Objective·To investigate the expression of protein tyrosine phosphatase receptor type N(PTPRN)in lung adenocarcinoma and its potential molecular mechanisms in promoting lung adenocarcinoma metastasis.Methods·A highly bone-metastatic A549-BM cell line was established through multiple rounds of intracardiac injection.RNA sequencing(RNA-seq),combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,was performed to identify PTPRN as a key metastasis-related gene.Subsequently,The Cancer Genome Atlas(TCGA)database was utilized to evaluate PTPRN expression in patients with lung adenocarcinoma and its correlation with clinical prognosis.Co-expression analysis based on TCGA data was conducted to identify and analyze key genes co-expressed with PTPRN.Small interfering RNA(siRNA)targeting PTPRN(siPTPRN)was transfected into A549-BM cells,and Transwell assays were performed to assess its effects on cell migration and invasion.Western blotting was used to detect the expression of epithelial-mesenchymal transition(EMT)-related proteins and the activation of the PI3K-AKT signaling pathway.siPTPRN-transfected A549-BM cells were injected into a mouse model via intracardiac injection,and in vivo metastasis was assessed.Additionally,multiple database analyses were integrated to predict BCL6 as an upstream transcription factor of PTPRN,and siBCL6 transfection experiments were performed to validate the regulatory effect of BCL6 on PTPRN expression.Results·RNA-seq and GO/KEGG enrichment analyses demonstrated that PTPRN was significantly upregulated in highly metastatic A549-BM cells and enriched in metastasis-associated pathways,including the PI3K-AKT signaling pathway and extracellular matrix(ECM)-receptor interactions.Analysis of the TCGA database further confirmed that PTPRN was highly expressed in lung adenocarcinoma patients and significantly associated with poor prognosis.Co-expression analysis based on TCGA data,combined with GO/KEGG enrichment analyses,revealed that PTPRN-associated genes were mainly enriched in biological processes such as neural signaling,endocrine regulation,cell communication,and ECM-receptor interactions.In vitro experiments demonstrated that siPTPRN transfection significantly inhibited the migration and invasion of A549-BM cells,accompanied by downregulation of EMT-related proteins and reduced activation of the PI3K-AKT signaling pathway.In vivo experiments further showed that PTPRN knockdown markedly suppressed the metastatic potential of A549-BM cells,confirming its pro-metastatic role.Additionally,siBCL6 transfection experiments demonstrated that BCL6 knockdown upregulated PTPRN expression.Conclusion·PTPRN is highly expressed in lung adenocarcinoma tissues and promotes tumor cell migration and metastasis by enhancing EMT and activating the PI3K-AKT signaling pathway.High PTPRN expression is significantly correlated with poor prognosis in lung adenocarcinoma patients,while PTPRN enhances lung adenocarcinoma cell invasiveness and metastatic potential.BCL6 may act as an upstream transcriptional regulator of PTPRN,influencing its expression levels.

Objective·To investigate the expression of protein tyrosine phosphatase receptor type N(PTPRN)in lung adenocarcinoma and its potential molecular mechanisms in promoting lung adenocarcinoma metastasis.Methods·A highly bone-metastatic A549-BM cell line was established through multiple rounds of intracardiac injection.RNA sequencing(RNA-seq),combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,was performed to identify PTPRN as a key metastasis-related gene.Subsequently,The Cancer Genome Atlas(TCGA)database was utilized to evaluate PTPRN expression in patients with lung adenocarcinoma and its correlation with clinical prognosis.Co-expression analysis based on TCGA data was conducted to identify and analyze key genes co-expressed with PTPRN.Small interfering RNA(siRNA)targeting PTPRN(siPTPRN)was transfected into A549-BM cells,and Transwell assays were performed to assess its effects on cell migration and invasion.Western blotting was used to detect the expression of epithelial-mesenchymal transition(EMT)-related proteins and the activation of the PI3K-AKT signaling pathway.siPTPRN-transfected A549-BM cells were injected into a mouse model via intracardiac injection,and in vivo metastasis was assessed.Additionally,multiple database analyses were integrated to predict BCL6 as an upstream transcription factor of PTPRN,and siBCL6 transfection experiments were performed to validate the regulatory effect of BCL6 on PTPRN expression.Results·RNA-seq and GO/KEGG enrichment analyses demonstrated that PTPRN was significantly upregulated in highly metastatic A549-BM cells and enriched in metastasis-associated pathways,including the PI3K-AKT signaling pathway and extracellular matrix(ECM)-receptor interactions.Analysis of the TCGA database further confirmed that PTPRN was highly expressed in lung adenocarcinoma patients and significantly