Objective This study conducted molecular biological identification of the viruses carried by Culex pipiens pallens specimens collected in Shunyi District, Beijing in 2023, and observed the changes in the infection rate of the viruses carried by Cx. pipiens pallens at different collection times. Methods Cx. pipiens pallens were collected using carbon dioxide mosquito traps. The mosquito samples were ground in batches and analyzed by molecular biology technologies. The virus infection rate at different collection times was analyzed statistically. Results 17 strains of Culex pipiens pallens densovirus(CppDNV)were identified from Cx. pipiens pallens samples collected in Shunyi District, Beijing, in 2023. The nucleotide sequence analysis of the virus genome coding region showed that CppDNV was a single-stranded DNA virus with a total length of 3 335 nt, encoding 2 non-structural proteins(NS1, NS2)and 1 capsid protein(VP). The nucleotide(amino acid)sequence lengths of the three proteins were 2 376 nt(791 aa),1 092 nt(363 aa)and 1 071 nt(356 aa), respectively. Phylogenetic analysis showed that CppDNV was located in genus Brevihamaparvovirus. Statistical analysis showed no significant difference in infection rates across collection times(χ2=4.429, P=0.194). Conclusions CppDNV was identified in Cx. pipiens pallens in Beijing, and it was stably maintained in this natural population.

AIM To investigate the protective effects of Shuangyi Qushi Tongluo Capsules(Shuangyi Capsules)on Dexamethasone(Dex)induced osteoporosis in mice.METHODS The C57BL/6J mice were randomly divided into the control group,the model group,the Xianling Gubao Capsules group(1.5 g/kg),and the low-dose,moderate-dose,and high-dose Shuangyi Capsules groups(0.6,1.2,and 2.4 g/kg).The mouse model of osteoporosis was induced by 8-week intraperitoneal injection of Dex sodium phosphate injection(5 mg/kg).The mice had their femur osteogenesis observed with hematoxylin and eosin(HE)staining and tartrate-resistant acid phosphatase(TRAP)staining;their serum alkaline phosphatase(ALP)and osteocalcin(BGP)activities detected by ELISA;their femoral mRNA expressions of Col-Ⅰ,OCN,and OPN detected by RT-qPCR;and their femoral protein expressions of OPG and RANKL detected by Western blot.Upon the MC3T3-E1 cells exposed to Dex and Shuangyi Capsules,their viability was evaluated by CCK-8 assay;their mineralization determined by alkaline phosphatase staining and alizarin red staining(ARS);and their intracellular ROS level detected using DCFH-DA probe.RESULTS Compared with the model group,Shuangyi Capsules groups demonstrated improved fracture of femoral trabeculae and reduced number of osteoclasts;increased serum ALP and BGP activities(P＜0.05,P＜0.01);increased femoral expressions of Col-Ⅰ mRNA and OPG protein(P＜0.05,P＜0.01);and decreased RANKL protein expression(P＜0.05).Compared with the MC3T3-E1 cells stimulated by Dex,those underwent further treatment of Shuangyi Capsules demonstrated increased cell viability and ALP activity(P＜0.05,P＜0.01);increased mineralization and calcium nodule formation;increased expressions of Col-Ⅰ,OCN,OPN mRNA and OPG protein(P＜0.05,P＜0.01);decreased RANKL protein expression(P＜0.05,P＜0.01);and reduced ROS levels.CONCLUSION Shuangyi Capsules ameliorate Dex-induced osteoporosis in mice by suppressing osteoclast overactivation,enhancing osteoblast activity,and stimulating bone formation through modulation of Col-Ⅰ,OCN,OPN mRNA and OPG/RANKL protein levels.

