Objective:To study the methylation level of miR-5194 promoter in bladder cancer tissues, and explore the effects of miR-5194 on the proliferation and migration of bladder cancer cells by targeting p21-activated protein kinase 2 (PAK2).Methods:The methylation level of miR-5194 promoter in bladder cancer tissues was analyzed using MethHC database. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-5194 in bladder cancer MGH-U3, EJ, J82, and UMUC3 cells. 5-aza-2′-deoxycytidine (5-Aza-CdR) was used to treat bladder cancer cell lines, and RT-qPCR was used to detect the changes in the expression of miR-5194 in bladder cancer cell lines after 5-Aza-CdR treatment. UMUC3 cells were divided into miR-5194 group and NC group, and miR-5194 or miR-NC were transfected into UMUC3 cells, respectively. Colony formation assay and scratch assay were used to detect the effect of overexpression of miR-5194 on the proliferation and migration of UMUC3 cells. The bioinformatics tool miRGator and dual-luciferase reporter gene experiments verified the targeting relationship between miR-5194 and PAK2. The effect of overexpression of miR-5194 on the expression of PAK2 mRNA in UMUC3 cells was detected by RT-qPCR. The effect of overexpression of miR-5194 on the expression of PAK2 protein, proliferation-related proteins (CDK1, Cyclin B) and migration-related proteins (FOXC2, E47) in UMUC3 cells was detected by Western blotting. The measurement data were expressed as mean ± standard deviation ( ± s), the independent sample t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison among multiple groups. Results:The methylation level of miR-5194 promoter in bladder cancer tissues was significantly higher than that in adjacent tissues ( P<0.01). Compared with the immortalized bladder epithelial cells SV-HUC-1, the expression of miR-5194 in bladder cancer cells was significantly down-regulated ( P<0.01). After 5-Aza-CdR treatment, the expression of miR-5194 in bladder cancer cells was significantly increased ( P<0.01). The number of colonies in miR-5194 group and NC group were 31.30 ± 8.09 and 99.98 ± 10.53, respectively, and the proliferation ability of UMUC3 cells in miR-5194 group was weakened ( P<0.01). The migration rates of UMUC3 cells in miR-5194 group and NC group were (31.50 ± 7.17)% and (76.06 ± 4.86)%, respectively, and the migration ability of UMUC3 cells in miR-5194 group was weakened ( P<0.01). miR-5194 can target bind PAK2 gene ( P<0.01). The relative expression of PAK2 mRNA in UMUC3 cells of miR-5194 group and NC group were 1.02 ± 0.34 and 5.43 ± 0.76, respectively, and miR-5194 could negatively regulate the expression of PAK2 mRNA ( P<0.01). Compared with the NC group, the expression of PAK2 protein, the expression of proliferation-related proteins CDK1 and Cyclin B, and the expression of migration-related proteins FOXC2 and E47 were down-regulated in UMUC3 cells with miR-5194 overexpression. Conclusion:The methylation level of miR-5194 promoter in bladder cancer tissue was significantly increased, and miR-5194 inhibited the proliferation and migration of bladder cancer cells by targeting down-regulation of PAK2 expression in bladder cancer UMUC3 cells.