associated with poor prognosis.Co-expression analysis based on TCGA data,combined with GO/KEGG enrichment analyses,revealed that PTPRN-associated genes were mainly enriched in biological processes such as neural signaling,endocrine regulation,cell communication,and ECM-receptor interactions.In vitro experiments demonstrated that siPTPRN transfection significantly inhibited the migration and invasion of A549-BM cells,accompanied by downregulation of EMT-related proteins and reduced activation of the PI3K-AKT signaling pathway.In vivo experiments further showed that PTPRN knockdown markedly suppressed the metastatic potential of A549-BM cells,confirming its pro-metastatic role.Additionally,siBCL6 transfection experiments demonstrated that BCL6 knockdown upregulated PTPRN expression.Conclusion·PTPRN is highly expressed in lung adenocarcinoma tissues and promotes tumor cell migration and metastasis by enhancing EMT and activating the PI3K-AKT signaling pathway.High PTPRN expression is significantly correlated with poor prognosis in lung adenocarcinoma patients,while PTPRN enhances lung adenocarcinoma cell invasiveness and metastatic potential.BCL6 may act as an upstream transcriptional regulator of PTPRN,influencing its expression levels.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

Objective:To investigate the efficacy of self-prescribed Huoxue Shengjing Prescription as adjuvant therapy for varicocele-induced infertility and its impact on the outcomes of in vitro fertilization-embryo transfer/intracytoplasmic sperm injection (IVF-ET/ICSI).Methods:A randomized controlled trial was conducted. A total of 99 patients with varicocele-induced infertility in our hospital from January 2022 to July 2023 were selected as observation subjects and divided into three groups using a random number table method, with 33 patients in each group. The low ligation group received low ligation of varicocele under a microscope, the low ligation + conventional Western medicine treatment group received low ligation + conventional Western medicine therapy, and the combined group received low ligation + conventional Western medicine therapy + a self-prescribed Huoxue Shengjing Prescription. Among them, the low ligation of varicocele under a microscope was followed by IVF-ET/ICSI assisted reproductive technology 3 months after surgery; the conventional Western medicine therapy involved continuous administration of L-carnitine oral solution for 3 months; the self-prescribed Huoxue Shengjing Prescription was started on the first day after surgery and continued for 3 months. TCM syndrome scores were assessed before and after treatment, and semen routine analysis was performed using an automated semen quality analyzer. Mitochondrial activity of granulosa cells was measured using the Hrudka extraction method, and sperm nuclear DNA integrity was assessed using a modified alkaline single-cell gel electrophoresis method. Follow-up was conducted for 1 year to observe and record the outcomes of IVF-ET/ICSI and evaluate the clinical efficacy.Results:The total effective rate was 93.9% (31/33) in the combined group, 69.7% (23/33) in the low ligation group, and 75.8% (25/33) in the low ligation + conventional Western medicine treatment group, with statistical significance ( χ 2=6.52, P=0.039). After treatment, the scores for mild abdominal pain, testicular heavy pain, impotence, mental fatigue, and the total score in the combined group were lower than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 89.29, 97.51, 136.36, 155.06, and 311.13, respectively, P<0.001). The sperm survival rate, sperm concentration, normal morphology rate, and progressive motility rate in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 19.23, 11.85, 35.97, and 52.21, respectively, P<0.001). Mitochondrial grade I cell activity of granulosa cells was higher than that of the low ligation + conventional treatment group and low ligation group ( F=23.23, P<0.001), and grade Ⅲ cell activity was lower than that of the low ligation + conventional treatment group and low ligation group ( F=20.28, P<0.001). After treatment, the detection of grade Ⅰ and Ⅱ sperm nuclear DNA integrity in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 17.73 and 18.39, respectively, P<0.001), while grades Ⅲ and Ⅳ were lower than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 29.07 and 10.36, respectively, P<0.001). During follow-up, the excellent embryo rate and the spouse's clinical pregnancy rate in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( χ2 values were 14.92 and 8.38, respectively; P values were 0.001 and 0.015, respectively). Conclusion:The adjuvant treatment with a self-prescribed Huoxue Shengjing Prescription can enhance sperm quality in patients with varicocele-related infertility, maintain DNA integrity, regulate seminal plasma mitochondrial function, increase the rate of high-quality embryos, and improve the spouse's pregnancy outcomes.

