Objective To investigate the effects of Danlong Xingnao Prescription on learning and memory ability and microglia activation in rats with vascular dementia(VD)based on HMGB1/RAGE/NF-κB pathway.Methods Ten rats were randomly selected from 72 rats as a sham-operation group.The remaining rats were treated with modified bilateral common carotid artery ligation method to prepare the VD model.The 50 successful model rats were randomly divided into model group,nimodipine group(10.8 mg/kg)and Danlong Xingnao Prescription low-,medium-and high-dosage groups(3.7,7.4,14.8 g/kg),with 10 rats in each group.The administration groups were given relevant solution for gavage,the sham-operation group and model group were given the same amount of normal saline for consecutive 28 d.Morris water maze test was performed to evaluate learning and memory abilities of rats,the morphology in the hippocampus were observed by HE staining,the contents of interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-α in hippocampal tissue were detect by ELISA,RT-PCR was used to detect high mobility group protein B1(HMGB1),receptor of advanced glycation end product(RAGE),nuclear factor(NF)-κB p65 and regulatory RNase-1(Regnase-1)mRNA expression in hippocampal tissue,immunohistochemistry and Western blot were used to detect the protein expressions of ion calcium binding adapter molecule 1(Iba1),HMGB1,RAGE,NF-κB p65 and Regnase-1 in hippocampal tissue.Results Compared with the sham-operation group,the escape latency of rats was prolonged,and the number of crossings through the original platform was increased in the model group(P<0.01),the pyramidal cells in the hippocampus were reduced and irregularly shaped,with unclear cell and nuclear membranes,and a significant number of necrotic neurons were visible,the contents of IL-1β,IL-6 and TNF-α in hippocampal tissue increased(P<0.01),the mRNA expressions of HMGB1,RAGE and NF-κB p65 in hippocampal tissue increased(P<0.01),while the mRNA expression of Regnase-1 decreased(P<0.01),the protein expressions of Iba1,HMGB1,RAGE and NF-κB p65 increased(P<0.01),while the protein expression of Regnase-1 decreased(P<0.01).Compared with the model group,the escape latency of rats was shortened in Danlong Xingnao Prescription groups,the number of crossings through the original platform was reduced(P<0.05,P<0.01),the neuronal structure of hippocampal tissue was significantly improved,the number of necrotic neurons was reduced,and the contents of IL-1β,IL-6 and TNF-α in hippocampal tissue reduced(P<0.05,P<0.01),the mRNA expressions of HMGB1,RAGE and NF-κB p65 in hippocampal tissue decreased,the mRNA expression of Regnase-1 increased(P<0.05,P<0.01),the protein expression of Iba1,HMGB1,RAGE and NF-κB p65 decreased,the protein expression of Regnase-1 increased(P<0.05,P<0.01).Conclusion Danlong Xingnao Prescription can improve the learning and memory ability of VD rats,and its mechanism may be related to inhibiting the activation of HMGB1/RAGE/NF-κB pathway and increasing Regnase-1 expression,thereby inhibiting the activation of microglia.