Objective:To compare the burden of benign prostatic hyperplasia（BPH）between China and the United States from 1990 to 2021.Methods:The prevalence，incidence，years lived with disability（YLD），and their age-standardized rates for BPH in China and the United States from 1990 to 2021 were obtained from the Global Burden of Disease Study 2021（GBD 2021）. The average annual percentage change（AAPC）of the age-standardized incidence rate（ASIR）and the age-standardized YLD rate（ASYR）was calculated using Joinpoint regression analysis. In addition，the YLD burden of BPH，prostate cancer，kidney cancer，bladder cancer，and three other urological diseases were compared between the two countries.Results:From 1990 to 2021，the number of BPH cases in China increased from 1.460 4 million to 3.244 5 million，the number of prevalent cases rose from 9.940 5 million to 23.111 2 million，and YLDs grew from 0.2 million person-years to 0.460 2 million person-years，with AAPCs of 2.63%，2.78%，and 2.75%，respectively. In 2021，the numbers of incident cases，prevalent cases，and YLDs were 0.577 9 million，4.930 3 million，and 0.095 9 million person-years in the United States，and 13.787 6 million，112.502 million，and 2.235 7 million person-years globally. China’s ASIR decreased from 363.07/100 000 to 299.14/100 000（AAPC -0.60%），and ASYR from 57.33/100 000 to 45.84/100 000（AAPC -0.70%），both of which were higher than those in the United States but lower than the global level. Age-specific analyses showed declining incidence and YLD rates across all age groups in China，while certain age groups in the United States demonstrated increasing trends. From 1990 to 2021，the proportion of YLDs attributable to BPH among seven urological diseases in China rose from 61.4% to 69.2%. In 2021，YLDs due to prostate cancer accounted for the highest proportion among seven urinary system diseases in the United States，reaching 54.5%. Projections indicate that although ASIR and ASYR in China will decline from 2022 to 2040，the absolute numbers of incident cases and YLDs are projected to continue to rise，reaching 4.97 million and 0.78 million，respectively，by 2040.Conclusions:Between 1990 and 2021，the number of incidence cases，prevalence cases，and YLDs of BPH in China increased markedly，while ASIR and ASYR declined. The disease burden of BPH remains substantial，with a higher proportion of YLDs among urological diseases compared with the United States. By 2040，the number of BPH cases and YLDs in China is projected to further increase，underscoring the need for greater public health attention.

Objective To explore the effects of eugenol combined with traditional Chinese medicine Wuxing music(EUG+MUSIC)on skin inflammation,behavioral characteristics,hormone and neurotransmitter levels,and the intestinal microbiota in a mouse model of 2,4-dinitrofluorobenzene(DNFB)-induced atopic dermatitis(AD).Methods A mouse model of AD was established by induction with DNFB for 21 consecutive days;throughout the study,changes in dorsal skin lesions and scratching behavior were recorded in mice and changes in dorsal skin lesions and pruritus were recorded.Mice underwent open-field,elevated 0 maze,tail-suspension,and forced-swimming tests on days 19 and 20.The mice were then sacrificed on day 21 and the organ index was calculated.Serum levels of immunoglobulin E,interleukin(IL)-4,and IL-10 were determined by enzyme-linked immunosorbent assay.Pathological changes in back-skin sections were detected by hematoxylin-eosin(HE)and toluidine blue staining,and brain levels of corticosterone,dopamine,and serotonin were measured.The composition of the intestinal microbiota was analyzed by 16S rRNA gene sequencing.Results The skin lesion score and skin inflammatory cell infiltration were significantly reduced in the EUG+MUSIC group.Regarding the behavioral tests,mice in the EUG+MUSIC group showed more active locomotion,an increased desire to explore unfamiliar environments,and increased struggling time in stressful situations.Brain levels of serotonin,dopamine,and corticosterone were also increased in the EUG+MUSIC group.16S rRNA gene sequencing showed that DNFB affected the diversity of the intestinal microbiota in AD mice,while EUG+MUSIC treatment remodeled the gut microbial composition,including increasing the relative abundance of gut microbes such as Ligilactobacillus and Lachnospiraceae and decreasing Parabacteroides and Bacteroides spp.Spearman's analysis showed significant correlations among altered gut microbiota,AD-associated skin manifestations,and anxiety-and depressive-like behaviors arising from comorbidities.Conclusions Eugenol combined with traditional Chinese medicine five-elements music therapy may have a positive effect on comorbid anxiety-and depression-like behaviors by regulating the intestinal microbiota and improving the behavioral performance in mice with AD,thus providing new ideas and method for improving AD and its associated anxiety-and depressive behaviors.

