GBA1 gene mutation is an important genetic risk factor for Parkinson’s disease (PD). This paper reports a case of a 43-year-old male PD patient carrying a rare heterozygous Thr408Met mutation in the GBA1 gene identified through whole-exome sequencing, leading to a diagnosis of GBA1-associated PD. The patient’s motor symptoms were primarily characterized by bradykinesia and rigidity, without significant cognitive decline. Treatment with low-dose levodopa combined with a dopamine agonist resulted in significant symptomatic improvement.

This study established an indirect ELISA method for antibody detection based on human rotavirus group A(RVA)VP6 protein,to provide technical support for RVA antibody detection.An ELISA method for detecting RVA antibodies was established through a checkerboard assay for optimization of the reaction conditions with VP6 protein of RVA as the diagnostic antigen.Serum samples from healthy children and patients infected with RVA were collected and detected with the ELISA method.The optimal work-ing conditions for ELISA involved coating the ELISA plate with VP6 protein at a concentration of 2 μg/mL and diluting the serum at a ratio of 1∶250.The critical value of negative and positive samples was 0.137.The ELISA method had good specificity,sensitivity,and repeatability,and showed a 95%consistency rate with the western blotting antibody detection method.We tested 370 serum samples collected from children,which showed an antibody positivity rate of 81.1%.We additionally tested the acute and recovery phase sera from 15 patients infected with RVA,and observed a significantly higher RVA antibody titer in the recovery phase serum than the acute infection phase serum.The ELISA antibody detection method showed good specificity,sensitivity,and reproducibility,and therefore can be used for RVA antibody detection and auxiliary diagnosis of RVA infection.

This study expressed the VP6 protein of human group A rotavirus in a prokaryotic expression system and prepared mouse polyclonal antibodies to this protein.The human rotavirus VP6 gene was amplified through RT-PCR from fecal samples infected with group A rotavirus and cloned into the pET-32a vector to construct the recombinant pET-32a-VP6 prokaryotic expression plas-mid.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).VP6 protein was expressed through induction with isopropyl-β-D-thiopyranoside(IPTG),purified with nickel nitrilotriacetic acid(Ni-NTA)affinity chromatography,and identified through SDS-PAGE and western blotting.Polyclonal antibodies to the VP6 protein were obtained by immunization of BALB/c mice with VP6 protein.Antibody titers were determined through indirect ELISA.The recombinant expression plasmid pET-32a-VP6 was con-structed,and VP6 protein was expressed primarily in inclusion bodies.Polyclonal antibodies to VP6 protein with a titer of 1∶32 000 were obtained by immunization of mice with VP6 protein purified by affinity chromatography.The antibodies specifically reacted with VP6 protein.The VP6 protein of human group A rotavirus was expressed and purified,and mouse polyclonal antibodies to this protein were prepared,thus providing a foundation for the preparation of a diagnostic antigen for human group A rotavirus and the establishment of serological detection methods.

