AIM:To investigate the potential impact of mitoNEET[mitochondrial protein containing Asn-Glu-Glu-Thr(NEET)sequence]on mitochondrial metabolism in brown adipocytes,and to elucidate its underlying mecha-nism.METHODS:An in vitro model of primary mouse brown adipocytes was established.Western blot were utilized to detect relevant proteins,and iron ion and ATP content was measured using kits.Mitochondrial membrane potential and re-active oxygen species(ROS)were assessed by fluorescence microscopy and flow cytometry.RESULTS:The expression of the ferroptosis-related protein ACSL4 increased by 1.13 times in ferroptosis inducer erastin treatment group,whereas the expression of SLC7A11 and GPX4 decreased by 27.33%and 25.33%,respectively,compared with control group(P<0.05).The expression of Nrf1,PGC-1α,MFN2 and UCP1 proteins,related to mitochondrial energy metabolism,de-creased by 20.98%,15.17%,15.03%and 34.22%,respectively(P<0.05).Additionally,the mitoNEET protein con-tent was significantly reduced by 42.14%(P<0.05).The iron ion content in erastin group was substantially increased by 1.80 times compared with control group.However,a notable decrease in ATP content of 14.95%was seen(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated a significant decrease in the mitochon-drial membrane potential of brown adipocytes in erastin group,with reductions of 52.18%and 61.31%(P<0.05),re-spectively.A substantial increase in mitochondrial ROS content of 80.97%was seen(P<0.05).Western blot analysis of overexpressed stable strains revealed a significant elevation in mitoNEET levels in brown adipocytes following lentivirus transfection,exhibiting an increase of 11.19 times(P<0.05),thus confirming successful transfection.The LV-mitoNEET group exhibited a significant decrease of 37.95%in the expression of ferroptosis-related protein ACSL4 in brown adipose cells compared with control group.Additionally,there was a notable increase of 77.82%and 66.3%in the expression of SLC7A11 and GPX4,respectively(P<0.05).Up-regulation was observed in the expression of MFN2(79.06%),PGC-1α(72.89%),Nrf1(40.14%),and UCP1(31.68%)(P<0.05).The test results demonstrated that the LV-mitoNEET group experienced a reduction of 43.5%in iron ion content compared with control group while exhibiting an increase of 33.5%in ATP content(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated that mitoNEET overexpression led to a significant increase in the mitochondrial membrane potential of erastin-induced brown adipocytes,with increments of 17.61%and 96.05%,respectively.Additionally,mitoNEET overexpression effec-tively reduced the production of mitochondrial ROS by 24.48%(P<0.05).CONCLUSION:Our findings suggest that mitoNEET overexpression can effectively inhibit the disruption of mitochondrial energy metabolism caused by ferroptosis-induced death of brown adipocytes.

Intersphincteric resection (ISR) is an advanced sphincter-preserving surgery for low rectal cancer. Accumulating evidences from clinical studies indicate that ISR can spare some pati-ents with low rectal cancer from the distress of anal amputation while ensuring oncological efficacy. However, due to the necessity of removing part or all of the internal sphincter during rectal resection and the extremely low anastomosis level, a subset of patients may experience low anterior resection syndrome (LARS) after surgery. LARS is characterized by symptoms such as anal incontinence, increased bowel frequency, urgency, incomplete evacuation, and obstructed defecation. Based on relevant literature and team practice, the authors provide an overview of the diagnosis and treat-ment progress of LARS following ISR.

Alveolar echinococcosis, caused by Echinococcus multilocularis infection, is a highly deadly zoonotic parasitic disease. As a benzimidazole compound, albendazole has a strong and broad-spectrum anti-parasitic action. For alveolar echinococcosis patients that are unwilling to receive surgical treatment, lose the timing for surgery, or are intolerant to surgery due to poor physical status, administration of albendazole may delay disease progression. Recently, a large number of advances have been achieved in experimental studies on alveolar echinococcosis. In order to increase the understanding of the therapeutic efficacy of albendazole for alveolar echinococcosis, this review summarizes the advances in albendazole treatment for alveolar echinococcosis, so as to provide insights into the clinical treatment of alveolar echinococcosis with albendazole.