The study was aimed at identifying the diversity of tick species in selected areas of Qinghai,to analyze the genetic differentiation characteristics of tick-borne spotted fever group rickettsiae(SFGR),and to provide the theoretical basis for SFGR prevention and control in the region.The 16S rRNA gene was used for molecular biological identification of 446 collected tick samples,and the infection characteristics of SFGR in tick samples were determined according to the SFGR outer membrane protein A(ompA)gene.Haplotype analysis,phylogenetic tree construction,and estimation of differentiation times for SFGR were conducted in DNASP v6,IQ-tree v2.2.0,and BEAST v2.7.4 software.The obtained 446 tick samples belonged to three categories:(1)Haemaphy-salis spp.,including Haemaphysalis qinghaiensis(n=192)and H.danieli(n=37);(2)Dermacentor spp.,including Dermacentor ever-estianus(n=121),D.nuttalli(n=55),and D.silvarum(n=36);and(3)Hyalomma marginatum(n=5).Rickettsia raoultii was de-tected in D.everestianus,D.silvarum,D.nuttalli,H.qinghaiensis,and H.danieli,with infection rates of 95.9%,80.6%,69.1%,4.1%,and 2.7%,respectively.R.sibirica subsp.sibirica BJ-90 was found only in D.silvarum and D.nuttalli,with infection rates of 5.6%and 1.8%,respectively.The Candidatus R.gannanii F107 was found in H.danieli and H.qinghaiensis,with infection rates of 16.2%and 7.8%,respectively.Ca.R.hongyuanensis was detected only in H.qinghaiensis,with a prevalence of 16.3%.The prevalence of R.aeschlimannii was 20%and 2.7%in Hy.marginatum and H.danieli,respectively.Haplotype and nucleotide polymorphism analy-ses revealed 13 haplotypes in R.raoultii,with haplotype H13 as the dominant haplotype(42/192);seven haplotypes in Ca.R.ganna-nii F107,with haplotype H4 as the dominant haplotype(4/18);and three haplotypes in Ca.R.hongyuanensis,with haplotype H1 as the dominant haplotype(11/13).The phylogenetic tree indicated that the sequences of R.raoultii in selected areas of Qinghai and R.rhipicephali clustered into one branch;Ca.R.hongyuanensis and Ca.R.gannanii F107 clustered into one branch;and R.sibirica subsp.sibirica BJ-90 clustered into one branch with R.sibirica.Estimates of differentiation time revealed that the mean differentiation time for the six Rickettsia was approximately 2 000 Mya(95%CI:1 999.08-2 001.02 Mya).The tick species distributed in selected ar-eas of Qinghai are diverse,and this study provides the first report of Hy.marginatum in Qinghai Province.SFGR significantly varied in prevalence among tick species and showed high genetic diversity.

Objective:To investigate the relationship between Ig class switch recombination(CSR)and mismatch repair(MMR)protein,microsatellite phenotype in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue(MALT lymphoma).Methods:Forty cases of MALT lymphoma archived in the Department of Pathology,Jiading District Central Hospital,Shanghai University of Medicine & Health Sciences were selected as the observation group,and twenty cases of benign lymphoid tissue hyperplasia were as the control group.The expressions of IgG,IgM,IgD,and IgA in both groups were detected by immunohistochemical double staining,and MMR proteins including MLH1,MSH2,MSH6,and PMS2 in both groups were detected by immunohistochemistry.Multiplex fluorescence PCR capillary electrophoresis was used to detect microsatellite phenotype in tumor and adjacent tissues of the experimental group.Results:In the observation group,the proportions of single Ig heavy chain expression(mode Ⅰ),negative expression(mode Ⅱ),and multiple expression(mode Ⅲ)were 65％(26/40),27.5％(11/40),and 7.5％(3/40),respectively,while in the control group were 0(0/20),5％(1/20),and 95％(19/20).The proportion of Ig heavy chain expression mode Ⅰ+Ⅱ in the observation group was 92.5％,which was significantly higher than 5％in the control group(P＜0.0 1).In the observation group,partial deletion of MMR protein was observed in 3 cases(7.5％),including 2 cases of MSH6 deletion and 1 case of both MSH6 and PMS2 deletion.In the control group,there was 1 case(5％)with PMS2 deletion.There was no significant difference in the deletion rate of MMR protein between the two groups(P＞0.05).A total of 5 cases of microsatellite instability(MSI)were detected in the observation group,including 1 case of low-frequency MSI(MSI-L),4 cases of high-frequency MSI(MSI-H),and 2 cases of MSI-H with MSH6 deletion.When the loss expression of MSI-H or MMR protein was counted as a positive result,the MSI-H rate detected by PCR capillary electrophoresis was 10％(4/40),which was slightly higher than the MMR protein deletion rate detected by immunohistochemistry(7.5％,3/40),but there was no statistically significant difference between the two groups(P＞0.05).The MMR protein deletion rates among the Ig heavy chain protein expression mode Ⅰ,mode Ⅱ,and mode Ⅲ groups were 0(0/26),18.2％(2/11),and 33.3％(1/3),respectively.There was a statistically significant difference in the constituent ratios among the three groups(P＜0.05).The MMR protein deletion rates among the MSS,MSI-L,and MSI-H groups were 2.9％(1/35),0(0/1),and 50％(2/4),respectively.There was a statistically significant difference in the constituent ratios among the three groups(P＜0.05).MMR protein deficiency was positively correlated with Ig heavy chain expression pattern and MSI(r=0.41,P＜0.05;r=0.48,P＜0.05),but Ig heavy chain expression pattern was not correlated with MSI(r=0.02,P＞0.05).Conclusion:Ig heavy chain CSR detection is helpful for the differential diagnosis of MALT lymphoma.Low frequency MMR protein deletion and MSI-H phenotype exist in MALT lymphoma,which may be of certain value for the study of its occurrence,development and clinical treatment.

