ObjectiveAngelicae Sinensis Radix， a commonly used medicinal herb with both medicinal and edible properties， is frequently adulterated in the market， severely affecting the clinical efficacy of preparations. While qualitative identification techniques for adulterants and counterfeits are now relatively mature， quantitative detection methods for adulterated processed products remain unexplored. Quantitative detection research of Angelicae Sinensis Radix and its primary closely related adulterant， "Tu Danggui" （Angelica gigas）， was conducted to establish a herbal quantitative molecular detection （Herb-Q） method for Angelicae Sinensis Radix and its processed products， providing a model for the establishment of quantitative detection technologies for Angelicae Sinensis Radix and related health products. MethodsThe specific single-nucleotide polymorphism （SNP） loci of Angelicae Sinensis Radix and Angelica gigas Nakai were screened based on the complete chloroplast genome sequence. The specific SNP loci of Angelicae Sinensis Radix were selected for quantitative methodological investigations （linearity， limit of quantification， limit of detection， and reproducibility） by mixing the powder of the herbs with different adulteration ratios. Huoxue Zhitong powder with three distinct adulteration ratios （15%， 25%， and 35%） was utilized to ascertain the precision of the Herb-Q method for the quantitative detection of Chinese patent medicines containing Angelicae Sinensis Radix. ResultsBy comparing the 123 chloroplast genome sequences of Angelicae Sinensis Radix， based on the principles of intraspecies conservation， interspecies specificity， and meeting the requirements of pyrophosphate high-throughput sequencing， it was determined that 9 674^th locus （A/G） in the chloroplast genome sequence NC_042826.1 and 38 592^nd locus （T/C） in the chloroplast genome sequence NC_029393.1 could be the exclusive molecular identification loci of Angelicae Sinensis Radix and Angelica gigas Nakai， respectively. The linear relationship R² of the Herb-Q method established by selecting the specific 9 674^th locus （A/G） of Angelicae Sinensis Radix was 0.997 4 （R²>0.99）， indicating an excellent linear relationship. The limits of quantification and detection were established at 2.0%， exhibiting excellent reproducibility ［relative standard deviation（RSD）<2.0%］. The established quantitative system based on the Herb-Q method detected the adulteration amount of counterfeit A. gigas in the Huoxue Zhitong powder， with an average deviation of 1.3% for three molecular quantitative replicates. ConclusionThis research demonstrates that the Herb-Q quantitative detection method established based on the 9 674^th locus （A/G） in the chloroplast genome sequence NC_042826.1 of Angelicae Sinensis Radix has good applicability， objectivity， and accuracy for Angelicae Sinensis Radix and A. gigas， and its processed products. This method has the capacity to provide technical support for the quantitative detection of commercially available Angelicae Sinensis Radix derivatives， including traditional Chinese medicinal preparations， dietary supplements， and nutraceuticals.