Objective To explore the effects of eugenol combined with traditional Chinese medicine Wuxing music(EUG+MUSIC)on skin inflammation,behavioral characteristics,hormone and neurotransmitter levels,and the intestinal microbiota in a mouse model of 2,4-dinitrofluorobenzene(DNFB)-induced atopic dermatitis(AD).Methods A mouse model of AD was established by induction with DNFB for 21 consecutive days;throughout the study,changes in dorsal skin lesions and scratching behavior were recorded in mice and changes in dorsal skin lesions and pruritus were recorded.Mice underwent open-field,elevated 0 maze,tail-suspension,and forced-swimming tests on days 19 and 20.The mice were then sacrificed on day 21 and the organ index was calculated.Serum levels of immunoglobulin E,interleukin(IL)-4,and IL-10 were determined by enzyme-linked immunosorbent assay.Pathological changes in back-skin sections were detected by hematoxylin-eosin(HE)and toluidine blue staining,and brain levels of corticosterone,dopamine,and serotonin were measured.The composition of the intestinal microbiota was analyzed by 16S rRNA gene sequencing.Results The skin lesion score and skin inflammatory cell infiltration were significantly reduced in the EUG+MUSIC group.Regarding the behavioral tests,mice in the EUG+MUSIC group showed more active locomotion,an increased desire to explore unfamiliar environments,and increased struggling time in stressful situations.Brain levels of serotonin,dopamine,and corticosterone were also increased in the EUG+MUSIC group.16S rRNA gene sequencing showed that DNFB affected the diversity of the intestinal microbiota in AD mice,while EUG+MUSIC treatment remodeled the gut microbial composition,including increasing the relative abundance of gut microbes such as Ligilactobacillus and Lachnospiraceae and decreasing Parabacteroides and Bacteroides spp.Spearman's analysis showed significant correlations among altered gut microbiota,AD-associated skin manifestations,and anxiety-and depressive-like behaviors arising from comorbidities.Conclusions Eugenol combined with traditional Chinese medicine five-elements music therapy may have a positive effect on comorbid anxiety-and depression-like behaviors by regulating the intestinal microbiota and improving the behavioral performance in mice with AD,thus providing new ideas and method for improving AD and its associated anxiety-and depressive behaviors.

Objective:To compare the burden of benign prostatic hyperplasia（BPH）between China and the United States from 1990 to 2021.Methods:The prevalence，incidence，years lived with disability（YLD），and their age-standardized rates for BPH in China and the United States from 1990 to 2021 were obtained from the Global Burden of Disease Study 2021（GBD 2021）. The average annual percentage change（AAPC）of the age-standardized incidence rate（ASIR）and the age-standardized YLD rate（ASYR）was calculated using Joinpoint regression analysis. In addition，the YLD burden of BPH，prostate cancer，kidney cancer，bladder cancer，and three other urological diseases were compared between the two countries.Results:From 1990 to 2021，the number of BPH cases in China increased from 1.460 4 million to 3.244 5 million，the number of prevalent cases rose from 9.940 5 million to 23.111 2 million，and YLDs grew from 0.2 million person-years to 0.460 2 million person-years，with AAPCs of 2.63%，2.78%，and 2.75%，respectively. In 2021，the numbers of incident cases，prevalent cases，and YLDs were 0.577 9 million，4.930 3 million，and 0.095 9 million person-years in the United States，and 13.787 6 million，112.502 million，and 2.235 7 million person-years globally. China’s ASIR decreased from 363.07/100 000 to 299.14/100 000（AAPC -0.60%），and ASYR from 57.33/100 000 to 45.84/100 000（AAPC -0.70%），both of which were higher than those in the United States but lower than the global level. Age-specific analyses showed declining incidence and YLD rates across all age groups in China，while certain age groups in the United States demonstrated increasing trends. From 1990 to 2021，the proportion of YLDs attributable to BPH among seven urological diseases in China rose from 61.4% to 69.2%. In 2021，YLDs due to prostate cancer accounted for the highest proportion among seven urinary system diseases in the United States，reaching 54.5%. Projections indicate that although ASIR and ASYR in China will decline from 2022 to 2040，the absolute numbers of incident cases and YLDs are projected to continue to rise，reaching 4.97 million and 0.78 million，respectively，by 2040.Conclusions:Between 1990 and 2021，the number of incidence cases，prevalence cases，and YLDs of BPH in China increased markedly，while ASIR and ASYR declined. The disease burden of BPH remains substantial，with a higher proportion of YLDs among urological diseases compared with the United States. By 2040，the number of BPH cases and YLDs in China is projected to further increase，underscoring the need for greater public health attention.