Objective To investigate the application value of ultrasonic artificial intelligence(AI)combined with serine/threonine-protein kinase(BRAF)V600E gene testing in differentiating benign-malignant and invasive thyroid nodules.Methods A total of 150 patients with malignant thyroid nodules(the malignant group)and 150 patients with benign thyroid nodules(the benign group)were selected.According to whether the pathological diagnosis of the malignant group involved capsule,vascular,nerve invasion or lymph node metastasis,patients were divided into the invasive group(66 cases)and the non-invasive group(84 cases).General clinical characteristics,ultrasonic AI parameters and BRAF V600E gene testing results were compared between groups.Discrepancies between ultrasonic AI,BRAF V600E gene testing and postoperative pathological diagnoses were analyzed.ROC curves and Delong tests were usd to evaluate the diagnostic efficacy of ultrasonic AI,BRAF V600E gene and their joint inspection.Results The malignant group exhibited higher probabilities of nodule maximum diameter(＞1 cm),solid structure,hypoechoic/very hypoechoic echogenicity,microcalcification,blurred margin,irregular shape,aspect ratio(＞1),internal and mixed blood flow distribution and high blood flow richness(gradesⅢ—Ⅴ)compared to those of the benign group(P＜0.05).The invasive subgroup showed higher probabilities of nodule maximum diameter(＞1 cm),solid structure,hypoechoic/very hypoechoic echogenicity,microcalcification,blurred margin,irregular shape,internal and mixed blood flow distribution,and high blood flow richness(grades Ⅲ—Ⅴ)than those of the non-invasive subgroup(P＜0.05).For diagnosing malignant thyroid nodules,ultrasonic AI demonstrated a sensitivity of 90.00%and specificity of 80.67%.For invasive malignant nodules,sensitivity was 84.85%and specificity was 83.33%.BRAF V600E gene testing showed a sensitivity of 72.67%,specificity of 90.00%for malignant nodules.For invasive nodules,sensitivity was 74.24%and specificity was 88.10%.Receiver operating characteristic curve(ROC)analysis revealed that the AUCs(95%CI)for ultrasonic AI,BRAF V600E gene and their joint inspection in diagnosing malignant thyroid nodules were 0.853(0.807-0.900),0.813(0.762-0.864)and 0.941(0.917-0.966),with the joint inspection outperforming individual tests(P＜0.05).For invasive malignant nodules,the AUCs were 0.841(0.773-0.909),0.812(0.737-0.886)and 0.924(0.880-0.967),respectively,with the joint inspection showing superior performance(P＜0.05).Conclusion The joint inspection ultrasound AI with BRAF V600E gene significantly improve the diagnostic efficacy for differentiating benign and malignant thyroid nodules,and assessing their invasive potential.

Objective To investigate the application value of ultrasonic artificial intelligence(AI)combined with serine/threonine-protein kinase(BRAF)V600E gene testing in differentiating benign-malignant and invasive thyroid nodules.Methods A total of 150 patients with malignant thyroid nodules(the malignant group)and 150 patients with benign thyroid nodules(the benign group)were selected.According to whether the pathological diagnosis of the malignant group involved capsule,vascular,nerve invasion or lymph node metastasis,patients were divided into the invasive group(66 cases)and the non-invasive group(84 cases).General clinical characteristics,ultrasonic AI parameters and BRAF V600E gene testing results were compared between groups.Discrepancies between ultrasonic AI,BRAF V600E gene testing and postoperative pathological diagnoses were analyzed.ROC curves and Delong tests were usd to evaluate the diagnostic efficacy of ultrasonic AI,BRAF V600E gene and their joint inspection.Results The malignant group exhibited higher probabilities of nodule maximum diameter(＞1 cm),solid structure,hypoechoic/very hypoechoic echogenicity,microcalcification,blurred margin,irregular shape,aspect ratio(＞1),internal and mixed blood flow distribution and high blood flow richness(gradesⅢ—Ⅴ)compared to those of the benign group(P＜0.05).The invasive subgroup showed higher probabilities of nodule maximum diameter(＞1 cm),solid structure,hypoechoic/very hypoechoic echogenicity,microcalcification,blurred margin,irregular shape,internal and mixed blood flow distribution,and high blood flow richness(grades Ⅲ—Ⅴ)than those of the non-invasive subgroup(P＜0.05).For diagnosing malignant thyroid nodules,ultrasonic AI demonstrated a sensitivity of 90.00%and specificity of 80.67%.For invasive malignant nodules,sensitivity was 84.85%and specificity was 83.33%.BRAF V600E gene testing showed a sensitivity of 72.67%,specificity of 90.00%for malignant nodules.For invasive nodules,sensitivity was 74.24%and specificity was 88.10%.Receiver operating characteristic curve(ROC)analysis revealed that the AUCs(95%CI)for ultrasonic AI,BRAF V600E gene and their joint inspection in diagnosing malignant thyroid nodules were 0.853(0.807-0.900),0.813(0.762-0.864)and 0.941(0.917-0.966),with the joint inspection outperforming individual tests(P＜0.05).For invasive malignant nodules,the AUCs were 0.841(0.773-0.909),0.812(0.737-0.886)and 0.924(0.880-0.967),respectively,with the joint inspection showing superior performance(P＜0.05).Conclusion The joint inspection ultrasound AI with BRAF V600E gene significantly improve the diagnostic efficacy for differentiating benign and malignant thyroid nodules,and assessing their invasive potential.