ObjectiveAngelicae Sinensis Radix， a commonly used medicinal herb with both medicinal and edible properties， is frequently adulterated in the market， severely affecting the clinical efficacy of preparations. While qualitative identification techniques for adulterants and counterfeits are now relatively mature， quantitative detection methods for adulterated processed products remain unexplored. Quantitative detection research of Angelicae Sinensis Radix and its primary closely related adulterant， "Tu Danggui" （Angelica gigas）， was conducted to establish a herbal quantitative molecular detection （Herb-Q） method for Angelicae Sinensis Radix and its processed products， providing a model for the establishment of quantitative detection technologies for Angelicae Sinensis Radix and related health products. MethodsThe specific single-nucleotide polymorphism （SNP） loci of Angelicae Sinensis Radix and Angelica gigas Nakai were screened based on the complete chloroplast genome sequence. The specific SNP loci of Angelicae Sinensis Radix were selected for quantitative methodological investigations （linearity， limit of quantification， limit of detection， and reproducibility） by mixing the powder of the herbs with different adulteration ratios. Huoxue Zhitong powder with three distinct adulteration ratios （15%， 25%， and 35%） was utilized to ascertain the precision of the Herb-Q method for the quantitative detection of Chinese patent medicines containing Angelicae Sinensis Radix. ResultsBy comparing the 123 chloroplast genome sequences of Angelicae Sinensis Radix， based on the principles of intraspecies conservation， interspecies specificity， and meeting the requirements of pyrophosphate high-throughput sequencing， it was determined that 9 674^th locus （A/G） in the chloroplast genome sequence NC_042826.1 and 38 592^nd locus （T/C） in the chloroplast genome sequence NC_029393.1 could be the exclusive molecular identification loci of Angelicae Sinensis Radix and Angelica gigas Nakai， respectively. The linear relationship R² of the Herb-Q method established by selecting the specific 9 674^th locus （A/G） of Angelicae Sinensis Radix was 0.997 4 （R²>0.99）， indicating an excellent linear relationship. The limits of quantification and detection were established at 2.0%， exhibiting excellent reproducibility ［relative standard deviation（RSD）<2.0%］. The established quantitative system based on the Herb-Q method detected the adulteration amount of counterfeit A. gigas in the Huoxue Zhitong powder， with an average deviation of 1.3% for three molecular quantitative replicates. ConclusionThis research demonstrates that the Herb-Q quantitative detection method established based on the 9 674^th locus （A/G） in the chloroplast genome sequence NC_042826.1 of Angelicae Sinensis Radix has good applicability， objectivity， and accuracy for Angelicae Sinensis Radix and A. gigas， and its processed products. This method has the capacity to provide technical support for the quantitative detection of commercially available Angelicae Sinensis Radix derivatives， including traditional Chinese medicinal preparations， dietary supplements， and nutraceuticals.

Objective To monitor the dynamic changes in liver stiffness and their correlation with clinical indicators among chronic hepatitis B virus (HBV) infected individuals in Wuwei City, Gansu Province, and to provide important evidence for the early detection and effective intervention of liver fibrosis (LF) progression. Methods Based on the Wuwei Hepatitis B Specialty Cohort, this study conducted annual serological and FibroScan ultrasonographic examinations for 3 882 chronic HBV-infected individuals. Over an average of 4 years of follow-up, the liver fibrosis outcome was monitored, and influencing factors were analyzed by constructing a logistic regression model. Results Among the 2 053 chronic HBV-infected individuals who completed at least one follow-up, baseline LF grades F0 to F4 were distributed as follows: 1 581 cases (77.0%), 164 cases (8.0%), 99 cases (4.8%), 110 cases (5.4%), and 99 cases (4.8%), respectively. Significant differences were observed among the five groups in terms of age, gender, smoking, antiviral treatment, liver function indicators, control attenuation index (CAP), and liver stiffness measurement (LSM) (P<0.05). After an average of 4 years of follow-up, 1 686 cases (17.9/100 person-years) showed no significant change in LF grade, 260 cases (2.8/100 person-years) demonstrated a decrease in LF grade, and 107 cases (1.1/100 person-years) exhibited an increase in LF grade. Stratified by baseline treatment status, among patients with chronic HBV infection who did not undergo treatment, baseline alanine aminotransferase (ALT) (OR=5.50, 95%CI：1.79-16.83, P=0.003) and LSM (OR=3.35, 95%CI：1.23-9.13, P=0.018) were identified as risk factors for LF progression. In contrast, among patients who underwent antiviral treatment, baseline aspartate aminotransferase (AST) (OR=2.23, 95%CI：1.41-3.53, P<0.001) and total bilirubin (TBIL) (OR=1.79, 95%CI：1.14-2.81, P=0.012) levels were identified as risk factors for LF progression. Conclusion LSM and liver function indicators, such as ALT, AST, and TBIL, are important influencing factors for LF progression. The monitoring of LSM and liver function indicators will be of great significance for the prevention and early diagnosis of liver cirrhosis.

