence),age-standardized mortality rate by World standard population(ASMRW)(referred to the World standard mortality)and other indicators of cervical canc-er in 2020 were statistically analyzed according to the urban and rural areas.The annual percent change(APC)was used to evaluate the changing trend of cervical cancer incidence and mortality from 2012 to 2020,and the GM(1,1)model was used to predict the crude incidence and ASIRW from 2021 to 2025.Results In 2020,there were 2426 new cases of cervical cancer in tumor registration area of Yunnan province,ranking fifth in the incidence of female malignant tumors.The crude incidence and ASIRW were 15.83/100,000 and 11.16/100,000,respectively.There were 831 deaths from cervical cancer,ranking sixth deaths of female malignant tumors.The crude mortality and ASMRW were 5.42/100,000 and 3.52/100,000,respectively.ASIRW was higher in rural areas(11.86/100,000)than that in urban areas(9.11/100,000).ASMRW was slightly higher in urban areas(3.63/100,000)than that in rural areas(3.48/100,000).The age-specific incidence of cervical cancer increased rapidly after the age of 20,and reached a peak in the 55-59 age group;The age-specific mortality of cervical cancer increased rapidly after the age of 35,reaching a peak in the 75-79 age group.ASIRW of cervical cancer in Yunnan province from 2012 to 2020 showed a downward trend with annual changes(APC=-7.54%,95%CI:-13.19%--1.53%),and the trend change was statistically significant(P<0.05).The prediction of the GM(1,1)model showed that the crude incidence and ASIRW of cervical cancer in Yunnan province would continue to decline from 2021 to 2025.Conclusion The incidence and mortality of cervical cancer are relatively low in Yunnan province,but it is still a common malignant tumor in women.The incidence of cervical cancer in Yunnan province is showing a trend of becoming younger,and rural women are the key population for prevention and treatment.

Objective The aim of this study was to analyze the incidence and mortality of liver cancer among residents in cancer registration areas of Yunnan province in 2020,as well as the trend from 2012 to 2020,in order to provide reference for the pre-vention and control of liver cancer in Yunnan province.Methods The incidence and death data of all liver cancer patients included in the national cancer registration annual report from 2012 to 2020 in tumor registration areas of Yunnan province were collected.The crude incidence and crude mortality of liver cancer in cancer registration areas of Yunnan province in 2020 were analyzed using Excel 2010 and SAS 9.4 statistical software,and the change trend of age-standardized incidence rate by World standard population(ASIRW)and age-standardized mortality rate by World standard population(ASMRW)of liver cancer in cancer registration areas of Yunnan province from 2012 to 2020 was analyzed using Joinpoint 4.8.0.1 software.Results In 2020,the crude incidence of liver cancer in tumor registration areas of Yunnan province was 20.32/100,000(male 29.41/100,000 and female 10.78/100,000),the crude incidence in urban areas was 19.45/100,000,the crude incidence in rural areas was 20.60/100,000.The crude mortality of liver cancer in tumor registration areas of Yunnan province was 20.54/100,000(male 29.60/100,000 and female 11.02/100,000),17.70/100,000 for urban areas,and 21.44/100,000 for rural areas.In 2020,the crude incidence in tumor registration areas of liver cancer in Yunnan province was at a low level before the age of 34,but rapidly increased at the age of 35.The peak rate of the 80-84 age group was 81.37/100,000,and the crude mortality was at a low level before the age of 34.After the age of 35,the mortality rapid-ly increased,and the peak rate of the 75-79 age group was 92.14/100,000.From 2012 to 2020,the annual change percentage of ASIRW of liver cancer in tumor registration areas of Yunnan province showed an upward trend(APC=1.82%,95%CI:0.07%-3.61%,t=2.45,P<0.05),and the annual change percentage of ASMRW showed an upward trend(APC=2.88%,95%CI:1.52%-4.26%,t=5.04,P<0.05).Conclusion In 2020,the crude incidence rate and crude mortality of liver cancer in tumor registration areas of Yunnan Province were higher in men than in women,and higher in rural areas than in urban areas.From 2021-2020,ASIRW and ASMRW of liver cancer are on the rise.We should continue to strengthen the prevention and treatment of liver cancer in Yunnan Province,strengthen the early screening and diagnosis of liver cancer through the combination of medicine and prevention,improve the tumor registration report detection system,strengthen health education,and improve the overall health awareness and cancer prevention knowledge of residents.

Objective To analyze the burden and changing trend of testicular cancer in China from 1990 to 2019.Methods Based on the 2019 Global Burden of Disease Database(GBD 2019),analyze the incidence,mortality,disability-adjusted life years(DALYs),years of life lost(YLLs),years lived with disability(YLDs)and their variation trend of testicular cancer in Chinese population from 1990 to 2019.Evaluating changes in age standardized rate(ASR)by calculating annual estimated percentage change(EAPC).According to the age grouping,analyze the age distribution characteristics of testicular cancer disease burden by age group.Results In 2019,the incident cases,deaths,age-standardized incidence rate,and age-standardized mortality rate of testicular cancer in China were 17.17×103,1.21×103,2.39/105,and 0.16/105,respectively.Compared to 1990,incident cases,deaths,and age-standardized incidence rate increased obviously in China,which was consistent with the global change trend,while the increase was higher than the global level.However,both Chinese and global age-standardized mortality rate showed a downward trend.From 1990 to 2019,DALYs,YLLs and YLDs of testicular cancer increased by 29.66%,9.83%and 720.91%respectively in China.The two age groups,0-15 years group and 30-35 years group,were with highest incidence of testicular cancer,while the highest disease burden of testicular cancer was 30-35 years.Conclusion From 1990 to 2019,the disease burden of testicular cancer in China showed an upward trend.Adolescents and young adults should be the priority population for screening and prevention due to their higher incidence and disease burden.