Objective To investigate the synergistic protective effects of WR-2721 combined with lentinan and cytokines against radiation damage in mice, and to provide a new treatment for acute radiation injury. Methods Seventy Institute of Cancer Research mice were divided into seven groups: a control group, a model group, WR-2721 group, Lentinan & cytokine group, WR-2721 & Lentinan group, WR-2721 & cytokine group and WR-2721 & Lentinan & cytokine group. All groups except the control group were irradiated with ⁶⁰Co γ-rays at a dose rate of 0.8 Gy/min and a cumulative dose of 5.0 Gy. The mice were sacrificed by cervical dislocation 14 d after irradiation to measure their spleen index, thymus index, and serum levels of superoxide dismutase (SOD), malondialdehyde (MDA), interleukin-11 (IL-11), and tumor necrosis factor-alpha (TNF-α). Results For the mice treated with WR-2721, lentinan, and cytokines, the spleen index was 7.33 ± 2.84, the thymus index was 1.70 ± 0.30, the serum SOD level was 114.0 ± 8.3, the MDA level was 7.33 ± 1.16, the IL-11 level was 155.8 ± 49.4, and the TNF-α level was 174.0 ± 37.8. All these indicators except the spleen index in the combination group significantly differed from those of the model group (P < 0.05 or 0.01), indicating the combined treatment promoted recovery from radiation damage. Conclusion WR-2721 combined with lentinan and cytokines has significant synergistic protective effects, which is a promising treatment for acute radiation injury.

OBJECTIVES: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types. RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations. CONCLUSIONS ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.
Child ; Humans ; Infant, Newborn ; Acyl-CoA Dehydrogenase/genetics* ; Genotype ; Phenotype ; Carnitine/metabolism* ; Mutation

Objective:To explore the effect of miR-1249-5p on the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer.Methods:The relationship between the expression level of miR-1249-5p and the overall survival of prostate cancer patients was analyzed using OncoMir Cancer Database (OMCD). The human prostate cancer cell line PC-3 was divided into two groups: miR-1249-5p group and negative control group. Mediated by Lipofectamine 2000, miR-1249-5p mimics liposome complex or negative miRNA liposome complex were transfected into PC-3 cell at logarithmic growth stage. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1249-5p in PC-3 cell of two groups. Colony formation assay was used to detect the changes of the proliferation ability of PC-3 cell in the two groups. Transwell experiment was used to detect the changes of PC-3 cell invasion in the two groups, and the cell cycle changes of the two groups of PC-3 were detected by flow cytometry. The miRNA prediction software miRGator was used to predict the target gene of miR-1249-5p. RT-qPCR and Western blotting were used to detect the target gene expression of miR-1249-5p. Measurement data were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between two groups. Results:Compared with prostate cancer patients with low miR-1249-5p expression, prostate cancer patients with higher miR-1249-5p expression had longer overall survival, and the difference was statistically significant ( P<0.01). The expression level of miR-1249-5p in the miR-1249-5p group (10.74±1.19) was significantly higher than that of the negative control group (1.56±0.27), the difference was statistically significant ( P<0.01). The number of colonies formed in the miR-1249-5p group (35.86±6.94) was significantly less than that in the negative control group (88.94±11.66), and the difference was statistically significant ( P<0.01). The number of transmembrane cells [(25.01±6.83)/high power field of view] in the miR-1249-5p group was significantly less than that of the negative control group [(82.76±8.35)/high power field of view], and the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the miR-1249-5p group [(50.79±6.61)%] was significantly higher than that in the negative control group [(27.09±2.30)%], the difference was statistically significant ( P<0.01), and PC-3 cell were inhibited in the G 0-G 1 phase. Neural precursor cell expressed developmentally down-regulated 9 ( NEDD9) may be the target gene of miR-1249-5p. Compared with the negative control group, the NEDD9 gene expression in the miR-1249-5p group was significantly lower than that of the negative control group, the difference was statistically significant ( P<0.01). Conclusion:miR-1249-5p can inhibit the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer, which may be achieved by negatively regulating the expression of proto-oncogene NEDD9.