Objective:To investigate and analyze the relationship between genotype and phenotype in patients with early-onset high myopia (eoHM) associated with hereditary eye diseases.Methods:A family-based study was conducted among 30 families diagnosed with eoHM at Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region from January 2022 to June 2023. Seven families (23.3%, 7/30), all probands, and their 14 parents were included. These seven families were unrelated. Detailed patient and family histories were collected. All participants underwent comprehensive ophthalmic examinations, including best-corrected visual acuity, color vision testing, fundus color photography, optical coherence tomography, fluorescein fundus angiography, and fundus autofluorescence imaging. Full-field electroretinography was performed in four cases. Peripheral venous blood samples were collected from patients and their parents for whole-genome DNA extraction and whole-exome sequencing. Potential pathogenic variants were identified, and their pathogenicity was analyzed and confirmed by Sanger sequencing. The pathogenicity of newly discovered gene variants was evaluated according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Literature on previously reported eoHM associated with hereditary eye diseases was reviewed to analyze the relationship between variant genes and clinical phenotypes.Results:Among the seven families, three exhibited X-linked inheritance, two showed autosomal recessive inheritance, and two demonstrated autosomal dominant inheritance. All the patients were male. Among the seven patients, one case each was identified with congenital stationary night blindness (CSNB), Bornholm eye disease, X-linked retinitis pigmentosa (XL-RP), cone-rod dystrophy, Knobloch syndrome, familial exudative vitreoretinopathy (FEVR), and Stickler syndrome. Genetic testing revealed nine gene variants highly correlated with the observed phenotypes. The genetic testing results revealed that all patients were found to carry nine gene variants highly associated with the phenotype, including: a hemizygous missense variant NYX c.647A>T (p.N216I) (M1), an OPN1LW LIAVA haplotype variant (M2), a hemizygous frameshift variant RPGR c.3096_3097del (p.E1033RfsTer45) (M3), compound heterozygous variants TTLL5 c.1588_1589del (p.L531EfsTer24) and c.850G>C (p.D284H) (M4, M5), compound heterozygous variants COL18A1 c.2118dup (p.G707RfsTer23) and c.3523_3524del (p.L1175VfsTer72) (M6, M7), a heterozygous missense mutation FZD4 c.1499C>T (p.T500I) (M8), and a heterozygous frameshift variant COL11A2 c.966dup (p.T323HfsTer19) (M9). Among them, M2, M4, M5, M8 and M9 were newly discovered mutation sites, and M1, M3, M6 and M7 were known mutation sites. According to the classification standards and guidelines of genetic variation issued by ACMG, M2, M3, M4, M6, M7, and M9 were judged to be pathogenic variants, while M1, M5, and M8 were of unknown clinical significance. Through literature review, it was found that eoHM was more common among the clinical phenotypes of 4 types of hereditary retinal diseases, including CSNB, Stickler syndrome, FEVR and XL-RP. Conclusion:eoHM is intricately associated with inherited eye diseases and may serve as the earliest indicator of such conditions.

