Metastatic dissemination is the major cause of death from breast-cancer (BC). Fusobacterium nucleatum (F.n) is widely enriched in BC and has recently been identified as one of the high-risk factors for promoting BC metastasis. Here, with an experimental model, we demonstrated that intratumoral F.n induced BC aggressiveness by transcriptionally activating Epithelial-mesenchymal transition-associated genes. Therefore, the F.n may be a potential target to prevent metastasis. Given the fact that cancer-associated fibroblasts (CAFs) are abundant in BC and located near blood vessels, we report an optogenetic system that drives CAF to in situ produce human antibacterial peptide LL37, with the characteristics of biosafety and freely intercellular trafficking, for depleting intratumoral F.n, leading to a 72.1% reduction in lung metastatic nodules number without affecting the balance of the systemic flora. Notably, mild photothermal treatment was found that could normalize CAF, contributing to synergistically inhibiting BC metastasis. In addition, the system can also simultaneously encode a gene of TNF-related apoptosis-inducing ligand to suppress the primary tumor. Together, our study highlights the potential of local elimination of tumor pathogenic bacteria to prevent BC metastasis.

Objective:To explore the clinical characteristics and genetic factors in four patients with Disorder of sex development (DSD).Methods:Four patients who visited Tianjin Medical University General Hospital between January 2023 and January 2024, presenting with short stature, abnormal external genitalia, or infertility as their chief complaints, were selected as the study subjects. Clinical data were collected, and peripheral or umbilical cord blood samples were obtained for karyotyping analysis and low-depth whole-genome sequencing (CNV-seq). Quantitative fluorescence PCR (QF-PCR) was used to detect the sex-determining region Y ( SRY) gene and azoospermia factor ( AZF) on the Y chromosome, while fluorescence in situ hybridization (FISH) was employed to determine the location of the SRY gene. Whole exome sequencing (WES) was performed for genetic testing, and Sanger sequencing was used for familial validation of the candidate variants. The study procedure and protocol were approved by the Medical Ethics Committee of Tianjin Medical University General Hospital (Ethics No.: IRB2024-WZ-006). Results:Case 1 had a karyotype of 45, X[22]/46, XY[8], with CNV-seq indicating a mosaic deletion of 7.44 Mb (copy number = 0.2) at Yp11.31-p11.2, a mosaic deletion of 5.32 Mb (copy number = 0.3) at Yq11.1-q11.221, and a deletion of 10.26 Mb (copy number = 0) at Yq11.221-q11.23. Y chromosome microdeletion analysis showed SRY and AZFa (+ ), AZFb+ c (-). Case 2 had a karyotype of 45, X[12]/46, X, del(X)(q26.3)[18], with CNV-seq indicating a mosaic deletion of 132.62 Mb (copy number = 1.4) at Xp22.33-q26.3 and a deletion of 19.62 Mb (copy number = 1) at Xq26.3-q28. Case 3 had a karyotype of 46, XX, with CNV-seq showing two copies of the X chromosome and no Y chromosome. Y chromosome microdeletion analysis showed SRY (+ ) and AZFa+ b+ c (-), and FISH confirmed a translocation of the SRY gene to the terminal end of the short arm of the X chromosome. Case 4 had a karyotype of 46, XY, with CNV-seq showing one copy each of the X and Y chromosomes. Y chromosome microdeletion analysis showed SRY(+ ) and AZFa+ b+ c (+ ), and WES revealed a c. 1103del variant in the AR gene (maternal origin), which was classified as a pathogenic variant based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) (PVS1+ PP1+ PM2_Supporting). Conclusion:The combined application of multiple detection techniques such as chromosomal karyotyping analysis, CNV-seq, QF-PCR, and WES can identify the genetic etiology of DSD patients, providing a basis for clinical consultation and treatment plan formulation.