Objective·To investigate the expression level of O-GlcNAc transferase(OGT)in non-small cell lung cancer(NSCLC)and its impact on lung cancer proliferation,as well as to explore the underlying mechanisms.Methods·The expression of OGT in NSCLC tumors and adjacent normal tissues was detected by immunohistochemistry(IHC).The dataset(GSE31210)from the GEO database was analyzed to assess the correlation between OGT expression and NSCLC patient prognosis.siRNA transfection was performed to knock down OGT expression in H460 and H1299 cells,followed by total RNA extraction and transcriptome sequencing.Pathway enrichment analysis was conducted on differentially downregulated genes in the knockdown group compared with the control group,and Western blotting was used to validate the enrichment results.The effects of OGT knockdown on cell proliferation and colony formation in H460 and H1299 cells were evaluated using the cell counting kit-8(CCK-8)assay and colony formation assay,respectively.The impact of overexpressing downstream genes was also examined.Stable OGT-knockdown cell lines were generated using shRNA and subcutaneously inoculated into nude mice to monitor tumor growth.Results·IHC revealed that OGT expression was significantly upregulated in NSCLC tumor tissues compared to adjacent normal tissues.Patients with high OGT expression exhibited shorter survival times and poorer prognoses than those with low expression.Transcriptome sequencing demonstrated that genes downregulated after OGT knockdown were primarily enriched in the mitogen-activated protein kinase(MAPK)signaling pathway.Western blotting showed that total extracellular regulated protein kinase 1/2(ERK1/2)levels remained unchanged in H460 and H1299 cells after OGT knockdown,while phosphorylated ERK1/2(p-ERK1/2)and its downstream proto-oncogene JUNB protein were markedly reduced.Suppression of OGT expression attenuated the proliferation rate and colony formation capacity of H460 and H1299 cells,whereas JUNB overexpression rescued the proliferation defects induced by OGT knockdown.Notably,H460 cells with stable OGT knockdown formed significantly smaller tumors in nude mice.Conclusion·OGT is highly expressed in NSCLC and correlates with poor prognosis.Knockdown of OGT inhibits NSCLC cell proliferation and clonogenicity in vitro,and tumor growth in vivo.Mechanistically,OGT appears to promote NSCLC progression by activating the ERK/JUNB signaling axis.

Objective·To investigate the expression level of O-GlcNAc transferase(OGT)in non-small cell lung cancer(NSCLC)and its impact on lung cancer proliferation,as well as to explore the underlying mechanisms.Methods·The expression of OGT in NSCLC tumors and adjacent normal tissues was detected by immunohistochemistry(IHC).The dataset(GSE31210)from the GEO database was analyzed to assess the correlation between OGT expression and NSCLC patient prognosis.siRNA transfection was performed to knock down OGT expression in H460 and H1299 cells,followed by total RNA extraction and transcriptome sequencing.Pathway enrichment analysis was conducted on differentially downregulated genes in the knockdown group compared with the control group,and Western blotting was used to validate the enrichment results.The effects of OGT knockdown on cell proliferation and colony formation in H460 and H1299 cells were evaluated using the cell counting kit-8(CCK-8)assay and colony formation assay,respectively.The impact of overexpressing downstream genes was also examined.Stable OGT-knockdown cell lines were generated using shRNA and subcutaneously inoculated into nude mice to monitor tumor growth.Results·IHC revealed that OGT expression was significantly upregulated in NSCLC tumor tissues compared to adjacent normal tissues.Patients with high OGT expression exhibited shorter survival times and poorer prognoses than those with low expression.Transcriptome sequencing demonstrated that genes downregulated after OGT knockdown were primarily enriched in the mitogen-activated protein kinase(MAPK)signaling pathway.Western blotting showed that total extracellular regulated protein kinase 1/2(ERK1/2)levels remained unchanged in H460 and H1299 cells after OGT knockdown,while phosphorylated ERK1/2(p-ERK1/2)and its downstream proto-oncogene JUNB protein were markedly reduced.Suppression of OGT expression attenuated the proliferation rate and colony formation capacity of H460 and H1299 cells,whereas JUNB overexpression rescued the proliferation defects induced by OGT knockdown.Notably,H460 cells with stable OGT knockdown formed significantly smaller tumors in nude mice.Conclusion·OGT is highly expressed in NSCLC and correlates with poor prognosis.Knockdown of OGT inhibits NSCLC cell proliferation and clonogenicity in vitro,and tumor growth in vivo.Mechanistically,OGT appears to promote NSCLC progression by activating the ERK/JUNB signaling axis.