Objective Itproposes a WISN staffing demand model for clinical physicians to address the challenge that current physicians staffing standards fail to meet the operational needs of general hospitals.Methods Collect the 2023 annual operation data of a tertiary general hospital,using inpatient visits and surgeries as benchmarks to measure the workload demands of physicians in internal medicine and surgery,respectively.Additionally,integrate non-clinical workloads of physicians and the workload of resident physicians in standardized training to address the limitation of traditional models that focus solely on clinical tasks.Results The good applicability and robustness of the model in practical applications are verified through model comparison and sensitivity analysis,accurately reflecting the demand for physicians.Conclusion The model accurately reflects the workload of physicians in general hospitals,providing a quantitative estimation formula for physicians staffing.It has broad applicability and significant potential for wider adoption.

Objective:To assess the clinicopathological characteristics of non-muscle-invasive bladder cancers (NMIBC) with high expression of human epidermal growth factor receptor 2 (HER2) and to examine the prognostic values of HER2 expression in NMIBC patients with intravesical anthracycline instillation.Methods:A total of 221 NMIBC samples diagnosed between January 1, 2017 and April 15, 2024 were collected. Their clinical, diagnostic and treatment features were analyzed. The expression of HER2 protein and the Ki-67 proliferation index were assessed using immunohistochemistry (IHC). For the patients with HER2 high-expression (IHC 3+), the clinical pathological features (age, gender, tumor grade, Ki-67 expression level, tumor size, and tumor number) were compared with those without (i.e., HER2 IHC 0/1+/2+). The impact of HER2 expression on the recurrence-free survival (RFS) of patients with intravesical anthracycline (epirubicin or pirarubicin) instillation after transurethral resection of bladder tumor (TURBT) was evaluated.Results:Among the 221 NMIBC patients, 30 (13.6%) were HER2 IHC 3+, 142 (64.3%) HER 2+, 46 (20.8%) HER2 1+, and 3 (1.4%) HER2 IHC 0. The proportion of high-grade tumors in patients with HER2 high-expression was higher than that in patients without (83.3% versus 44.5%, P<0.001). Additionally, a high Ki-67 index (≥20%) was more commonly noted in HER2 high-expression tumors ( P=0.003). In the patients treated with intravesical anthracycline instillation, HER2 high-expression was associated with a shorter RFS ( P<0.001). Conclusion:HER2 high-expression seems to be not only associated with worse clinicopathological features of NMIBC but also a poor RFS in NMIBC patients treated with anthracycline instillation after TURBT.

Objective:To investigate the mutation of PIK3CA, AKT1 and PTEN genes in hormone receptor (HR)-positive and HER2-negative invasive breast cancer.Methods:A total of 44 formalin-fixed paraffin-embedded samples from HR-positive/HER2-negative female patients with breast cancer obtained between January 2020 and July 2024 in Peking Union Medical College Hospital were selected. The mutations of PIK3CA, AKT1 and PTEN genes were analyzed by next-generation sequencing (NGS), and the related clinicopathological characteristics were summarized.Results:In the cohort, 31 out of 44 cases (70.5%) exhibited alterations in the PIK3CA, AKT1 and PTEN genes. Of these, 83.9% (26/31) tumors harbored genetic abnormalities involving one gene, including 21 (47.7%, 21/44) PIK3CA, 2 (4.5%, 2/44) PTEN and 3 (6.8%, 3/44) AKT1 gene mutations. Mutations of both PIK3CA and PTEN genes were found in 16.1% (5/31) of specimens. Among the 26 cases with PIK3CA gene mutations, 13 variants were identified, including E542K, E545K, Q546K, H1047R, H1047L, G1049R, M1043I, C420R, P447_L455del, N345K, N345I, K711N and H1047L/V346G. In addition, 7 mutants of PTEN gene were determined (T319 *, T321Qfs *23, Q245 *, Q171H, L108P, Y68Ifs *6 and V343fs). For AKT1 gene mutation, only E17K was observed.Mutations of PIK3CA/AKT1/PTEN genes are more likely to occur over 40 year-old patients.In this cohort, the PIK3CA V346G mutation (co-existent PIK3CA H1047L) and the PTEN V343fs mutation were not found in previous publications. Conclusion:In addition to the predominance of common loci, PIK3CA and PTEN gene mutations also have rare loci mutations in the breast cancer, warranting further analysis with an expanded sample size.

