Objective:To discuss the effect of berberine hydrochloride(BBR)on autophagy in the HeLa cells infected with herpes simplex virus type 1(HSV-1),and to clarify its related mechanism.Methods:The HeLa cells at logarithmic growth phase were added with 0,10,20,40,80,120,and 160 μmol·L-1 BBR,respectively,and cultured for 24 h.In addition,the HeLa cells were divided into HSV-1 group(the cells were infected with HSV-1),L-BBR group(were infected with HSV-1 and then treated with 20 μmol·L-1 BBR for 24 h),M-BBR group(were infected with HSV-1 and then treated with 40 μmol·L-1 BBR for 24 h),H-BBR group(were infected with HSV-1 and then treated with 80 μmol·L-1 BBR for 24 h),and 740 Y-P group(were infected with HSV-1 and then treated with 80 μmol·L-1 BBR and 10 μmol·L-1 740 Y-P for 24 h)for viral infection and corresponding drug treatment.MTT method was used to detect the activities of microtubule-associated protein 1 light chain 3(LC3)the HeLa cells after treated with different concentrations of BBR;plaque reduction assay was used to detect the proliferation ability of HSV-1 in the HeLa cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of virus replication-related genes in the HeLa cells in various groups;immunofluorescence method was used to detect the formation of autophagosomes in the HeLa cells in various groups;flow cytometry was used to detect the apoptotic rate of the HeLa cells in various groups;Western blotting method was used to detect the expression levels of autophagy and the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway pathway-related proteins in the HeLa cells in various groups.Results:The MTT method results showed that compared with 0 μmol·L-1 BBR group,the activities of the HeLa cells in 120 and 160 μmol·L-1 BBR groups were significantly decreased(P<0.05),which had certain toxicity to the cells;20,40,and 80 μmol·L-1 BBR were selected for subsequent experiments.The plaque reduction assay results showed that compared with HSV-1 group,the plaque forming units(PFUs)in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the PFUs in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).The RT-qPCR results showed that compared with HSV-1 group,the mRNA expression levels of infected cell protein 0(ICP0),infected cell protein 22(ICP22),infected cell protein 8(ICP8),thymidine kinase(TK),glycoprotein B(gB),and glycoprotein D(gD)in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the expression levels of ICP0,ICP22,ICP8,TK,gB,and gD mRNA in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).The immunofluorescence method results showed that compared with HSV-1 group,the number of LC3 autophagosomes formed in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group was increased;compared with H-BBR group,the number of LC3 autophagosomes formed in the HeLa cells in 740 Y-P group was decreased.The flow cytometry results showed that compared with HSV-1 group,the apoptotic rates of the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the apoptotic rate of the HeLa cells in 740 Y-P group was significantly decreased(P<0.05).The Western blotting method results showed that compared with HSV-1 group,the expression levels of Beclin-1 and B-cell lymphoma 2(Bcl-2)/adenovirus E1B 19kDa protein interacting protein protein(BNIP)2 proteins and the ratios of LC3-Ⅱ/LC3-Ⅰ in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the expression levels of Beclin-1 and BNIP proteins and the LC3-Ⅱ/LC3-Ⅰ ratio in the HeLa cells in 740 Y-P group were significantly decreased(P<0.05);compared with HSV-1 group,the expression levels of cysteine-containing aspartate protein hydrolase 1(Caspase-1),cysteine-containing aspartate protein hydrolase 3(Caspase-3),and Bcl-2 associated X protein(Bax)proteins in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05),and the expression level of Bcl-2 protein was significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the expression levels of Caspase-1,Caspase-3,and Bax proteins in the HeLa cells in 740 Y-P group were significantly decreased(P<0.05),and the expression level of Bcl-2 protein was significantly increased(P<0.05);compared with HSV-1 group,the ratios of phosphorylated PI3K(p-PI3K)/PI3K,phosphorylated AKT(p-AKT)/AKT,and phosphorylated mTOR(p-mTOR)/mTOR in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).Conclusion:BBR can promote the autophagy process in the HeLa cells infected with HSV-1 by regulating the PI3K/AKT/mTOR pathway,induce autophagy-dependent apoptosis,and significantly inhibit the virus replication.