Objective:To explore the genetic causes of abnormal isovaleryl carnitine (C5) metabolism in newborns.Methods:Retrospective study.The screening and clinical follow-up data of 34 neonates with elevated C5 levels shown by the tandem mass spectrometry test in Children′s Hospital, Zhejiang University School of Medicine from January 2018 to December 2021 were collected.Afterwards, their ethylenediaminetetraacetic acid (EDTA) anticoagulant venous blood was collected to extract genomic DNA.A total of 79 genes related to genetic metabolic diseases, such as ACADSB, IVD and ACADM, were captured by liquid-phase capture technology.High-throughput sequencing and bioinformatics analysis were used to acquire gene variation information and the genes were categorized by American College of Medical Genetics and Genomics classification standard.According to the results of genetic analysis, the newborns with C5 elevation were divided into 3 groups: non-mutation group(11 cases), ACADSB mutation group(16 cases) and IVD mutation group(7 cases). Wilcoxon rank sum test was performed to analyze the difference between these groups. Results:Among 34 neonates, 6 ACADSB variants were detected in 16 cases, and 2 of them [c.461G>A (p.G154E), c.746delC(p.P249Lfs*15)] were novel variants.Eleven IVD variants were detected in 7 cases, and 7 of them [c.118A>G(p.N40D), c.296-10C>G, c.302A>G(p.Y101C), c.537G>A(p.M179I), c.667C>T(p.R223W), c.983A>G(p.K328R), c.1147+ 5G>A] were never reported before.There was no significant difference in the C5 concentration in initial screening among the three groups ( P>0.05). Conclusions:Mutations in ACADSB and IVD genes are the main causes of augmented C5 levels in neonatal screening.For newly discovered genetic variants, functional prediction by multiple bioinformatics analysis software is recommended.And it is also important to carry out clinical follow-up and evaluation.

Objective:To investigate influencing factors of dental caries among 3 to 5-year-old children in the Yuhua District of Changsha by Cariostat detection and provide evidence to prevent caries.Methods:1 032 children aged 3-5 years old from 6 kindergartens in Yuhua District of Changsha were randomly selected for oral health examination. 565 children in 6 large classes, 6 middle classes and 6 small classes were randomly selected from 6 kindergartens were perfected caries activity test (CAT) by Cariostat detection.Results:Among 1 032 children, 626 had caries, the caries rate was 60.66%, the caries was 2.87±3.67, and the caries filling rate was 8.53%. The caries rate of maxillary central incisors was the highest (37.89%), followed by mandibular second primary molars (32.56%) and mandibular first primary molars (32.36%). There was significant difference in caries rate among different age groups (χ 2=9.231, P<0.05). The caries rate of 5-year-old children was the highest (66.50%). 1 032 children were divided into 4 groups according to dmft (decayed, missing, filled teeth) value, and the number of caries free group was the largest; the number of caries in the middle caries group was the least. The number of missing teeth in 1 032 children with caries was 2 959. The number of missing teeth in the high caries group was the most, up to 1 861, accounting for 61.89% of the total number of missing teeth. The analysis of 565 children by CAT showed that the caries rate was different in different CAT grades (χ 2=27.390, P<0.05). The caries rate in the high-risk group was the highest, 90.48%. The CAT value was positively correlated with caries ( r=0.261, P<0.05). Conclusions:The incidence of dental caries in children aged 3-5 years in Yuhua District of Changsha was 60.66%. Cariostat method can truly reflect the situation of caries in children and is helpful to screen the population susceptible to caries in children.