Objective To investigate the effect of exosomes derived from Echinococcus multilocularis on macrophage polarization after treatment for different durations and concentrations. Methods A total of 60 BALB/c mice were used for modeling, among which 4 mice were selected to observe the growth of abdominal lesions on 7.0T MRI. The mice for modeling were dissected, and the protoscoleces was taken from the abdominal lesion and cultured in vitro ; ultracentrifugation was used to extract the exosomes from the supernatant, and transmission electron microscopy and Western blotting were used for the characterization of exosomes. The macrophages without exosome treatment were established as control group, and the macrophages co-cultured with different concentrations of exosomes derived from Echinococcus multilocularis were established as experimental group (10 μg/mL group and 50 μg/mL group) and were cultured for 48 and 72 hours. The morphological changes of macrophages were observed under a microscope, and flow cytometry and ELISA were used to observe polarization state. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results The results of 7.0T MRI showed the formation of diffuse lesions with different sizes in the abdominal cavity of mice, and the exosomes derived from Echinococcus multilocularis were approximately 100 nm in diameter and were cup-shaped or saucer-shaped, with the positive expression of the surface markers CD9, TSG101, and CD63. After co-culture, most of the cells in the experimental group were elongated with an irregular and polygonal shape. Flow cytometry showed that after 48 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.53%±0.06%, 90.27%±0.21%, and 2.40%±0.20%, respectively; compared with the control group, except that the 10 μg/mL exosome group had a significant reduction in the positive rate of CD369 (0.80%±0.00%) ( P < 0.05), all the other groups had a significant increase in the positive rates of CD16/32, CD206, and CD369 (all P < 0.000 1); after 72 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.67%±0.06%, 85.47%±0.55%, and 6.60%±0.20%, respectively, and compared with the control group, the experimental group had significant increases in the positive rates of CD16/32, CD206, and CD369 (all P < 0.05). ELISA showed that after 48 hours of co-culture, the levels of IL-6 and TNFα in the control group were 58.53±15.52 pg/mL and 320.70±5.30 pg/mL, respectively, and when the exosome concentration was 50 μg/mL, the level of IL-6 in the experimental group was 98.81±15.55 pg/mL, which was higher than that in the control group ( P < 0.05); after 72 hours of co-culture, the levels of IL-6 and TNFα in the control group were 76.22±9.68 pg/mL and 323.90±87.37 pg/mL, respectively, and when the exosome concentration was 10 μg/mL, the level of TNFα was 164.20±14.17 pg/mL, which was significantly lower than that in the control group ( P < 0.05); when the exosome concentration was 50 μg/mL, the level of IL-6 was 99.52±8.35 pg/mL, which was significantly higher than that in the control group ( P < 0.05). Conclusion Exosomes derived from Echinococcus multilocularis can regulate macrophage polarization and induce M2-like polarization of macrophages after co-culture at a concentration of 10 μg /mL for 72 hours, and further studies are needed to clarify the specific method.

Purpose To investigate the diagnostic criteria and prognostic significance of squamous cell components in pa-pillary thyroid carcinoma(PTC).Methods Twenty-three cases of PTC containing squamous cell components were collect-ed,and pathological sections were reviewed.p40,CK5/6,Ki-67,BRAF V600E,p53,PD-L1(22C3),PAX8,and CD10,markers related to diagnosis,prognosis,and treatment,were de-tected by immunohistochemistry.Histological characteristics and immunophenotype of squamous cell components were comprehen-sively evaluated.The squamous cell components were classified histologically.The relationship between squamous cell classifica-tion and clinicopathological parameters,and their prognostic im-pact were analyzed.Results The squamous cell components were divided into squamous differentiation(19 cases)and ana-plastic carcinoma(4 cases).In the latter,the squamous cell components were diffusely distributed in sheets;the cell atypia was moderate to severe;and the Ki-67 index was at least 30%.High expression of p53 was found only in anaplastic carcinoma.Compared with squamous differentiation,anaplastic carcinoma had a larger diameter,higher BRAF V600E positive rate,high PD-L1 combined positive score,and extremely worse progression-free survival and overall survival.Squamous differentiation had inconspicuous impact on survival,and only one case relapsed af-ter secondary surgery.Conclusion The cell atypia,distribu-tion,maximum diameter,Ki-67 and p53 expression of squamous cell components are helpful in differentiating squamous compo-nents from anaplastic carcinoma.Positive BRAF V600E and PD-L1 expression suggest the feasibility of targeted therapy and im-munotherapy for anaplastic carcinoma.

The arising of biliary and pancreatic ampulla diseases is almost subtle, parti-cularly the symptoms are not readily apparent in the early stages, and patient condition become worse when jaundice and other symptoms manifest. This region′s imaging evaluation is highly intricate. There are significant similarities in the clinical symptoms of biliary and pancreatic ampulla disorders due to their similar imaging findings, complicated architecture, and close proximity to neighboring tissue structures, which make qualitative diagnosis difficult. In order to assist doctors in making treatment choices, the authors provide an overview of the application of endoscopic ultra-sound in the diagnosis of biliary and pancreatic ampulla diseases.