AIM:To establish an optimal protocol for isolating and extracting mouse hepatocellular carcinoma H22 cell-detrived microparticles(H22-MPs),evaluate their stimulating effect on dendritic cells(DC),and investigate the cytotoxic capacity of CD8+T cells induced by H22-MPs on hepatocellular carcinoma cells.METHODS:The H22-MPs were extracted by high-speed differential centrifugation,and the morphology of H22-MPs was observed by transmis-sion electron microscopy.The particle size distribution of H22-MPs was detected by dynamic light scattering,and the ex-pression of vesicle surface markers was detected by Western blot.The protein components and pathways involved by MPs were analyzed by labeling free quantitative proteomics combining with bioinformatics GO and KEGG enrichment methods.The effect of MPs antigen on the expression of CD11c,a mature marker of DC,was detected by laser confocal microscopy.Next,CD8+T cells were sorted by magnetic beads,and the secretion of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and interleukin-2(IL-2)in the supernatant of CD8+T cells stimulated by DC loaded with H22-MPs was detected by ELISA.Lactate dehydrogenase(LDH)test was conducted to evaluate the specific killing effect of CD8+T cells induced by DC loaded with H22-MPs on hepatocellular carcinoma cells.RESULTS:The results showed that the morphology,size and vesicle markers of H22-MPs met the requirements.Most of the H22-MPs protein antigens were derived from its parent cell H22 and coordinated various signal transduction pathways in the cells.Subsequently,it was detected that H22-MPs increased the expression of CD11c on the surface of DC,and H22-MPs were mainly localized in the lysosomes of DC.In addition,DC loaded with H22-MPs stimulated CD8+T to release high levels of TNF-α,IFN-γ and IL-2,and it also pro-moted CD8+T to overexpress CD69,thus inducing CD8+T to killhepatocellular carcinoma cells specifically.CONCLU-SION:H22-MPs promote the maturation of DC,and the mature DC may induce CD8+T cells to play an anti-hepatocellu-lar carcinoma immune response by promoting the activation of CD8+T cells.

This study established an indirect ELISA method for antibody detection based on human rotavirus group A(RVA)VP6 protein,to provide technical support for RVA antibody detection.An ELISA method for detecting RVA antibodies was established through a checkerboard assay for optimization of the reaction conditions with VP6 protein of RVA as the diagnostic antigen.Serum samples from healthy children and patients infected with RVA were collected and detected with the ELISA method.The optimal work-ing conditions for ELISA involved coating the ELISA plate with VP6 protein at a concentration of 2 μg/mL and diluting the serum at a ratio of 1∶250.The critical value of negative and positive samples was 0.137.The ELISA method had good specificity,sensitivity,and repeatability,and showed a 95%consistency rate with the western blotting antibody detection method.We tested 370 serum samples collected from children,which showed an antibody positivity rate of 81.1%.We additionally tested the acute and recovery phase sera from 15 patients infected with RVA,and observed a significantly higher RVA antibody titer in the recovery phase serum than the acute infection phase serum.The ELISA antibody detection method showed good specificity,sensitivity,and reproducibility,and therefore can be used for RVA antibody detection and auxiliary diagnosis of RVA infection.

This study expressed the VP6 protein of human group A rotavirus in a prokaryotic expression system and prepared mouse polyclonal antibodies to this protein.The human rotavirus VP6 gene was amplified through RT-PCR from fecal samples infected with group A rotavirus and cloned into the pET-32a vector to construct the recombinant pET-32a-VP6 prokaryotic expression plas-mid.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).VP6 protein was expressed through induction with isopropyl-β-D-thiopyranoside(IPTG),purified with nickel nitrilotriacetic acid(Ni-NTA)affinity chromatography,and identified through SDS-PAGE and western blotting.Polyclonal antibodies to the VP6 protein were obtained by immunization of BALB/c mice with VP6 protein.Antibody titers were determined through indirect ELISA.The recombinant expression plasmid pET-32a-VP6 was con-structed,and VP6 protein was expressed primarily in inclusion bodies.Polyclonal antibodies to VP6 protein with a titer of 1∶32 000 were obtained by immunization of mice with VP6 protein purified by affinity chromatography.The antibodies specifically reacted with VP6 protein.The VP6 protein of human group A rotavirus was expressed and purified,and mouse polyclonal antibodies to this protein were prepared,thus providing a foundation for the preparation of a diagnostic antigen for human group A rotavirus and the establishment of serological detection methods.