OBJECTIVE: To explore the clinical characteristics and genetic factors in four patients with Disorder of sex development (DSD). METHODS: Four patients who visited Tianjin Medical University General Hospital between January 2023 and January 2024, presenting with short stature, abnormal external genitalia, or infertility as their chief complaints, were selected as the study subjects. Clinical data were collected, and peripheral or umbilical cord blood samples were obtained for karyotyping analysis and low-depth whole-genome sequencing (CNV-seq). Quantitative fluorescence PCR (QF-PCR) was used to detect the sex-determining region Y (SRY) gene and azoospermia factor (AZF) on the Y chromosome, while fluorescence in situ hybridization (FISH) was employed to determine the location of the SRY gene. Whole exome sequencing (WES) was performed for genetic testing, and Sanger sequencing was used for familial validation of the candidate variants. The study procedure and protocol were approved by the Medical Ethics Committee of Tianjin Medical University General Hospital (Ethics No.: IRB2024-WZ-006). RESULTS: Case 1 had a karyotype of 45,X[22]/46,XY[8], with CNV-seq indicating a mosaic deletion of 7.44 Mb (copy number = 0.2) at Yp11.31-p11.2, a mosaic deletion of 5.32 Mb (copy number = 0.3) at Yq11.1-q11.221, and a deletion of 10.26 Mb (copy number = 0) at Yq11.221-q11.23. Y chromosome microdeletion analysis showed SRY and AZFa (+), AZFb+c (-). Case 2 had a karyotype of 45,X[12]/46,X,del(X)(q26.3)[18], with CNV-seq indicating a mosaic deletion of 132.62 Mb (copy number = 1.4) at Xp22.33-q26.3 and a deletion of 19.62 Mb (copy number = 1) at Xq26.3-q28. Case 3 had a karyotype of 46,XX, with CNV-seq showing two copies of the X chromosome and no Y chromosome. Y chromosome microdeletion analysis showed SRY (+) and AZFa+b+c (-), and FISH confirmed a translocation of the SRY gene to the terminal end of the short arm of the X chromosome. Case 4 had a karyotype of 46,XY, with CNV-seq showing one copy each of the X and Y chromosomes. Y chromosome microdeletion analysis showed SRY(+) and AZFa+b+c (+), and WES revealed a c.1103del variant in the AR gene (maternal origin), which was classified as a pathogenic variant based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) (PVS1+PP1+PM2_Supporting). CONCLUSION The combined application of multiple detection techniques such as chromosomal karyotyping analysis, CNV-seq, QF-PCR, and WES can identify the genetic etiology of DSD patients, providing a basis for clinical consultation and treatment plan formulation.
Humans ; Male ; Female ; Chromosomes, Human, Y/genetics* ; Disorders of Sex Development/genetics* ; Sex-Determining Region Y Protein/genetics* ; Karyotyping ; In Situ Hybridization, Fluorescence ; Exome Sequencing ; Adult ; Child

Objective: To establish a method for determining the molecular weight and molecular weight distribution of Poly(Lactide-co-Glycolide Acid) (PLGA) using Size Exclusion Chromatography-Refractive Index-Multiangle Laser Light Scattering (SEC-RI-MALLS) and Size Exclusion Chromatography-Refractive Index (SEC-RID), and to compare the results obtained from these two methods.
Methods: For SEC-RI-MALLS, tetrahydrofuran was used as the mobile phase, Shodex GPC KF-803L was employed as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, and an injection volume of 100 μL. For SEC-RID, tetrahydrofuran was also used as the mobile phase, Agilent PLgel 5 μm MIXD-D was used as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, differential detector temperature at 35 ℃, and an injection volume of 20 μL. The molecular weight and molecular weight distribution were calculated using Agilent’s GPC software. The newly established methods were validated methodologically, and the molecular weight and molecular weight distribution of 13 batches of samples were determined.
Results: The precision, accuracy, stability, and repeatability tests for SEC-RI-MALLS showed RSD values of 1.35%, 1.58%, 1.53%, and 1.26%, respectively. The SEC-RID method exhibited good linearity (r=0.999 9), with RSD values for precision, accuracy, stability, and repeatability tests (n=6) of 2.05%, 1.62%, 1.30%, and 2.97%, respectively. The results obtained from SEC-RI-MALLS were lower than those from SEC-RID, and the molecular weight distribution coefficient was smaller, but the results from the paired T-test performed with the value measured by SEC-RID method and the value measured by SEC-RI-MALLS method multiplied a conversion coefficient of 1.5 showed no significant difference between the two methods.
Conclusion: Both methods are stable and reliable, and can be used for the determination of PLGA molecular weight and molecular weight distribution based on the specific situations.