Aim To investigate the therapeutic effect of newly formulated Tadalafil tablets on liver fibrosis in mice induced by carbon tetrachloride(CCl4)and its impact on the activation of hepatic stellate cells(HSCs).Methods Liver fibrosis model was estab-lished by intraperitoneally injecting 20％CCl4 corn oil solution twice a week for eight weeks.After four weeks of modeling,the treatment group was administered ei-ther the newly formulated Tadalafil tablets(1.0 mg·kg-1)or the Cialis(2.5 mg·kg-1)via gavage for the remaining four weeks.We assessed the effects of Tadalafil on collagen deposition,tissue structural dam-age,and HSCs activation markers in the fibrotic liver of mice using serum biochemical analysis,histopathologi-cal staining,and Western blotting following the treat-ment period.LX-2 cells were cultured and treated with tadalafil after TGF β1 stimulation,and the effects of tadalafil on LX-2 cell activation were assessed via Western blot.Results Compared to the normal mice,the model group mice exhibited a significantly higher liver-specific index,increased liver function indicators,and notable hepatocyte necrosis.Additionally,liver lobules were damaged,accompanied by severe infiltra-tion of inflammatory cells.Both smooth muscle actin(α-SMA)and fibronectin(Fn)were elevated,serving as markers of HSCs activation.As a result of treatment with the newly formulated Tadalafil tablets,liver tissue damage was significantly reduced,transaminase levels decreased,necrosis and inflammatory cell infiltration were reduced,and collagen fiber deposition was allevia-ted,and α-SMA and Fn expression was reduced.It was worth noting that low-dose newly formulated Tadalafil tablets were found to be as effective as high-dose Cia-lis.In a cellular model,Tadalafil significantly inhibited the activation of LX-2 cells and reduced the expression of proteins related to cell activation.Conclusions The newly formulated Tadalafil tablets can significantly inhibit HSCs activation,reduce extracellular matrix(ECM)deposition,improve liver fibrosis and liver function damage caused by CCl4.This new formulation offers a significant advantage over Cialis in terms of ef-fectiveness,with a lower effective dose.

AIM To investigate the protective effects of Shuangyi Qushi Tongluo Capsules(Shuangyi Capsules)on Dexamethasone(Dex)induced osteoporosis in mice.METHODS The C57BL/6J mice were randomly divided into the control group,the model group,the Xianling Gubao Capsules group(1.5 g/kg),and the low-dose,moderate-dose,and high-dose Shuangyi Capsules groups(0.6,1.2,and 2.4 g/kg).The mouse model of osteoporosis was induced by 8-week intraperitoneal injection of Dex sodium phosphate injection(5 mg/kg).The mice had their femur osteogenesis observed with hematoxylin and eosin(HE)staining and tartrate-resistant acid phosphatase(TRAP)staining;their serum alkaline phosphatase(ALP)and osteocalcin(BGP)activities detected by ELISA;their femoral mRNA expressions of Col-Ⅰ,OCN,and OPN detected by RT-qPCR;and their femoral protein expressions of OPG and RANKL detected by Western blot.Upon the MC3T3-E1 cells exposed to Dex and Shuangyi Capsules,their viability was evaluated by CCK-8 assay;their mineralization determined by alkaline phosphatase staining and alizarin red staining(ARS);and their intracellular ROS level detected using DCFH-DA probe.RESULTS Compared with the model group,Shuangyi Capsules groups demonstrated improved fracture of femoral trabeculae and reduced number of osteoclasts;increased serum ALP and BGP activities(P＜0.05,P＜0.01);increased femoral expressions of Col-Ⅰ mRNA and OPG protein(P＜0.05,P＜0.01);and decreased RANKL protein expression(P＜0.05).Compared with the MC3T3-E1 cells stimulated by Dex,those underwent further treatment of Shuangyi Capsules demonstrated increased cell viability and ALP activity(P＜0.05,P＜0.01);increased mineralization and calcium nodule formation;increased expressions of Col-Ⅰ,OCN,OPN mRNA and OPG protein(P＜0.05,P＜0.01);decreased RANKL protein expression(P＜0.05,P＜0.01);and reduced ROS levels.CONCLUSION Shuangyi Capsules ameliorate Dex-induced osteoporosis in mice by suppressing osteoclast overactivation,enhancing osteoblast activity,and stimulating bone formation through modulation of Col-Ⅰ,OCN,OPN mRNA and OPG/RANKL protein levels.