AIM:To establish an optimal protocol for isolating and extracting mouse hepatocellular carcinoma H22 cell-detrived microparticles(H22-MPs),evaluate their stimulating effect on dendritic cells(DC),and investigate the cytotoxic capacity of CD8+T cells induced by H22-MPs on hepatocellular carcinoma cells.METHODS:The H22-MPs were extracted by high-speed differential centrifugation,and the morphology of H22-MPs was observed by transmis-sion electron microscopy.The particle size distribution of H22-MPs was detected by dynamic light scattering,and the ex-pression of vesicle surface markers was detected by Western blot.The protein components and pathways involved by MPs were analyzed by labeling free quantitative proteomics combining with bioinformatics GO and KEGG enrichment methods.The effect of MPs antigen on the expression of CD11c,a mature marker of DC,was detected by laser confocal microscopy.Next,CD8+T cells were sorted by magnetic beads,and the secretion of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and interleukin-2(IL-2)in the supernatant of CD8+T cells stimulated by DC loaded with H22-MPs was detected by ELISA.Lactate dehydrogenase(LDH)test was conducted to evaluate the specific killing effect of CD8+T cells induced by DC loaded with H22-MPs on hepatocellular carcinoma cells.RESULTS:The results showed that the morphology,size and vesicle markers of H22-MPs met the requirements.Most of the H22-MPs protein antigens were derived from its parent cell H22 and coordinated various signal transduction pathways in the cells.Subsequently,it was detected that H22-MPs increased the expression of CD11c on the surface of DC,and H22-MPs were mainly localized in the lysosomes of DC.In addition,DC loaded with H22-MPs stimulated CD8+T to release high levels of TNF-α,IFN-γ and IL-2,and it also pro-moted CD8+T to overexpress CD69,thus inducing CD8+T to killhepatocellular carcinoma cells specifically.CONCLU-SION:H22-MPs promote the maturation of DC,and the mature DC may induce CD8+T cells to play an anti-hepatocellu-lar carcinoma immune response by promoting the activation of CD8+T cells.

Background and Aims:Laparoscopic anatomical liver segmentectomy has been widely applied in the surgical treatment of hepatic tumors due to its safety,feasibility,and effectiveness.The combination of indocyanine green(ICG)fluorescence-guided positive staining and intraoperative laparoscopic ultrasound has become an important technique for precision liver resection,particularly in accurately delineating hepatic segment/subsegment boundaries and achieving negative surgical margins.This study reports a case of anatomical resection of the right posterior segment and the dorsal subsegment of the right anterior segment of the liver,guided by laparoscopic ultrasound combined with ICG positive staining,to evaluate its clinical feasibility and outcomes.Methods:A retrospective analysis was conducted on an elderly female patient with a hepatic space-occupying lesion who underwent laparoscopic anatomical resection of the right posterior segment and right anterior dorsal subsegment using intraoperative ultrasound combined with ICG fluorescence-guided positive staining.Results:Preoperative three-dimensional reconstruction revealed that the tumor was located in the right posterior segment and right anterior dorsal subsegment.Intraoperatively,under laparoscopic ultrasound guidance,the anterior-ventral branch of the right portal vein was punctured and injected with ICG to achieve precise staining of the right anterior-ventral subsegment.The resection was performed along the fluorescent boundary,enabling accurate anatomical removal of the targeted liver segments.Intraoperative blood loss was approximately 100 mL without transfusion.Pathology confirmed a moderately differentiated small-duct type intrahepatic cholangiocarcinoma with negative margins(R0 resection).The patient recovered well and was discharged on postoperative day 19.Follow-up CT at 6 months showed no evidence of recurrence.Conclusion:During anatomical resection of the right posterior segment and right anterior dorsal subsegment of the liver,laparoscopic ultrasound combined with ICG fluorescence-guided positive staining can accurately define segmental boundaries,enhance surgical safety,and ensure complete tumor resection,thus offering significant value in achieving R0 resection.