Objective:This study aims to explore the application value of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors.Methods:We retrospectively analyzed the clinical data of 15 patients with complex adrenal tumors treated at the General Hospital of Southern Theater Command from May 2022 to June 2023. The cohort comprised 5 males and 10 females with an average age of (47.6±7.8) years and a body mass index (BMI) of 26.5 (23.8-27.9) kg/m 2. Among the patients, 3 had a BMI ≥28 kg/m 2, 2 had diabetes, 6 had hypertension, and 1 had coronary heart disease. Preoperative endocrine hormone examination revealed abnormal blood catecholamines in 5 cases and abnormal blood cortisol in 2 cases. Ultrasound and CT scans indicated that 9 tumors were located on the left side and 6 on the right, with 4 cases showing tumor compression on adjacent large blood vessels or organs. The average tumor diameter was (7.61±2.79) cm, with 10 cases having a diameter ≥ 6 cm. All patients underwent laparoscopic adrenalectomy assisted by robots through the transperitoneal approach. The surgeries were performed in a lateral position under general anesthesia. The "liftoff" modular method was utilized to separate the treatment of adrenal tumors into lateral, medial, dorsal, cephalic, and adrenal renal plane sides. Tumors were appropriately manipulated during the operations to achieve a "liftoff" shape. Different modular dissociation steps were adopted based on the size and location of the left and right adrenal tumors. The left adrenal gland was dissected in the order of medial and dorsal, adrenal renal plane side, and lateral and cephalic sides, while the right adrenal gland was dissected in the order of lateral and dorsal, adrenal renal plane side, and medial and cephalic sides. Postoperative related indicators and follow-up status of patients were recorded and analyzed. Results:All 15 surgeries were successfully completed without any conversions to open adrenalectomy, with an average operation time of 118 (102-130) minutes and an average intraoperative blood loss of 102 (69-163) ml. The postoperative drainage time was 4 (3-5) days, and the postoperative hospital stay was 6 (4-7) days. The postoperative pathological diagnoses included 5 cases of pheochromocytoma, 3 cases of macronodular adrenal hyperplasia, 6 cases of adrenocortical adenoma, and 1 case of myelolipoma. Follow-up for 6-12 months after surgery showed good recovery and no recurrence.Conclusions:The application of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors is safe and feasible. It efficiently aids in tumor removal and holds significant clinical application value.