Objective:To characterize the minimal inhibitory concentration (MIC) of macrolides against clinical Mycoplasma pneumoniae (MP) isolates and to investigate the significance of 23S rRNA mutations. Methods:Cross-sectional study.A total of 288 clinical MP strains preserved in the laboratory from 2016 to 2021 were taken for macrolide resistance gene testing and the evaluation of in vitro susceptibility to Azithromycin and Erythromycin.MIC 50 and MIC 90 values were calculated separately for macrolide-susceptible and -resistant strains. Results:All 288 MP strains underwent the test of in vitro susceptibility to Azithromycin, while 86 of them were additionally tested for Erythromycin.Among these strains, 22 strains were Azithromycin-sensitive, and 266 strains were Azithromycin-resistant.A2063G mutations were detected in 260 (97.7%) strains, while A2064G mutations were detected in 6 (2.3%) strains.Azithromycin-resistant strains had an MIC 50 of 128.000 μg/mL and an MIC 90 of 512.000 μg/mL, with the MIC ranging between 16.000 and 512.000 μg/mL.Seven strains were sensitive and 79 strains were resistant to Erythromycin.Among Erythromycin-resistant strains, A2063G mutations were detected in 73 (92.4%) strains, while A2064G mutations were detected in 6 (7.6%) strains.Erythromycin-resistant strains had an MIC 50 of 256.000 μg/mL and an MIC 90 of 512.000 μg/mL, with the MIC ranging between 64.000 and 1 024.000 μg/mL. Conclusions:A2063G and A2064G mutations in the 23S rRNA gene of MP are associated with high-level in vitro resistance to Azithromycin and Erythromycin, significantly limiting the clinical effectiveness of these antibiotics.Early resistance gene testing is recommended for suspected MP patients, which can help optimize the treatment, improve prognosis, and prevent resistance spread.

Objective:To characterize the minimal inhibitory concentration (MIC) of macrolides against clinical Mycoplasma pneumoniae (MP) isolates and to investigate the significance of 23S rRNA mutations. Methods:Cross-sectional study.A total of 288 clinical MP strains preserved in the laboratory from 2016 to 2021 were taken for macrolide resistance gene testing and the evaluation of in vitro susceptibility to Azithromycin and Erythromycin.MIC 50 and MIC 90 values were calculated separately for macrolide-susceptible and -resistant strains. Results:All 288 MP strains underwent the test of in vitro susceptibility to Azithromycin, while 86 of them were additionally tested for Erythromycin.Among these strains, 22 strains were Azithromycin-sensitive, and 266 strains were Azithromycin-resistant.A2063G mutations were detected in 260 (97.7%) strains, while A2064G mutations were detected in 6 (2.3%) strains.Azithromycin-resistant strains had an MIC 50 of 128.000 μg/mL and an MIC 90 of 512.000 μg/mL, with the MIC ranging between 16.000 and 512.000 μg/mL.Seven strains were sensitive and 79 strains were resistant to Erythromycin.Among Erythromycin-resistant strains, A2063G mutations were detected in 73 (92.4%) strains, while A2064G mutations were detected in 6 (7.6%) strains.Erythromycin-resistant strains had an MIC 50 of 256.000 μg/mL and an MIC 90 of 512.000 μg/mL, with the MIC ranging between 64.000 and 1 024.000 μg/mL. Conclusions:A2063G and A2064G mutations in the 23S rRNA gene of MP are associated with high-level in vitro resistance to Azithromycin and Erythromycin, significantly limiting the clinical effectiveness of these antibiotics.Early resistance gene testing is recommended for suspected MP patients, which can help optimize the treatment, improve prognosis, and prevent resistance spread.