Objective:To investigate and analyze the relationship between genotype and phenotype in patients with early-onset high myopia (eoHM) associated with hereditary eye diseases.Methods:A family-based study was conducted among 30 families diagnosed with eoHM at Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region from January 2022 to June 2023. Seven families (23.3%, 7/30), all probands, and their 14 parents were included. These seven families were unrelated. Detailed patient and family histories were collected. All participants underwent comprehensive ophthalmic examinations, including best-corrected visual acuity, color vision testing, fundus color photography, optical coherence tomography, fluorescein fundus angiography, and fundus autofluorescence imaging. Full-field electroretinography was performed in four cases. Peripheral venous blood samples were collected from patients and their parents for whole-genome DNA extraction and whole-exome sequencing. Potential pathogenic variants were identified, and their pathogenicity was analyzed and confirmed by Sanger sequencing. The pathogenicity of newly discovered gene variants was evaluated according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Literature on previously reported eoHM associated with hereditary eye diseases was reviewed to analyze the relationship between variant genes and clinical phenotypes.Results:Among the seven families, three exhibited X-linked inheritance, two showed autosomal recessive inheritance, and two demonstrated autosomal dominant inheritance. All the patients were male. Among the seven patients, one case each was identified with congenital stationary night blindness (CSNB), Bornholm eye disease, X-linked retinitis pigmentosa (XL-RP), cone-rod dystrophy, Knobloch syndrome, familial exudative vitreoretinopathy (FEVR), and Stickler syndrome. Genetic testing revealed nine gene variants highly correlated with the observed phenotypes. The genetic testing results revealed that all patients were found to carry nine gene variants highly associated with the phenotype, including: a hemizygous missense variant NYX c.647A>T (p.N216I) (M1), an OPN1LW LIAVA haplotype variant (M2), a hemizygous frameshift variant RPGR c.3096_3097del (p.E1033RfsTer45) (M3), compound heterozygous variants TTLL5 c.1588_1589del (p.L531EfsTer24) and c.850G>C (p.D284H) (M4, M5), compound heterozygous variants COL18A1 c.2118dup (p.G707RfsTer23) and c.3523_3524del (p.L1175VfsTer72) (M6, M7), a heterozygous missense mutation FZD4 c.1499C>T (p.T500I) (M8), and a heterozygous frameshift variant COL11A2 c.966dup (p.T323HfsTer19) (M9). Among them, M2, M4, M5, M8 and M9 were newly discovered mutation sites, and M1, M3, M6 and M7 were known mutation sites. According to the classification standards and guidelines of genetic variation issued by ACMG, M2, M3, M4, M6, M7, and M9 were judged to be pathogenic variants, while M1, M5, and M8 were of unknown clinical significance. Through literature review, it was found that eoHM was more common among the clinical phenotypes of 4 types of hereditary retinal diseases, including CSNB, Stickler syndrome, FEVR and XL-RP. Conclusion:eoHM is intricately associated with inherited eye diseases and may serve as the earliest indicator of such conditions.

Abstract: Objective To express Mycobacterium tuberculosis Rv2626c protein in Escherichia coli (E. coli) and study the antigenicity of the purified recombinant Rv2626c protein. Methods The amino acid sequence of Rv2626c protein from Mycobacterium tuberculosis H37Rv strain (accession number: CCP45424.1) in GenBank was retrieved and converted into the corresponding DNA sequence according to the codon preference of E. coli. This DNA sequence was synthesized and cloned into pET24a(+) plasmid to construct pET24a(+)-Rv2626c recombinant plasmid. This plasmid was transformed into E. coli BL21(DE3) cells, and the expression of Rv2626c protein was induced under various conditions of isopropyl β-D-thiogalactopyranoside (IPTG) concentrations, temperature, and period. The recombinant Rv2626c protein was identified by SDS-PAGE and Western Blot. The recombinant Rv2626c protein was purified by nickel chelate affinity chromatography and used to immunize violet blue rabbits to prepare anti-Rv2626c anti-serum. The specificity and titer of the serum were respectively detected by Western Blot and enzyme-linked immunosorbent assay (ELISA). Results The recombinant plasmid pET24a(+)-Rv2626c was successfully constructed. SDS-PAGE analysis showed that recombinant Rv2626c was expressed in the recombinant plasmid transformed E. coli with IPTG induction, with a molecular weight of about 14 500, and the size was consistent with the expectation. The optimal expression condition for recombinant Rv2626c protein was at 31 ℃ with 1.0 mmol/L IPTG for 6 hours. The target protein was mainly present in a soluble form, which was consistent with the results of Western blot. The hyperimmunized serum with recombinant Rv2626c protein vaccination showed good specificity, with a titer of 1∶ 256 000 detected by ELISA. Conclusions Mycobacterium tuberculosis Rv2626c protein is successfully expressed in E. coli, and the purified protein has good purity and antigenic activity, laying the foundation for further reveals of its biological functions.