Objective:To investigate the abnormalities of mesenchymal-epithelial transition factor (MET) gene in early-stage lung adenocarcinomas and to provide genetic bases for related clinical studies.Methods:A total of 630 cases of formalin-fixed and paraffin-embedded lung adenocarcinoma specimens with ALK and EGFR double-negativities were collected at Peking Union Medical College Hospital, Beijing, China between July 2020 and April 2022. Forty-three stage Ⅰ-ⅢA tumors with MET exon 14 skipping mutation identified by reverse transcription droplet digital PCR (RT-ddPCR) were identified and then evaluated for MET amplification using fluorescence in situ hybridization (FISH). MET amplification was determined using the ratio of MET to chromosome 7 enumeration probe (CEP7) or the mean of MET gene copy number (GCN).Results:Among the 43 samples with MET exon 14 skipping mutation, MET amplification was detected in 9 cases (9/43, 20.93%), including 1 case of MET/CEP7 ≥2 and GCN ≥5 (1/9), 8 cases of GCN≥5 (8/9), as well as 10 cases with high level of CEP7 (7.00-9.72) which included 5 cases with MET amplification. There were no significant differences in clinicopathological features between the two subgroups of tumors which harbored MET exon 14 skipping mutation with MET amplification versus those without ( P>0.05). Conclusions:Co-occurrence of MET exon 14 skipping mutation and MET amplification or high level of CEP7 is frequently observed in early-stage lung adenocarcinomas. The most common pattern of MET gene amplification is GCN ≥5.

Objective:To investigate the mutation of PIK3CA, AKT1 and PTEN genes in hormone receptor (HR)-positive and HER2-negative invasive breast cancer.Methods:A total of 44 formalin-fixed paraffin-embedded samples from HR-positive/HER2-negative female patients with breast cancer obtained between January 2020 and July 2024 in Peking Union Medical College Hospital were selected. The mutations of PIK3CA, AKT1 and PTEN genes were analyzed by next-generation sequencing (NGS), and the related clinicopathological characteristics were summarized.Results:In the cohort, 31 out of 44 cases (70.5%) exhibited alterations in the PIK3CA, AKT1 and PTEN genes. Of these, 83.9% (26/31) tumors harbored genetic abnormalities involving one gene, including 21 (47.7%, 21/44) PIK3CA, 2 (4.5%, 2/44) PTEN and 3 (6.8%, 3/44) AKT1 gene mutations. Mutations of both PIK3CA and PTEN genes were found in 16.1% (5/31) of specimens. Among the 26 cases with PIK3CA gene mutations, 13 variants were identified, including E542K, E545K, Q546K, H1047R, H1047L, G1049R, M1043I, C420R, P447_L455del, N345K, N345I, K711N and H1047L/V346G. In addition, 7 mutants of PTEN gene were determined (T319 *, T321Qfs *23, Q245 *, Q171H, L108P, Y68Ifs *6 and V343fs). For AKT1 gene mutation, only E17K was observed.Mutations of PIK3CA/AKT1/PTEN genes are more likely to occur over 40 year-old patients.In this cohort, the PIK3CA V346G mutation (co-existent PIK3CA H1047L) and the PTEN V343fs mutation were not found in previous publications. Conclusion:In addition to the predominance of common loci, PIK3CA and PTEN gene mutations also have rare loci mutations in the breast cancer, warranting further analysis with an expanded sample size.