Objective To investigate the effects of ischemia time and storage periods on RNA quality in fresh-frozen breast cancer(BC)and esophageal cancer(EC)tissue samples in order to establish evidence-based protocols for biobank sample management.Methods The tumor(T)and paired normal(N)tissue samples from 6 cases of BC and 6 cases of EC were collected and cryopreserved in Biobank,Shantou Central Hospital.Mirror paraffin-embedded tissues were simultaneously prepared into sections for morphological analysis.The samples were divided into two groups of<15 min and 15-30 min according to ischemia time,and RNA quality was analyzed at 4 storage periods of 8-10 months(T1),14-16 months(T2),26-28 months(T3)and 38-40 months(T4).Results In 96 analyzed samples,93.8%(90/96)exhibited high quality(RIN≥6),with 89.6%(43/48)in BC and 97.9%(47/48)in EC.Significant differences in RIN were observed between BC group and EC group(8.050 vs.8.600,P=0.009).In EC group,RIN value was significantly negatively correlated with RNA yield(P<0.001).Moreover,RIN values of tumor-normal pairs exhibited markedly significant differences(7.550 vs.9.000,P<0.001).In contrast,no significant difference was detected in BC group(8.200 vs.7.700,P=0.348).Statistical analysis showed that RIN value was positively correlated with 28S/18S(P<0.001),but had no correlation with tumor content(P=0.676)and necrotic content(P=0.055).Neither ischemia time(<15 min vs.15-30 min:8.200 vs.8.300,P=0.932)nor storage periods(T1-T4:8.400,7.700,8.450,8.600,P=0.163)compromised RNA quality.Conclusion Organ origin and tissue type could influence RNA quality of fresh-frozen tissue samples.However,limited ischemia time(≤30 min)and long-term storage period(38-40 months)do not adversely affect RNA quality in fresh-frozen breast cancer and esophageal cancer tissue samples.

Aim To investigate the therapeutic effect of newly formulated Tadalafil tablets on liver fibrosis in mice induced by carbon tetrachloride(CCl4)and its impact on the activation of hepatic stellate cells(HSCs).Methods Liver fibrosis model was estab-lished by intraperitoneally injecting 20％CCl4 corn oil solution twice a week for eight weeks.After four weeks of modeling,the treatment group was administered ei-ther the newly formulated Tadalafil tablets(1.0 mg·kg-1)or the Cialis(2.5 mg·kg-1)via gavage for the remaining four weeks.We assessed the effects of Tadalafil on collagen deposition,tissue structural dam-age,and HSCs activation markers in the fibrotic liver of mice using serum biochemical analysis,histopathologi-cal staining,and Western blotting following the treat-ment period.LX-2 cells were cultured and treated with tadalafil after TGF β1 stimulation,and the effects of tadalafil on LX-2 cell activation were assessed via Western blot.Results Compared to the normal mice,the model group mice exhibited a significantly higher liver-specific index,increased liver function indicators,and notable hepatocyte necrosis.Additionally,liver lobules were damaged,accompanied by severe infiltra-tion of inflammatory cells.Both smooth muscle actin(α-SMA)and fibronectin(Fn)were elevated,serving as markers of HSCs activation.As a result of treatment with the newly formulated Tadalafil tablets,liver tissue damage was significantly reduced,transaminase levels decreased,necrosis and inflammatory cell infiltration were reduced,and collagen fiber deposition was allevia-ted,and α-SMA and Fn expression was reduced.It was worth noting that low-dose newly formulated Tadalafil tablets were found to be as effective as high-dose Cia-lis.In a cellular model,Tadalafil significantly inhibited the activation of LX-2 cells and reduced the expression of proteins related to cell activation.Conclusions The newly formulated Tadalafil tablets can significantly inhibit HSCs activation,reduce extracellular matrix(ECM)deposition,improve liver fibrosis and liver function damage caused by CCl4.This new formulation offers a significant advantage over Cialis in terms of ef-fectiveness,with a lower effective dose.