ObjectiveThe correlation of Pueraria lobata producing areas， climate factors， total flavonoids of P. lobata， polysaccharide content of P. lobata， and antioxidant activity of P.lobata for medicinal application was analyzed， and the relationship between climate factors and the formation of P. lobata quality was evaluated. MethodThe scavenging rates of 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl（DPPH） and 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)（ABTS） radicals by total flavonoids and polysaccharides of P. lobata were detected， and the correlation between the contents of each component and the information of producing areas and climate factors was analyzed. ResultThe ABTS⁺ scavenging rate by total flavonoids of P.lobata was negatively correlated with altitude （P<0.05） and positively correlated with annual sunshine hours （P<0.05）. The altitude was positively correlated with the total flavonoid content， while the annual sunshine hours were negatively correlated with the total flavonoid content. There was a negative correlation between total flavonoid content and ABTS⁺ scavenging rate by total flavonoids. In other words， lower altitude and longer annual sunshine hours indicated lower total flavonoid content and higher ABTS⁺ scavenging rate by total flavonoids. The ABTS⁺ scavenging rate by polysaccharides of P. lobata was negatively correlated with the frost-free period （P<0.05） and the mean temperature in July （P<0.01）. There was a positive correlation between the polysaccharide content of P. lobata and the frost-free period. The mean temperature in July was positively correlated with the polysaccharide content of P. lobata （P<0.05）. The polysaccharide content of P. lobata was negatively correlated with the ABTS⁺ scavenging rate by polysaccharides of P. lobata. In other words， a shorter frost-free period in the producing area and lower mean temperature in July indicated lower polysaccharide content of P. lobata and higher ABTS⁺ scavenging rate by polysaccharides of P. lobata. The mean temperature in July was significantly correlated with the contents of total flavonoids and polysaccharides in P. lobata samples （P<0.05）. The lower mean temperature in July was often accompanied by lower total flavonoid content of P. lobata， lower polysaccharide content of Pueraria lobata， and stronger antioxidant activity of P. lobata samples. ConclusionThe ability of P. lobata to remove ABTS⁺ is stronger than that of DPPH⁺. There is a significant correlation between climate factors， content， and antioxidant capacity in each producing area. Further research on the internal law of the formation of medicinal active components of P. lobata induced by core climate factors will provide a scientific basis for revealing the formation mechanism of genuine P. lobata and the subsequent control of P. lobata quality according to the environment of producing areas.

Abstract Vitamin D, as an important nutrient, has been widely recognized for its significant role in the growth and development of children and adolescents, but its association with mental health is still under exploration. The article reviews and summarizes the related researches on vitamin D and common mental health problems of children and adolescents, including depression symptoms, anxiety symptoms, and suicidal ideation and behaviors, and possible biological mechanisms of vitamin D influencing mental health, to provide scientific evidence and ideas for improving the mental health of children and adolescents in China, as well as insights for future studies.

Objective To explore the value of free mitochondrial deoxyribonucleic acid(DNA)and micro ribonucleic acid(miRNA,miR)-146a expression in peripheral blood in assessing short-term prognosis of sepsis.Methods Totally 145 patients with sepsis admitted to the hospital from March 2021 to February 2023 were selected to detect the free mitochondrial DNA and miR-146a expression in peripheral blood.The incidence of poor prognosis after 28 days was counted,and the patients were then divided into poor prognosis group and good prognosis group.The general data and the free mitochondrial DNA and miR-146a expression in peripheral blood of the two groups were compared.Logistic regression analysis was used to explore the influencing factors of poor prognosis in patients.Receiver operating characteristic(ROC)curve was drawn to analyze the predictive efficacy of free mitochondrial DNA and miR-146a expressionin assessing the short-term prognosis of sepsis.Results The incidence of poor short-term prognosis in the 139 patients who completed the study was 28.78％.Concurrent diabetes(OR=1.765,95％CI:1.181～2.637),acutephysiology and chronic health evaluation(APACHE Ⅱ)score(OR=1.972,95％CI:1.317～2.953),free mitochondrial DNA in peripheral blood(OR=2.416,95％CI:1.524-3.829),and miR-146a expres-sion(OR=2.462,95％CI:1.431～4.237)were risk factors for poor short-term prognosis of patients with sepsis(P＜0.05).The sensitivity,specificity and area under curve(AUC)of free mitochondrial DNA and miR-146a expression in peripheral blood to predict poor short-term prognosis were higher than those of APACHE Ⅱ score(P＜0.05),and the sensitivity and AUC of free mitochondrial DNA and miR-146a expressions in peripheral blood to predict poor short-term prognosis were higher than those of both alone(P＜0.05).Conclusions Free mitochondrial DNA and miR-146a expression in peripheral blood are related to poor short-term prognosis of sepsis.The efficacy of both of them in assessing poor short-term prognosis of sepsis is better than that of APACHE Ⅱ score,and their combined prediction efficacy is even better.