Objective:To investigate the abnormalities of mesenchymal-epithelial transition factor (MET) gene in early-stage lung adenocarcinomas and to provide genetic bases for related clinical studies.Methods:A total of 630 cases of formalin-fixed and paraffin-embedded lung adenocarcinoma specimens with ALK and EGFR double-negativities were collected at Peking Union Medical College Hospital, Beijing, China between July 2020 and April 2022. Forty-three stage Ⅰ-ⅢA tumors with MET exon 14 skipping mutation identified by reverse transcription droplet digital PCR (RT-ddPCR) were identified and then evaluated for MET amplification using fluorescence in situ hybridization (FISH). MET amplification was determined using the ratio of MET to chromosome 7 enumeration probe (CEP7) or the mean of MET gene copy number (GCN).Results:Among the 43 samples with MET exon 14 skipping mutation, MET amplification was detected in 9 cases (9/43, 20.93%), including 1 case of MET/CEP7 ≥2 and GCN ≥5 (1/9), 8 cases of GCN≥5 (8/9), as well as 10 cases with high level of CEP7 (7.00-9.72) which included 5 cases with MET amplification. There were no significant differences in clinicopathological features between the two subgroups of tumors which harbored MET exon 14 skipping mutation with MET amplification versus those without ( P>0.05). Conclusions:Co-occurrence of MET exon 14 skipping mutation and MET amplification or high level of CEP7 is frequently observed in early-stage lung adenocarcinomas. The most common pattern of MET gene amplification is GCN ≥5.

Objective:To investigate the variations and co-alteration of KRAS and HER-family genes in the patients with ampullary carcinoma.Methods:A total of 37 formalin-fixed paraffin-embedded primary ampullary carcinoma specimens, which were collected at Peking Union Medical College Hospital from April 2019 to October 2024 were analyzed for KRAS and HER-family gene mutations using next-generation sequencing (NGS). Immunohistochemistry (IHC) was performed for HER2 protein expression in HER2 mutation cases and fluorescence in situ hybridization (FISH) for further gene status in HER2 IHC 2+cases.Results:In our cohort (22 males, 15 females; 31-82 years old), KRAS gene mutations were detected in 51.4% (19/37) of cases, with G12D being the most frequent abnormality (7/19), followed by G12V (5/19) and Q61R (3/19). Other variants of KRAS gene included G12C, A146T, N116H, and Q61H (each 1/19). In this cohort, 27.0% (10/37) of cases harbored HER-family gene alterations with most frequently in HER2 (6/10) and HER3 genes (missense mutations mainly). Notably, 3 cases (8.1%, 3/37) with coexistence of KRAS and HER-family genes mutations were recognized in our series, including KRAS p.G12D/HER2 p.V842I/HER2 p.V777L (c.2329 G>T)/HER3 p.Asp581Asn, KRAS p.Q61R/HER4 p.D1018H and KRAS p.G12C/HER2 p.R678Q. Additionally, a mutation of HER3 p.V104L (c.310 G>C) was identified in our population. Moreover, 4 novel mutations including HER3 p.V296E, HER3 p.V920L (c.2758 G>T), HER3 p.Asp581Asn, and HER4 p.D1018H were detected. In 6 tumors with HER2 gene changes (16.2%, 6/37), 5 variants with the high proportion of HER2 p.S310Y (3/6) were revealed. A tumor (HER2 IHC 2+) with HER2 p.S310Y presented HER2 gene amplification confirmed by NGS and FISH, and another one (also HER2 IHC 2+) with HER2 p.L755S possessed HER2 gene amplification determined by FISH assay.Conclusion:In ampullary carcinoma, co-alteration of KRAS and HER-family genes is observed, and HER2 gene mutations account for more than half of HER-family gene abnormities, which may be accompanied by gene amplification.