Objective:To explore the surgical intervention strategy for metastatic cervical lymph nodes surrounding the carotid artery in head and neck squamous cell carcinoma.Methods:A total of 62 patients with advanced head and neck tumors and carotid wrap by disease treated in Department of Otorhinolaryngology and Head and Neck Surgery, the Affiliated BenQ Hospital of Nanjing Medical University between June 2019 and December 2023 were reviewed, of whom 9 patients presented with metastatic squamous cell carcinoma in cervical lymph nodes of unknown primary or with no recurrence of primary lesion and all the 9 patients were males, aged from 48 to 79 years old, with≤level 2 of Eastern Cooperative Oncology Group-Performance Status (ECOG-PS). Radiographically common carotid artery (CCA) and/or internal carotid artery (ICA) were surrounded by≥270° with tumor. All the 9 patients received implantation of covered stent in carotid artery and radical resection of metastatic cervical lymph nodes. The success rate, complications, surgery-related complications, local recurrence rate, quality of life (QOL) and overall survival (OS) were analyzed. The QOL of patients was compared by paired rank sum test, and P<0.05 indicated statistically significant difference. The OS was analyzed by Kaplan-Meier. Results:The success rate of stent implantation was 100%, with no implantation-related complications. R0 resection was performed in 8 cases and R1 resection in 1 case. The QOL of patients after surgery was improved, and the improvements in "pain", "mood" and "anxiety" were statistically significant( Z values were -2.236, -2.460 and -2.200, respectively, and all P values were<0.05). Follow-up was 1-18 months, with a median of 7 months, and 1 case was lost to follow-up. Local recurrence occurred in 3 patients with an incidence of 37.5% (3/8). OS was 59.9% at 12 months after surgery. Conclusion:Implantation of covered stent in carotid artery combined with radical resection is an effective method for the treatment of cervical lymph node metastasis.

Objective:To analyze the clinical characteristics, muscle imaging and pathological features of patients with anti-signal recognition particle positive immune-mediated necrotizing myopathy (SRP-IMNM).Methods:Nine patients with SRP-IMNM were collected in the Neuromuscular Disease Center of Jiaozuo People′s Hospital from May 2018 to May 2023, who were confirmed by skeletal muscle pathology and myositis-specific autoantibodies detection, and their clinical manifestations, muscle imaging and muscle pathology characteristics were systematically summarized.Results:Among the 9 patients with SRP-IMNM, there were 7 females and 2 males. The age of onset ranged from 18 to 59 years. All the patients presented proximal muscle weakness. Seven patients experienced neck weakness, and dysphagia was present in 5 patients. Laboratory examinations showed elevated serum creatine kinase levels in all 9 patients (1 866-6 725 U/L). Eight patients were combined with other antibodies positivity, except for anti-SRP antibody. Among them, 7 patients were combined with anti-Ro-52 antibody positivity, 4 patients combined with anti-Ro-52 antibody positivity alone, and 3 patients combined with 3 or more positive antibodies simultaneously. Those patients who presented with interstitial lung disease and cardiac involvement were all combined with other antibodies positivity. Seven patients completed thigh muscle magnetic resonance imaging (MRI), which showed diffuse skeletal muscle oedema, partial muscle atrophy and fatty replacement, primarily affecting the posterior thigh muscle group. Two patients underwent shank muscle MRI. The soleus involvement was evident, while the tibialis anterior muscle and gastrocnemius muscles were involved in 1 patient. All 9 patients showed varying degrees of scattered muscle fiber necrosis and regeneration on muscle biopsies. In 1 patient, a small amount of inflammatory cell infiltration was observed. Pipestem capillaries were observed in 4 patients. Immunohistochemical staining revealed a small number of CD68-positive lymphocytes in 8 patients. Additionally, 5 patients showed upregulation of major histocompatibility complex Ⅰ expression on the muscle fiber membrane, while 6 patients showed deposition of membrane attack complex (C5b-9) on non-necrotic muscle fibers and capillaries. P62 staining showed homogeneous fine-granular in sarcoplasm in 6 patients.Conclusions:In addition to proximal muscle weakness, patients with SRP-IMNM often experience neck weakness and dysphagia. Those with multiple antibodies are more likely to develop interstitial lung disease and cardiac involvement. SRP-IMNM patients have diffuse oedema in the affected muscles, and the posterior thigh muscles are more prone to atrophy and fatty tissue formation. C5b-9 deposition and pipestem capillaries are significant pathological features of SRP-IMNM, which provide additional evidence for clinical diagnosis.