Objective:To explore the value of ultrasound imaging characteristics combined with clinical indicators in assessing the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC).Methods:A retrospective analysis was conducted for patients who underwent pancreatic contrast-enhanced ultrasound (CEUS) from September 2017 to October 2023 at Peking Union Medical College Hospital and were diagnosed with PDAC based on pathological findings. Various parameters were recorded, including CA19-9 levels, tumor size, location, morphologic features, echogenicity, presence of internal cystic components, dilatation of the main pancreatic duct, peripheral vascular invasion, CEUS characteristics, presence or absence of liver metastasis, and treatment methods. In April 2024, patient survival information was obtained through telephone follow-up or review of medical records. Based on the results of the cox regression model analysis, a nomogram model of the risk of death was developed. The receiver operating characteristic (ROC) curves were applied to evaluate the predictive efficacy of the model. The calibration curves were plotted to evaluate the accuracy of the model, and clinical decision curves were used to evaluate the clinical benefit of the model.Results:This study included a total of 207 patients with PDAC. As of April 2024, 71 patients were alive and 136 died, with a median survival time of 14 months (95% CI: 12 -17). Multivariate analysis confirmed that the elevated CA19-9 ( HR=1.689, 95% CI: 1.102-2.588), tumor size >4 cm ( HR=1.641, 95% CI: 1.159-2.322), taller-than-wide shapes ( HR=1.450, 95% CI: 1.019-2.065), incomplete hypo-enhancement ( HR=1.618, 95% CI: 1.100-2.380), and liver metastasis ( HR=1.687, 95% CI: 1.175-2.423) were independent risk factors for survival in patients with PDAC. A nomogram model was further constructed for 6-month, 12-month and 3-year survival of patients with PDAC. The areas under the ROC curve were 0.679, 0.705 and 0.815, respectively. The calibration curves suggested that the model was more accurate, and the clinical decision curves showed that the model had a better clinical benefit. Conclusion:The combined use of ultrasound imaging characteristics and clinical indicators could effectively predict the prognosis of PDAC patients. Specifically, tumor size >4 cm, taller-than-wide shapes, incomplete hypo-enhancement, elevated CA19-9, and the presence of liver metastasis are correlated with poorer survival outcomes. The nomogram model constructed on the basis of these factors can be used to assess the survival of patients with PDAC.

Objective:To evaluate the clinical efficacy of a novel surgical approach of debridement, antibiotics, and implant retention (DAIR) with flap transfer, for treating chronic implant infections in bone tumor patients.Methods:A retrospective review was conducted on nine consecutive patients [6 males, 3 females; median age 35(27, 51) years, range 9-71] who underwent a modified procedure of DAIR plus flap transfer between November 2022 and January 2024. The cohort included six cases of chronic periprosthetic joint infection and three cases of chronic plate and screw infection. Tumor diagnoses included seven primary malignant tumors (osteosarcoma=5, undifferentiated pleomorphic sarcoma of bone=1, synovial sarcoma=1) and two bone metastasis of renal cell carcinoma. The procedure involved wide, radical debridement, meticulous removal of biofilm from implants and surrounding soft tissue, followed by the transfer of a well vascularized musculocutaneous flap to fully envelope the contaminated interface. Pre-operative clinicopathological data, surgical details, postoperative complications and infection recurrence were analyzed.Results:The median interval between initial implantation and debridement was 10.0(3.3, 14.8) months. Median follow-up after debridement was 15.9(15.4, 18.2) months. All nine surgeries were completed as planned: six musculocutaneous flaps, two fasciocutaneous flaps and one muscle-only flap. Implants were preserved in six patients; two required subsequent removal for recurrent infection, and one patient later underwent amputation for tumor recurrence. Infection-free implant survival at 3, 6 and 12 months was 88.9%, 87.5% and 87.5%, respectively. Major complications included one donor-site hematoma, one donor-site sensory deficit and one wound healing delay. All the complications were well management. Both reinfections occurred in proximal tibial prostheses, likely due to limited flap coverage options and local anatomical constraints.Conclusion:Although reinfections happened in two cases DAIR with flap transfer provides promising short-term infection control in patients with chronic implant-associated infections following bone tumor surgery.