OBJECTIVE To investigate the impact of vesicular stomatitis virus envelope glycopro-tein-G(VSV-G)modification on the mucosal immune efficacy of antigen-loaded engineered exosome vaccines.METHODS In vitro experiments:Dendritic cells(DCs)were divided into three groups:cell-control(treated with culture medium),receptor binding domain(RBD)(transfected with plasmid RBD),and RBD+VSV-G(co-transfected with plasmids RBD and VSV-G).Expression levels of RBD and VSV-G were assessed using Western blotting,flow cytometry,and immunofluorescence.Exosomes were extracted via ultracentrifugation,whose morphology,size distribution,and marker proteins were analyzed using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting that confirmed the expressions of RBD and VSV-G in the exosomes.In vivo experiments:① Female BALB/c mice were divided into the control group Mock exosomes(Mock-Exo)(derived from the supernatant of cell-control),RBD decorated exosomes(RBD-Exo)(derived from the RBD cell supernatant),and RBD and VSV-G decorated exosomes(RBD+VSV-G-Exo)(derived from RBD+VSV-G cell supernatant).Follow-ing intranasal immunization with the respective vaccines,the nasal retention effects were evaluated using in vivo imaging.Flow cytometry was used to assess the ability to recruit immune cells to the nasal tissue.Serum RBD-specific immunoglobulin G(IgG)and mucosal immunoglobulin A(IgA)(bronchoal-veolar lavage fluid/nasal wash)were quantified at 7 and 21 d post-immunization by enzyme-linked immuno-sorbent assay.Body weight changes were monitored and key serum biochemical parameters along with histopathological damage to major organs were analyzed following immunization.② Female BALB/c mice were divided into the Mock-Exo group(intranasally inoculated with Mock-Exo),RBD+VSV-G-Exo group(intranasally inoculated with RBD+VSV-G-Exo),and RBD+VSV-G-Exo(im)group(intramus-cularly injected with RBD+VSV-G-Exo).RESULTS In vitro experiments:RBD and VSV-G were successfully expressed in cells,with positive rates of RBD+and VSV-G+cells at 64.4%and 31.2%,respectively.The extracted exosomes exhibited regular morphology and qualified purity,with a particle size of approximately 138 nm and successfully loaded RBD and VSV-G proteins.In vivo experiments:Compared to Mock-Exo and RBD-Exo,RBD+VSV-G-Exo prolonged nasal retention time to 96 h and markedly increased the numbers of CD49B+natural killer cells,CD11c+dendritic cells,and F4/80+macrophages in nasal tissues.RBD+VSV-G-Exo induced robust RBD-specific immune responses,with serum IgG titers,BALF IgA titers,and nasal wash IgA titers reaching 1∶5 215,1∶2 560,1∶1 114,respec-tively.In contrast,no RBD-specific IgA antibody titers were detected in the BALF and nasal wash of mice treated with RBD+VSV-G-Exo(im).Mice showed stable body weight gain during 30 d post-immu-nization.Major serum biochemical indices were within normal reference ranges,and no obvious patho-logical changes were observed in major organs or olfactory bulbs 7 d after immunization.CONCLU-SION VSV-G modification extends the retention time of engineered exosome vaccines in nasal tissues,enhance their ability to recruit immune cells,and induce a high-level antigen-specific respiratory mucosal immune response.

OBJECTIVE To investigate the impact of vesicular stomatitis virus envelope glycopro-tein-G(VSV-G)modification on the mucosal immune efficacy of antigen-loaded engineered exosome vaccines.METHODS In vitro experiments:Dendritic cells(DCs)were divided into three groups:cell-control(treated with culture medium),receptor binding domain(RBD)(transfected with plasmid RBD),and RBD+VSV-G(co-transfected with plasmids RBD and VSV-G).Expression levels of RBD and VSV-G were assessed using Western blotting,flow cytometry,and immunofluorescence.Exosomes were extracted via ultracentrifugation,whose morphology,size distribution,and marker proteins were analyzed using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting that confirmed the expressions of RBD and VSV-G in the exosomes.In vivo experiments:① Female BALB/c mice were divided into the control group Mock exosomes(Mock-Exo)(derived from the supernatant of cell-control),RBD decorated exosomes(RBD-Exo)(derived from the RBD cell supernatant),and RBD and VSV-G decorated exosomes(RBD+VSV-G-Exo)(derived from RBD+VSV-G cell supernatant).Follow-ing intranasal immunization with the respective vaccines,the nasal retention effects were evaluated using in vivo imaging.Flow cytometry was used to assess the ability to recruit immune cells to the nasal tissue.Serum RBD-specific immunoglobulin G(IgG)and mucosal immunoglobulin A(IgA)(bronchoal-veolar lavage fluid/nasal wash)were quantified at 7 and 21 d post-immunization by enzyme-linked immuno-sorbent assay.Body weight changes were monitored and key serum biochemical parameters along with histopathological damage to major organs were analyzed following immunization.② Female BALB/c mice were divided into the Mock-Exo group(intranasally inoculated with Mock-Exo),RBD+VSV-G-Exo group(intranasally inoculated with RBD+VSV-G-Exo),and RBD+VSV-G-Exo(im)group(intramus-cularly injected with RBD+VSV-G-Exo).RESULTS In vitro experiments:RBD and VSV-G were successfully expressed in cells,with positive rates of RBD+and VSV-G+cells at 64.4%and 31.2%,respectively.The extracted exosomes exhibited regular morphology and qualified purity,with a particle size of approximately 138 nm and successfully loaded RBD and VSV-G proteins.In vivo experiments:Compared to Mock-Exo and RBD-Exo,RBD+VSV-G-Exo prolonged nasal retention time to 96 h and markedly increased the numbers of CD49B+natural killer cells,CD11c+dendritic cells,and F4/80+macrophages in nasal tissues.RBD+VSV-G-Exo induced robust RBD-specific immune responses,with serum IgG titers,BALF IgA titers,and nasal wash IgA titers reaching 1∶5 215,1∶2 560,1∶1 114,respec-tively.In contrast,no RBD-specific IgA antibody titers were detected in the BALF and nasal wash of mice treated with RBD+VSV-G-Exo(im).Mice showed stable body weight gain during 30 d post-immu-nization.Major serum biochemical indices were within normal reference ranges,and no obvious patho-logical changes were observed in major organs or olfactory bulbs 7 d after immunization.CONCLU-SION VSV-G modification extends the retention time of engineered exosome vaccines in nasal tissues,enhance their ability to recruit immune cells,and induce a high-level antigen-specific respiratory mucosal immune response.