The liver reserve function refers to the compensatory ability to maintain liver function after damage, providing implication for the resection of hepatic malignant tumor. Hepatobiliary scintigraphy imaging can provide quantitative evaluation of liver blood perfusion, and has advantages on the evaluation of liver reserve function and the prediction of postoperative complications. 99Tc m-galactosyl serum albumin (GSA) and 99Tc m-mebrofenin are commonly used imaging agents for hepatobiliary scintigraphy imaging assessment of liver reserve function. This article reviews the application and progress of hepatobiliary scintigraphy in liver reserve function assessment.

Objective:To summarize the characteristics of clinical, muscle pathology and gene mutation of late-onset reducing body myopathy caused by FHL1 gene mutation, in order to improve clinicians′ understanding of this disorder. Methods:The clinical, muscle pathology and muscle magnetic resonance imaging data of the proband from a family diagnosed as reducing body myopathy in Jiaozuo People′s Hospital in December 2021 were collected. Genetic tests and pedigree verification were conducted on the proband and her son.Results:The proband was a 59-year-old female with progressive, asymmetrical limb weakness and muscular atrophy. Her mother, sister and brother had similar symptoms. Electromyography showed myogenic and neurogenic damage. Muscle magnetic resonance imaging indicated that the lesion mainly involved the posterior muscles of the thigh and calf, as well as the gluteus maximus. The muscle pathology showed eosinophilic granular inclusion bodies and rimmed vacuoles in the muscle fibers of the lesion. The structure of myofibrils was disordered and abnormal protein deposition was observed. The gene sequencing showed the FHL1 gene p.C150S heterozygous variation. Conclusions:Late-onset reducing body myopathy is characterized by progressive asymmetric proximal limb muscle weakness, partially involving distal limb muscles and gluteus maximus. Muscle pathology shows the characteristic pathological changes of many kinds of myofibrillar myopathies. FHL1 gene mutation is an important basis for diagnosis.

Objective:To summarize the clinical, imaging, muscle pathological and gene mutational features of patients with late-onset mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS).Methods:Three patients with late-onset MELAS, admitted to Department of Neurology, Jiaozuo People's Hospital Affiliated of Xinxiang Medical University from January 1997 to December 2021 were chosen; all patients were screened for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) mutations by second-generation gene sequencing. The clinical, imaging, muscle pathological and gene mutational features of patients with late-onset MELAS were analyzed retrospectively.Results:The main clinical manifestations of these late-onset MELAS patients included stroke-like attacks, headache, hearing and vision loss, cognitive decline and mental disorder. The muscle tension and muscle strength of both upper extremities in these 3 patients were normal. Increased muscle tension and active tendon reflexes, and positive pathological signs in both lower extremities were noted in 2 patients. Head MRI showed abnormal long T1 and long T2 signals in temporal occipital parietal cortex and subcortex in 3 patients, and CT showed calcification in bilateral globus pallidus in 1 patient. Ragged red fibers (RRF) and ragged blue fibers (RBF) were found in the muscle biopsies of 3 patients, and cytochrome oxidase (COX)-negative muscle fibers were found in 2 patients. MT-TL1 gene m.3243A>G mutation was detected in all 3 patients by genetic testing, among which mutation in the blood of 2 patients was 15% and 17%, respectively, and mutation in the muscle tissues of 1 patient was 73%. Conclusion:Muscle pathology indicates high RRF percentage in patients with late-onset MELAS; and m.3243A>G spot mutation is the most common mutation type in late-onset MELAS, and m.3243A>G mutation ratio in muscle tissues is obviously higher than that in blood.