Objective:To investigate the differences in acoustic characteristics between older patients with dysarthria resulting from anterior and posterior circulation cerebral infarctions.Methods:A case-control study was conducted.Sixty hospitalized older patients with dysarthria were selected and divided into two groups: the anterior circulation cerebral infarction group and the posterior circulation cerebral infarction group, each comprising 30 cases.Additionally, thirty healthy individuals aged 65 and above were included as a control group.The subjective evaluation of the patients' overall phonetic function was conducted using the GRBAS scale.Objective parameters, including fundamental frequency(F0), Jitter, Shimmer, maximum phonation time(MPT), maximum sound pressure level(SPLmax), minimum sound pressure level(SPLmin), and the dysphonia severity index(DSI), were collected using the DIVAS2.5 voice analysis system.We analyzed the acoustic characteristics across the three groups: patients with dysarthria and healthy subjects.Results:The grade(G), roughness(R), breathiness(B), asthenia(A), and strain(S)scores of patients in both the anterior and posterior circulation cerebral infarction groups were significantly higher than those of the healthy control group( F=16.574, 39.793, 46.309, 52.154, 25.603; all P<0.001).Furthermore, the roughness(R)and strain(S)of the voice in the anterior circulation cerebral infarction group were significantly elevated compared to the posterior circulation cerebral infarction group, whereas the breathiness(B), asthenia(A), and grade(G)scores in the posterior circulation cerebral infarction group were significantly higher than those in the anterior circulation cerebral infarction group(all P<0.001).The fundamental frequency value(F0)of the voice in patients with anterior circulation cerebral infarction was significantly greater than that of both the posterior circulation cerebral infarction group and the healthy control group( F=39.050, P<0.001).In contrast, the fundamental frequency value(F0)of patients with posterior circulation cerebral infarction was lower than that of the healthy control group( P=0.003).Additionally, the Jitter value in the anterior circulation cerebral infarction group was higher than in both the posterior circulation cerebral infarction group and the healthy control group( F=64.976, P<0.001).The Shimmer value in the anterior circulation cerebral infarction group was lower than that in the posterior circulation cerebral infarction group but higher than that in the healthy control group(both P<0.001).Finally, the values of MPT, SPLmin and SPL max, DSI in the anterior circulation cerebral infarction group were higher than those in the posterior circulation cerebral infarction group and lower than those in the healthy control group( F=90.406, 24.003, 16.164; all P<0.001); the value of DSI in the anterior circulation cerebral infarction group was lower than in both the posterior circulation cerebral infarction group and the healthy control group( F=87.921, P<0.001). Conclusions:There are notable differences in the acoustic characteristic parameters of dysarthria resulting from injuries at various anatomical sites in older patients with cerebral infarction.In practical clinical settings, a comprehensive evaluation of dysarthria in these patients should integrate the anatomical location of the injury, subjective symptom assessment, and objective analysis of acoustic characteristics to inform precise and personalized rehabilitation strategies.