Objective:To explore the clinical characteristics and genetic factors in four patients with Disorder of sex development (DSD).Methods:Four patients who visited Tianjin Medical University General Hospital between January 2023 and January 2024, presenting with short stature, abnormal external genitalia, or infertility as their chief complaints, were selected as the study subjects. Clinical data were collected, and peripheral or umbilical cord blood samples were obtained for karyotyping analysis and low-depth whole-genome sequencing (CNV-seq). Quantitative fluorescence PCR (QF-PCR) was used to detect the sex-determining region Y ( SRY) gene and azoospermia factor ( AZF) on the Y chromosome, while fluorescence in situ hybridization (FISH) was employed to determine the location of the SRY gene. Whole exome sequencing (WES) was performed for genetic testing, and Sanger sequencing was used for familial validation of the candidate variants. The study procedure and protocol were approved by the Medical Ethics Committee of Tianjin Medical University General Hospital (Ethics No.: IRB2024-WZ-006). Results:Case 1 had a karyotype of 45, X[22]/46, XY[8], with CNV-seq indicating a mosaic deletion of 7.44 Mb (copy number = 0.2) at Yp11.31-p11.2, a mosaic deletion of 5.32 Mb (copy number = 0.3) at Yq11.1-q11.221, and a deletion of 10.26 Mb (copy number = 0) at Yq11.221-q11.23. Y chromosome microdeletion analysis showed SRY and AZFa (+ ), AZFb+ c (-). Case 2 had a karyotype of 45, X[12]/46, X, del(X)(q26.3)[18], with CNV-seq indicating a mosaic deletion of 132.62 Mb (copy number = 1.4) at Xp22.33-q26.3 and a deletion of 19.62 Mb (copy number = 1) at Xq26.3-q28. Case 3 had a karyotype of 46, XX, with CNV-seq showing two copies of the X chromosome and no Y chromosome. Y chromosome microdeletion analysis showed SRY (+ ) and AZFa+ b+ c (-), and FISH confirmed a translocation of the SRY gene to the terminal end of the short arm of the X chromosome. Case 4 had a karyotype of 46, XY, with CNV-seq showing one copy each of the X and Y chromosomes. Y chromosome microdeletion analysis showed SRY(+ ) and AZFa+ b+ c (+ ), and WES revealed a c. 1103del variant in the AR gene (maternal origin), which was classified as a pathogenic variant based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) (PVS1+ PP1+ PM2_Supporting). Conclusion:The combined application of multiple detection techniques such as chromosomal karyotyping analysis, CNV-seq, QF-PCR, and WES can identify the genetic etiology of DSD patients, providing a basis for clinical consultation and treatment plan formulation.

Objective:To established a rapid method for the determination of lactose(C12 H22 O11)content.Methods:The specific rotation([α]20D)of pure lactose was measured with lactose extract product,and the correc-tion coefficient was calculated.The measured rotatory value of the sample was multiplied by the correction coeffi-cient to obtain the mass(g)of lactose in the test product,and the lactose content was calculated.The equivalence of the newly established polarimetric method and the legal test method(HPLC-RID method)for the determination of lactose content was studied.Results:The linear relation of established method was excellent with the range of 0.05-0.20 g·mL-1(r=1.000 0)and the method also had good reproducibility(RSD=0.07％(n=6)).The lactose content of 141 batches samples measured by polarimetry was compared with the results determined by official analytical procedure,and the results showed the relative deviation between the same batches was less than 1.0％.The results of one-way ANOVA also showed that there was no significant difference between two groups(Sig.＞0.05).Conclusion:The performance of polarimetry is comparable to HPLC-RID in the Chinese Pharma-copoeia 2020 Vol Ⅳ.Meanwhile,the polarimetry can reduce the test cost,shorten the test time and meet the requirements of the quality control.

OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8−1.2, the detection concentration of bivalirudin was 0.1 μg·mL−1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL−1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL−1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.

Objective:To explore the clinical and genetic characteristics of a fetus diagnosed with Congenital myasthenic syndrome type 16 (CMS16).Methods:A couple who had visited Tianjin Medical University General Hospital in February 2018 due to "adverse outcome of two pregnancies" was selected as the study subject. Clinical data was gathered. Peripheral blood and amniotic fluid samples were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Low-depth whole-genome sequencing was carried out to detect copy number variation (CNV) in the fetus.Results:The couple′s first pregnancy had resulted in a miscarriage at 27 + 5 weeks, when ultrasound had revealed pleural effusion and polyhydramnios in the fetus. Their second pregnancy was terminated at 30 + 5 weeks due to fetal hand malformations, polyhydramnios and pleural fluid. Both couple had denied family history of genetic conditions. For their third pregnancy, no CNV abnormality was detected, whilst a compound heterozygous variants, including a maternally derived c. 3172C>T (p.R1058W) and paternal c. 1431delG (p.K477fs*89) in the SCN4A gene were detected. Based on the guidelines from the American College of Medical Genetics and Genomics, the c. 3172C>T (p.R1058W) was predicted as a likely pathogenic variant (PM1+ PM2_supporting+ PP3+ PP4), whilst the c. 1431delG (p.K477fs*89) was predicted as a pathogenic variant (PVS1+ PM2_supporting+ PP4). Conclusion:The c. 3172C>T (p.R1058W) and c. 1431delG (p.K477fs*89) compound heterozygous variants of the SCN4A gene probably underlay the CMS16 in the third fetus.