Objective:To investigate the clinical characteristics and electron transfer flavoprotein dehydrogenase ( ETFDH) genetic mutations in patients with riboflavin responsive lipid storage myopathy (RR-LSM). Methods:A retrospective analysis was performed. The clinical data and muscular pathology of 26 patients with RR-LSM, admitted to our hospital from January 2009 to June 2021, were collected. Peripheral venous blood DNA was extracted, and the mutations of ETFDH gene were detected and analyzed by whole exome sequencing. Results:These 26 patients had onset of proximal limb myasthenia, 17 patients had difficulty in raising their head, 12 patients had mastication weakness, 6 had dysphagia, 5 had nausea and vomiting, and one was complicated with rhabdomyolysis and one was with reversible splenic lesion syndrome. Muscle biopsy indicated pathological deposition of lipid droplet, which type I fibers were involved mainly; degenerative necrotic muscle fibers were seen in a few cases. ETFDH gene mutations were detected in 26 patients; 23 patients had compound heterozygous mutation, two had single heterozygous mutation and one had homozygous mutation; 25 different mutation sites were found, mainly missense mutations; the C.770A>G frequency was the highest, accounting for 20% alleles (10/50); two novel mutation sites were found: c.1115A>G and c.1781T>C. Conclusion:RR-LSM is mainly characterized by proximal limb muscle weakness and fatigue intolerance, often accompanied by neck extensor and masticatory weakness; c. 770A>G is the hot site of ETFDH genetic mutations in RR-LSM patients.

Objective:To investigate the clinical characteristics, skeletal muscle pathologies and gene variations of chronic progressive external ophthalmoplegia (CPEO).Methods:Sixteen patients with conformed CPEO, admitted to our hospital from January 1997 to December 2021, were chosen. Their clinical data such as onset age and course of diseases and muscle pathological examination results were collected and their gene variation characteristics were analyzed.Results:The initial symptom in all 16 patients was ptosis of varying degrees; 15 patients were with eye movement disorder, 6 with diplopia, 4 with proximal limb weakness, and 3 with dysphagia and dysarthria. Among the 16 patients, electromyography showed myogenic damage in 7 patients, myogenic combined with neurogenic damage in 1 patient, neurogenic damage in 1 patient, and normal in 7 patients. Skeletal muscle biopsies indicated that 14 patients were with ragged red fibers (RRF), 11 patients had cytochrome C oxidase (COX)-negative muscle fibers, 3 patients had a small amount of degenerated and necrotic myofibers with mononuclear phagocytic infiltration. Immunohistochemical staining indicated infiltration of CD8 and CD68 positive lymphocytes. Ten patients accepted genetic test, indicating 6 patients with single large fragment deletion of mitochondrial DNA (mtDNA), 1 patient with mtDNA point mutation, 1 patient with nucleosomal DNA (nDNA) point mutation, and 2 patients without pathogenicity variation clearly associated with clinical phenotype. Electron microscopy in 5 patients showed that abnormal mitochondrial aggregation was noted in 4 patients under the sarcolemma and among the myofibrils.Conclusion:In addition to ptosis and eye movement disorders, a small number of patients with CPEO may be accompanied by dysphagia and limb weakness; and single large fragment deletion of mtDNA is the main mutation form of CPEO.

