Neuronal intranuclear inclusion disease（NIID）is a rare hereditary neurodegenerative disorder that can affect multiple systems. However，it is uncommon for urinary dysfunction to be the initial symptom. This article reports five cases. The five patients began to experience voiding dysfunction such as frequent urination，weak urination，and incomplete urination at the mean ages of 55.4（47 - 65）years old. Four months to twelve years after urinary onset，neurological symptoms such as headache，memory decline，transient loss of consciousness，and unsteady gait began to appear. Four of the five cases had a family history. Brain MRI revealed the “ribbon sign” or “crest sign” in all cases. Skin biopsy revealed eosinophilic inclusions in the cell nuclei，and NOTCH2NLC gene testing identified abnormal GGC mutations. Three of the five patients underwent cystostomy due to secondary hydronephrosis，while the other two received no special treatment. After a follow-up of 18 to 35 months since diagnosis，the patients who underwent cystostomy had normal renal function. Neurological symptoms in all five patients worsened to varying degrees.

Mannheimia varigena(M.varigena)is a significant pathogen causing bovine respiratory disease.Transferrin binding protein B(TbpB)is a lipoprotein directly exposed to the outer mem-brane of the cell,which is not only involved in the bacterial iron metabolism pathway,but also an important virulence factor.This study aims to lay the groundwork for developing novel subunit vaccines by conducting site-directed mutagenesis on M.varigena TbpB binding-related residues and evaluating their immunogenicity.Based on whole-genome sequencing of a bovine M.varigena i-solate,its iron uptake pathway was predicted.Key amino acid residues of M.varigena TbpB that play a role for binding to bovine transferrin(bTf)was identified by bioinformatics.We constructed two M.varigena TbpB mutants(Y205A and Y258A)and assessed their bTf binding activity and immunogenicity through dot blot assays and mouse immunization studies.Dot blot assays result showed that the Y258A mutation caused TbpB to lose its ability to bind to bTf.Mouse immuniza-tion studies showed that,compared to wild-type TbpB,the mutants of TbpB induced higher levels of specific antibodies.In challenge experiments,mice immunized with mutant TbpB exhibited high-er survival rates.These results demonstrate that site-directed mutagenesis can enhance the immu-nogenicity of TbpB.This study provides a novel approach for developing new subunit vaccines a-gainst M.varigena.

Pathogenic microorganisms are direct causative agents of zoonotic infectious diseases,po-sing severe threats to the livestock industry by inducing massive animal mortality,economic losses in livestock products,and significant risks to human health.The CRISPR/Cas system has been widely adopted in nucleic acid detection of pathogenic microorganisms due to its unique trans-cleavage activity.By leveraging the superior optical properties of nanomaterials,researchers have integrated them with CRISPR/Cas systems to develop numerous visual biosensors,which not only significantly enhance signal output but also substantially reduce detection time and cost.This re-view focuses on five nanomaterials-graphene oxide(GO),gold nanoparticles(AuNPs),MoS2 nanosheets,metal-organic frameworks(MOFs),and quantum dots(QDs)—that have been exten-sively integrated with CRISPR/Cas systems in recent years.We systematically summarize their distinct physical characteristics and specific applications in CRISPR/Cas-based pathogen detection,followed by a concise comparison of the advantages and limitations of different methodologies.Fi-nally,we discuss the prospects for nanomaterials in CRISPR/Cas detection systems,aiming to pro-vide a valuable reference for advancing molecular diagnostics of pathogenic microorganisms.

Neuronal intranuclear inclusion disease（NIID）is a rare hereditary neurodegenerative disorder that can affect multiple systems. However，it is uncommon for urinary dysfunction to be the initial symptom. This article reports five cases. The five patients began to experience voiding dysfunction such as frequent urination，weak urination，and incomplete urination at the mean ages of 55.4（47 - 65）years old. Four months to twelve years after urinary onset，neurological symptoms such as headache，memory decline，transient loss of consciousness，and unsteady gait began to appear. Four of the five cases had a family history. Brain MRI revealed the “ribbon sign” or “crest sign” in all cases. Skin biopsy revealed eosinophilic inclusions in the cell nuclei，and NOTCH2NLC gene testing identified abnormal GGC mutations. Three of the five patients underwent cystostomy due to secondary hydronephrosis，while the other two received no special treatment. After a follow-up of 18 to 35 months since diagnosis，the patients who underwent cystostomy had normal renal function. Neurological symptoms in all five patients worsened to varying degrees.