Objective To investigate the effects of ischemia time and storage periods on RNA quality in fresh-frozen breast cancer(BC)and esophageal cancer(EC)tissue samples in order to establish evidence-based protocols for biobank sample management.Methods The tumor(T)and paired normal(N)tissue samples from 6 cases of BC and 6 cases of EC were collected and cryopreserved in Biobank,Shantou Central Hospital.Mirror paraffin-embedded tissues were simultaneously prepared into sections for morphological analysis.The samples were divided into two groups of<15 min and 15-30 min according to ischemia time,and RNA quality was analyzed at 4 storage periods of 8-10 months(T1),14-16 months(T2),26-28 months(T3)and 38-40 months(T4).Results In 96 analyzed samples,93.8%(90/96)exhibited high quality(RIN≥6),with 89.6%(43/48)in BC and 97.9%(47/48)in EC.Significant differences in RIN were observed between BC group and EC group(8.050 vs.8.600,P=0.009).In EC group,RIN value was significantly negatively correlated with RNA yield(P<0.001).Moreover,RIN values of tumor-normal pairs exhibited markedly significant differences(7.550 vs.9.000,P<0.001).In contrast,no significant difference was detected in BC group(8.200 vs.7.700,P=0.348).Statistical analysis showed that RIN value was positively correlated with 28S/18S(P<0.001),but had no correlation with tumor content(P=0.676)and necrotic content(P=0.055).Neither ischemia time(<15 min vs.15-30 min:8.200 vs.8.300,P=0.932)nor storage periods(T1-T4:8.400,7.700,8.450,8.600,P=0.163)compromised RNA quality.Conclusion Organ origin and tissue type could influence RNA quality of fresh-frozen tissue samples.However,limited ischemia time(≤30 min)and long-term storage period(38-40 months)do not adversely affect RNA quality in fresh-frozen breast cancer and esophageal cancer tissue samples.

Objective To explore the application value of MRI diaphragmatic navigation technology combined with three dimensional liver acquisition with volume acceleration-flexible(3D LAVA-FLEX)sequence in abdominal enhanced imaging of infants and young children.Methods A retrospective analysis was conducted on imaging data of 84 infants and young children who underwent abdominal enhanced MRI examination in our hospital between January 2021 and December 2023.All 84 infants and young children initially underwent conventional dynamic contrast-enhanced 3D LAVA-FLEX sequence scanning;the delayed phase images obtained were included in the dynamic enhancement group.Subsequently,diaphragmatic navigation combined with 3D LAVA-FLEX sequence examination was implemented,and the obtained images were included in the diaphragm navigation group.Subjective scoring was performed for images in both groups,while the signal to noise ratio(SNR),contrast to noise ratio(CNR),and artifact quantification(AQ)were measured and compared between the two groups.Results The respiratory motion artifacts,the clarity of liver parenchyma enhancement,the clarity of liver vascular enhancement,the clarity of spleen parenchyma enhancement and the overall image quality score in the diaphragm navigation group were higher than those in the dynamic enhancement group,and the differences were statistically significant(P<0.05).There were statistically significant differences in SNR and AQ between the two groups of images(P<0.000 1),while there was no statistically significant difference in CNR between the two groups of images(P>0.05).Conclusion Diaphragmatic navigation technology combined with 3D LAVA-FLEX sequence imaging can improve the image quality of abdominal MRI enhanced imaging in infants and young children,and provide a reference for clinical diagnosis and treatment.