Objective:To investigate the clinical characteristics, skeletal muscle pathologies and gene variations of chronic progressive external ophthalmoplegia (CPEO).Methods:Sixteen patients with conformed CPEO, admitted to our hospital from January 1997 to December 2021, were chosen. Their clinical data such as onset age and course of diseases and muscle pathological examination results were collected and their gene variation characteristics were analyzed.Results:The initial symptom in all 16 patients was ptosis of varying degrees; 15 patients were with eye movement disorder, 6 with diplopia, 4 with proximal limb weakness, and 3 with dysphagia and dysarthria. Among the 16 patients, electromyography showed myogenic damage in 7 patients, myogenic combined with neurogenic damage in 1 patient, neurogenic damage in 1 patient, and normal in 7 patients. Skeletal muscle biopsies indicated that 14 patients were with ragged red fibers (RRF), 11 patients had cytochrome C oxidase (COX)-negative muscle fibers, 3 patients had a small amount of degenerated and necrotic myofibers with mononuclear phagocytic infiltration. Immunohistochemical staining indicated infiltration of CD8 and CD68 positive lymphocytes. Ten patients accepted genetic test, indicating 6 patients with single large fragment deletion of mitochondrial DNA (mtDNA), 1 patient with mtDNA point mutation, 1 patient with nucleosomal DNA (nDNA) point mutation, and 2 patients without pathogenicity variation clearly associated with clinical phenotype. Electron microscopy in 5 patients showed that abnormal mitochondrial aggregation was noted in 4 patients under the sarcolemma and among the myofibrils.Conclusion:In addition to ptosis and eye movement disorders, a small number of patients with CPEO may be accompanied by dysphagia and limb weakness; and single large fragment deletion of mtDNA is the main mutation form of CPEO.

Objective:To develop an approach for the automatic diagnosis of bone metastasis and to design a parameter of quantitative evaluation for tumor burden on bone scans based on deep learning technology.Methods:A total of 621 cases (389 males, 232 females, age: 12-93 years) of bone scan images from the Department of Nuclear Medicine in Tenth People′s Hospital of Tongji University from March 2018 to July 2019 were retrospectively analyzed. Images were divided into bone metastasis group and non-bone metastasis group. Eighty percent of the cases were randomly extracted from both groups as the training set, and the rest of cases were used as the test set. A deep residual convolutional neural network ResNet34 was used to construct the classification model and the segmentation model. The sensitivity, specificity and accuracy were calculated and the performance differences of the classification model in different age groups (15 cases of <50 years, 75 cases of ≥50 and <70 years, 33 cases of ≥70 years) were analyzed. The regions of metastatic bone lesions were automatically segmented by the segmentation model. The Dice coefficient was used to evaluate the effect of the segmentation model and the manual labeled results. Finally, the bone scans tumor burden index (BSTBI) was calculated to assess the tumor burden of bone metastases.Results:There were 280 cases with bone metastases and 341 cases with non-bone metastases, including 498 in training set and 123 in test set. The classification model could accurately identify bone metastases, with the sensitivity, specificity and accuracy of 92.59%(50/54), 85.51%(59/69) and 88.62%(109/123), respectively, and it performed best in the <50 years group (sensitivity, 2/2; specificity, 12/13; accuracy, 14/15). The specificity in the ≥70 years group (8/12) was the lowest. The Dice coefficient of bone metastatic area and bladder area were 0.739 and 0.925 in the segmentation model, which performed similarly in the three age groups. Preliminary results showed that the value of BSTBI increased with the increase of the number of bone metastatic lesions and the degree of 99Tc m-MDP uptake. The machine learning model in this study took (0.48±0.07) s for the entire analysis process from input to the final BSTBI calculation. Conclusions:The deep learning based on automatic diagnosis framework for bone metastases can automatically and accurately identify segment bone metastases and calculate tumor burden. It provides a new way for the interpretation of bone scans. The proposed BSTBI may be used as a quantitative evaluation indicator in the future to assess the tumor burden of bone metastases based on bone scans.