Objective:To investigate the clinical value of three-dimensional (3D) visualization technology in the precise drainage through endoscopic retrograde biliary drainage (ERBD) for hilar cholangiocarcinoma.Methods:Clinical data of 42 patients with highly suspected hilar cholangiocarcinoma who underwent ERBD in Qinghai University Affiliated Hospital from September 2019 to August 2022 were retrospectively collected. Twenty patients underwent 3D biliary tract reconstruction before surgery (the reconstruction group) and 22 others did not undergo 3D biliary tract reconstruction before surgery (the non-reconstruction group). The surgery time, X-ray exposure time, the technical success rate, the clinical success rate, incidence of postoperative complications, recent and short-term endoscopic retrograde cholangiopancreatography (ERCP) reintervention rate of the two groups were compared.Results:There was no significant difference in preoperative baseline data between the two groups ( P>0.05). ERBD was conducted successfully in all 42 patients. The operation time in the reconstruction group [35.00 (25.00, 57.50) min] was significantly shorter than that in the non-reconstruction group [60.00 (33.75, 60.00) min] with significant difference ( Z=-2.251, P=0.024). There was no significant difference in the X-ray exposure time between the two groups [10.00 (5.00, 12.00) min VS 10.55 (9.50, 17.50) min, Z=-1.552, P=0.121]. The technical success rates of both groups were 100.0%, and the clinical success rate of the reconstruction group was higher than that of the non-reconstruction group [70.0% (14/20) VS 31.8% (7/22)] with significant difference ( χ 2=6.109, P=0.013). There was no significant difference in the incidence of postoperative complications between the two groups [20.0% (4/20) VS 22.7% (5/22), χ 2=0.141, P=0.708]. All patients were followed up for 6 months after the procedure. The median survival time was 3.91 months in the reconstruction group and 2.78 months in the non-reconstruction group. There was no ERCP intervention in the reconstruction group within 2 weeks after the procedure, while 4 cases (18.2%) in the non-reconstruction group received 6 ERCP interventions due to cholangitis and postoperative pancreatitis. Within 2 weeks to 3 months, 2 patients (10.0%) in the reconstruction group received 4 ERCP interventions for cholangitis, and 2 patients (9.1%) in the non-reconstruction group received 3 ERCP interventions for cholangitis. There was no significant difference in recent ( χ 2=2.183, P=0.140) or short-term ( χ 2=0.000, P=1.000) ERCP reintervention rate between the reconstruction group and the non-reconstruction group. Conclusion:3D visualization biliary duct reconstruction technology can measure the volume of liver drainage for hilar cholangiocarcinoma, shorten the operation time and improve the clinical success rate through precise preoperative planning, which is worth of promotion.

Objective:To study the virulence-related functions and the pathogenic molecular mechanism of the transcription factor OmpR in Vibrio vulnificus. Methods:The Vibrio vulnificus ompR mutant (Δ ompR) strain was constructed using a markerless gene deletion system. Changes in the tolerance to osmotic pressure, swarming mobility and biofilm synthesis were detected by growth curve, swimming assay and crystal violet assay, respectively. Colony counting methods were used to analyze the cytoadherence of the Δ ompR strain. The cytotoxicity of the Δ ompR strain was measured by lactate dehydrogenase (LDH) releasing test. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes including ompN, ompU, ompA and hcp2 at mRNA level in the Δ ompR. Results:The Δ ompR strain was constructed successfully. Compared with the wild-type strain, the mutant strain showed decreased tolerance to high osmotic pressure, suppressed capability of biofilm formation and reduced cytoadherence and cytotoxicity, whereas no significant difference in motility was detected. The expression of ompN gene at mRNA level in the Δ ompR strain was down-regulated ( P<0.05), while the expression of other target genes showed no significant changes. Conclusions:The transcription factor OmpR regulated the tolerance to high osmotic pressure, biofilm formation, cytoadherence and cytotoxicity in Vibrio vulnificus.

Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.

Colorectal cancer is a common tumor of the digestive system with a high degree of malignancy. Most patients are in the advanced stages of the disease when they develop symptoms. Although the detection rate of traditional screening methods is improving with advances in imaging and the popularity of colonoscopy, the diagnosis of early, asymptomatic colon cancer is still unsatisfactory. Therefore, it is particularly important to further study the occurrence and development mechanism of colorectal cancer and obtain new ideas for the diagnosis and treatment.? In recent years, with the deepening of epigenetics research, the role of DNA methylation in the pathogenesis of colorectal cancer has gradually attracted attention. This paper will review the research progress of DNA methylation in colorectal cancer′s diagnosis and treatment.