Pathogenic microorganisms are direct causative agents of zoonotic infectious diseases,po-sing severe threats to the livestock industry by inducing massive animal mortality,economic losses in livestock products,and significant risks to human health.The CRISPR/Cas system has been widely adopted in nucleic acid detection of pathogenic microorganisms due to its unique trans-cleavage activity.By leveraging the superior optical properties of nanomaterials,researchers have integrated them with CRISPR/Cas systems to develop numerous visual biosensors,which not only significantly enhance signal output but also substantially reduce detection time and cost.This re-view focuses on five nanomaterials-graphene oxide(GO),gold nanoparticles(AuNPs),MoS2 nanosheets,metal-organic frameworks(MOFs),and quantum dots(QDs)—that have been exten-sively integrated with CRISPR/Cas systems in recent years.We systematically summarize their distinct physical characteristics and specific applications in CRISPR/Cas-based pathogen detection,followed by a concise comparison of the advantages and limitations of different methodologies.Fi-nally,we discuss the prospects for nanomaterials in CRISPR/Cas detection systems,aiming to pro-vide a valuable reference for advancing molecular diagnostics of pathogenic microorganisms.

Mannheimia varigena(M.varigena)is a significant pathogen causing bovine respiratory disease.Transferrin binding protein B(TbpB)is a lipoprotein directly exposed to the outer mem-brane of the cell,which is not only involved in the bacterial iron metabolism pathway,but also an important virulence factor.This study aims to lay the groundwork for developing novel subunit vaccines by conducting site-directed mutagenesis on M.varigena TbpB binding-related residues and evaluating their immunogenicity.Based on whole-genome sequencing of a bovine M.varigena i-solate,its iron uptake pathway was predicted.Key amino acid residues of M.varigena TbpB that play a role for binding to bovine transferrin(bTf)was identified by bioinformatics.We constructed two M.varigena TbpB mutants(Y205A and Y258A)and assessed their bTf binding activity and immunogenicity through dot blot assays and mouse immunization studies.Dot blot assays result showed that the Y258A mutation caused TbpB to lose its ability to bind to bTf.Mouse immuniza-tion studies showed that,compared to wild-type TbpB,the mutants of TbpB induced higher levels of specific antibodies.In challenge experiments,mice immunized with mutant TbpB exhibited high-er survival rates.These results demonstrate that site-directed mutagenesis can enhance the immu-nogenicity of TbpB.This study provides a novel approach for developing new subunit vaccines a-gainst M.varigena.

To develop vaccines for the prevention and control of porcine circovirus 2d genotype(PCV2d)and pseudorabies virus(PRV),the PCV2d ORF2 gene was amplified by PCR,and cloned into the BamH Ⅰ site of PRV transfer plasmid pG vector harboring the enhanced green fluorescent protein(EGFP)gene.The resulting recombinant transfer plasmid pG-PCV2d-EGFP was transfect-ed into ST cells infected with the three gene deleted PRV variant strain gE-/g-/TK-PRV NY to generate a recombinant virus rPRV-PCV2d-EGFP+,and then the EGFP gene was knocked out to harvest the rPRV-PCV2d using gene-editing technology termed CRISPR/Cas9 system.The recom-binant virus rPRV-PCV2d had similar genetic stability to the parental PRV as indicated by PCR and one-step growth curve test,and the expression of PCV2d capsid(Cap)protein was validated by Western blot.In animal experiment,higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with the recombinant vi-rus rPRV-PCV2d compared to commercial PCV2 inactivated vaccine.rPRV-PCV2d significantly re-duced the PCV2d loads in tissues such as the heart,liver and spleen of mice following virulent PCV2d challenge.Moreover,rPRV-PCV2d elicits PRV-specific immune responses in mice and can prevent PRV virulent infection in mice,indicating the recombinant virus rPRV-PCV2d has strong immunogenicity.

Objective To evaluate the effect of Helicobacter pylori(H.pylori)infection on colorectal adenoma(CRA)recurrence after polypectomy and to study other potential prognosis factors associated with CRA recurrence.Methods This single-centered retrospective cohort study included 808 patients with CRA who underwent colonoscopy,polypectomy,and gastroscopy between January 2005 and October 2022.The patients were classified into three groups based on H.pylori infection status:persistently negative(group A,n=626),initially positive but turned negative(group B,n=141),and persistently positive(group C,n=41).The CRA recurrence and high-risk CRA or colorectal cancer(CRC)occurrence were assessed,and potential prognosis factors for recurrence were analyzed.Results During a median follow-up period of 1.6(1.1,2.4)years,the recurrence rate was 56.4%(456/808),including 124 cases(15.3%)of high-risk CRA/CRC[of which 5 cases(0.6%)were CRC]and 332 cases(41.1%)of low-risk CRA.The recurrence rates in the three groups were 55.4%(347/626),60.3%(85/141),and 58.5%(24/41),respectively,with no statistically significant difference(log-rank χ2=0.525,P=0.769).The high-risk CRA/CRC recurrence rates in the three groups were 14.9%(93/626),17.7%(25/141),and 14.6%(6/41),respectively,showing no significant intergroup differences(log-rank χ2=0.340,P=0.844).Multivariate analysis identified increasing age(HR=1.011,95%CI:1.002-1.021,P=0.020)and baseline high-risk CRA(HR=1.428,95%CI:1.183-1.724,P<0.001)as independent prognosis factors for CRA recurrence.Conclusions This study did not find a significant correlation between H.pylori infection and CRA recurrence after polypectomy.Increasing age and baseline high-risk CRA are prognosis factors for CRA recurrence.