Objective:To explore the diagnostic value of laboratory examination in bronchoalveolar lavage fluid(BALF) for interstitial lung disease (ILD) and its application value in assessing the degree of fibrosis in the disease.Methods:Retrospective analysis. The clinical data of ILD patients treated in Shanghai First People′s Hospital from January 1, 2021 to December 31, 2023[104 cases, male︰female=48︰56, (62.79±1.24) years] were collected. According to the imaging scores, they were divided into a mild fibrosis ILD group [53 cases, male︰female=26︰27, (61.32±1.71) years] and a moderate to severe ILD fibrosis group [51 cases, male︰female=22︰29, (64.31±1.88) years]. Patients with community-acquired pneumonia without fibrotic lesions by HRCTduring the same period were selected as the control group [49 cases, male︰female=25︰24, (65.37±1.65)years]. The clinical information of all study subjects, as well as BALF lymphocyte subset analysis, cytokine and cytology count detection results were collected. Furthermore, the Kruskal-Wallis H test was used to screen the differential indexes, and the receiver operating characteristic (ROC) curve was used to evaluate the differential indexes to assess the degree of ILD pulmonary fibrosis.Results:Compared with the non-fibrotic pneumonia group, IL-6, IL-8, CD4+CD45RO+cells and macrophages (M%) were significantly upregulated in the mild fibrosis ILD group (P<0.05), and significantly higher in the moderate to severe fibrosis ILD group ( P<0.05). Compared with the non-fibrotic pneumonia group, IL-1β and white blood cell (WBC) count were significantly upregulated only in the moderate to severe fibrotic ILD group ( P<0.05). The correction model was constructed by stepwise logistic regression analysis, and the differential indexes were combined, and the proportion of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages was finally screened as the optimal combined diagnostic mode, with an area under the curve of 0.925, sensitivity of 92.3%, and specificity of 80.0%. Conclusion:Compared with the non-fibrotic pneumonia group, BALF-derived IL-1β, IL-6, IL-8, CD4+CD45RO+cells, WBC count and M% can be used as potential biomarkers to assess the degree of fibrosis, and the combination of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages has a better diagnostic efficacy for moderate to severe fibrotic ILD.

Objective:To verify the effect of Fu′s massage combined with physical therapy technology in rapidly relieving neck and low back pain caused by long-haul flight.Methods:Forty-two male volunteers were divided into 8, 12, 16, 24 and 48 h groups according to the simulated flying hours. After the completion of the simulated long-haul flight, the 5 groups of volunteers were immediately treated with Fu′s massage combined with physical factors for neck and low back pain. The Visual Analogue Scale (VAS) was used to evaluate the cervical and lumbar pain of volunteers after simulated flight and after treatment, and the treatment effect was before treatment analyzed.Results:There were significant differences in the VAS scores of cervical and lumbar regions before treatment among volunteers with different flying hours ( H=-30.15, -28.25, both P<0.001). The VAS scores of cervical and lumbar regions of volunteers in the 24 h and 48 h groups were higher than those in the 8 h group ( P=0.002, <0.001, =0.002, <0.001). The VAS scores of cervical and lumbar regions of volunteers in the 48 h group were higher than those in the 12 h group ( P=0.016, 0.001). The VAS score of cervical region of volunteers in the 48 h group was higher than that in the 16 h group ( P=0.033). After the intervention of Fu's massage combined with physical therapy, the VAS scores of cervical and lumbar regions in each group were lower than those before treatment, and the differences were significant ( t/ Z=-3.00-17.00, all P<0.05 or 0.01). Conclusions:Fu′s massage manipulation combined with physical treatment can effectively relieve the neck and low back pain caused by long-haul flight, which can be widely used as a relief method.

Objective:To verify the effect of Fu′s massage combined with physical therapy technology in rapidly relieving neck and low back pain caused by long-haul flight.Methods:Forty-two male volunteers were divided into 8, 12, 16, 24 and 48 h groups according to the simulated flying hours. After the completion of the simulated long-haul flight, the 5 groups of volunteers were immediately treated with Fu′s massage combined with physical factors for neck and low back pain. The Visual Analogue Scale (VAS) was used to evaluate the cervical and lumbar pain of volunteers after simulated flight and after treatment, and the treatment effect was before treatment analyzed.Results:There were significant differences in the VAS scores of cervical and lumbar regions before treatment among volunteers with different flying hours ( H=-30.15, -28.25, both P<0.001). The VAS scores of cervical and lumbar regions of volunteers in the 24 h and 48 h groups were higher than those in the 8 h group ( P=0.002, <0.001, =0.002, <0.001). The VAS scores of cervical and lumbar regions of volunteers in the 48 h group were higher than those in the 12 h group ( P=0.016, 0.001). The VAS score of cervical region of volunteers in the 48 h group was higher than that in the 16 h group ( P=0.033). After the intervention of Fu's massage combined with physical therapy, the VAS scores of cervical and lumbar regions in each group were lower than those before treatment, and the differences were significant ( t/ Z=-3.00-17.00, all P<0.05 or 0.01). Conclusions:Fu′s massage manipulation combined with physical treatment can effectively relieve the neck and low back pain caused by long-haul flight, which can be widely used as a relief method.