Objective:To evaluate the safety and efficacy of combined inflatable mediastinoscopy with laparoscopy guided by the concept of “reduced field and port” during esophagectomy for esophageal cancer.Methods:This is a retrospective cohort study. The clinical data of 497 patients with esophageal squamous cell carcinoma who underwent minimally invasive esophagectomy at the Center of Minimally Invasive Thoracic Surgery, the Second Affiliated Hospital of Naval Medical University, between January 2017 and December 2024 were retrospectively analyzed. There were 416 male and 81 female patients, with an age of (68.3±8.0) years (range: 44 to 89 years). Patients were divided into the traditional video-assisted thoracoscopic surgery group (Group A, n=354) and the combined inflatable mediastinoscopy with laparoscopic surgery group(Group B, n=143) based on the surgical approach. Furthermore, Group B was subdivided into the multiport laparoscopic group (Group B1, n=81) and the single-incision laparoscopic surgery plus one port group (Group B2, n=62). Perioperative indicators and postoperative survival differences were compared between the groups. Inter-group comparisons were performed using the independent sample t-test, χ2 test, or Fisher′s exact probability test. Survival curves were plotted using the Kaplan-Meier method, and the Log-rank test was used to analyze the survival differences between groups. Results:Compared with Group A, Group B demonstrated a significantly shorter operative time ((181.8±11.4) minutes vs. (196.7±8.1)minutes, t=16.09, P<0.01), a lower incidence of postoperative pulmonary complications (8.4% (12/143) vs. 17.8% (63/354), χ2=6.27, P=0.012), lower perioperative mortality (0 vs. 3.1%(11/354), P=0.039), and a shorter postoperative hospital stay ((16.2±2.2)days vs. (18.9±4.1)days, t=8.56, P<0.01). There was no significant difference in the anastomotic leak rate, number of lymph nodes dissected, or intraoperative blood loss between the two groups (all P>0.05). Overall survival time and recurrence-free survival time showed no significant difference between the two groups (all P>0.05). Subgroup analysis revealed no significant differences in perioperative indicators or postoperative complication rates between Group B1 and Group B2. Conclusions:Compared with traditional thoracoscopic combined with laparoscopic surgery, inflatable mediastinoscopy offered advantages in terms of lower postoperative pulmonary complication rates, shorter operative time, reduced postoperative hospital stay, and lower perioperative mortality. The “reduced field and port” concept could further minimize surgical trauma during the transmediastinal approach for esophagectomy while ensuring surgical safety and efficacy.

To develop vaccines for the prevention and control of porcine circovirus 2d genotype(PCV2d)and pseudorabies virus(PRV),the PCV2d ORF2 gene was amplified by PCR,and cloned into the BamH Ⅰ site of PRV transfer plasmid pG vector harboring the enhanced green fluorescent protein(EGFP)gene.The resulting recombinant transfer plasmid pG-PCV2d-EGFP was transfect-ed into ST cells infected with the three gene deleted PRV variant strain gE-/g-/TK-PRV NY to generate a recombinant virus rPRV-PCV2d-EGFP+,and then the EGFP gene was knocked out to harvest the rPRV-PCV2d using gene-editing technology termed CRISPR/Cas9 system.The recom-binant virus rPRV-PCV2d had similar genetic stability to the parental PRV as indicated by PCR and one-step growth curve test,and the expression of PCV2d capsid(Cap)protein was validated by Western blot.In animal experiment,higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with the recombinant vi-rus rPRV-PCV2d compared to commercial PCV2 inactivated vaccine.rPRV-PCV2d significantly re-duced the PCV2d loads in tissues such as the heart,liver and spleen of mice following virulent PCV2d challenge.Moreover,rPRV-PCV2d elicits PRV-specific immune responses in mice and can prevent PRV virulent infection in mice,indicating the recombinant virus rPRV-PCV2d has strong immunogenicity.