Objective To investigate the predictive value of depth of invasion (DOI) of tongue squamous cell carcinoma (TSCC) for cervical lymph node metastasis and prognosis. Methods We retrospectively analyzed the clinical and pathological data of 73 patients with T_1/2 TSCC. ROC curve was used to determine the optimal cut-off value of DOI for predicting cervical lymph node metastasis, and logistic regression analysis was performed to analyze the related factors affecting cervical lymph node metastasis of TSCC. Kaplan-Meier method and Cox regression analysis were used for survival analysis. Results Among 73 patients, 18 patients were with lymph node metastasis and 55 patients were without lymph node metastasis. The median DOI with and without lymph node metastasis were 8.00 and 5.00 mm, respectively (P=0.003). The optimal cut-off value for DOI was 6.15 mm, with AUC 0.75 (95%CI: 64.1%~87.1%, P=0.001), sensitivity 77.8% and specificity 63.6%. DOI and pathological differentiation were independent prognostic factors for cervical lymph node metastasis in multivariate analysis. DOI, nerve invasion and pathological differentiation were independent prognostic factors of survival in Cox regression analysis. Conclusion DOI of TSCC patients has important predictive value for both cervical lymph node metastasis and prognosis. Neck lymph node dissection is recommended for patients with DOI > 6.15 mm to improve survival rate and reduce recurrence rate.

Objective:To summarize the clinical and genetic characteristics of catecholaminergic polymorphic ventricular tachycardia (CPVT) in children caused by CASQ2 gene variants. Methods:The clinical data of 8 children (4 males and females, respectively) with CPVT caused by CASQ2 gene variants admitted to Beijing Children′s Hospital, Capital Medical University from January 2017 to November 2018 were retrospectively analyzed.The targeted next generation sequencing was employed to identify CASQ2 variants and Sanger sequencing was conducted to conform the candidate variants and determine the parental origin. Results:As for 8 children in this study, the average age of onset was 6.4 years, the mean age at diagnosis was 9.4 years, and the average interval from onset to diagnosis was 3 years.Only 2 cases had clearly diagnosis at onset, other 6 cases had a delay to diagnosis and 3 cases of them were diagnosed at other hospitals as having epilepsy and did not respond to anti-epileptic therapy.During physical activity and/or emotional stress, 8 cases presented with recurrent syncope and were able to regain consciousness after a few minutes.They had no a history of sudden cardiac death or family history.There was no abnormality on resting electrocardiogram during the paroxysmal interval in 6 cases and mild sinus bradycardia in 2 cases.Typical bidirectional ventricular tachycardia (VT) and/or polymorphic VT were detected in 8/8 cases and 5/5 cases, respectively, based on Holter electrocardiography and cardiac stress test.The CASQ2 gene variant was found in all children, with 6 cases carrying compound heterozygous variants and 2 cases carrying homozygous variants.A total of 9 different CASQ2 variants were detected in 8 cases, of which 5 had not been previously reported.According to the family-line verification, all of them had a familial variant, with no novel variants.All 8 cases were treated orally with β-blockers, with asymptomatically recurrent episodes, with a mean follow-up of 1.5 years, during which implantable cardioverter defibrillation was performed in 1 case owing to severe sinus bradycardia.There was no death case among them. Conclusions:CPVT with CASQ2 variants is characterized by early onset before preschool age, recurrent syncope after exercise or emotional stress and bidirectional/polymorphic VT.Early diagnosis of CPVT remains challenging due to delayed diagnosis or misdiagnosis.Treatment with β-blockers can achieve favorable effectiveness and safety.Five novel variants in this study would further expand the database of CASQ2 genes.

Mycophenolic acid (MPA, ) and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases. Despite chemical synthetic achievements, the biosynthetic formation of a seven-carbon carboxylic acid pharmacophore side chain of , especially the processes involving the cleavage of the prenyl side chain between DHMP () and DMMPA (), remains unknown. In this work, we identified a membrane-bound prenyltransferase, PgMpaA, that transfers FPP to to yield FDHMP (). Compound undergoes the first cleavage step a new globin-like enzyme PgMpaB to form a cryptic intermediate . Heterologous expression of genes in demonstrates that the second cleavage step (from to ) of is a cluster-independent process . Our results, especially the discovery of the broad tolerance of substrates recognized by PgMpaB, set up a strategy for the formation of "pseudo-isopentenyl" natural products using fungal globin-like enzymes.

Objective To investigate the diagnostic value of IL-27, ADA and TB-Ab in TB pleurisy. Methods The data of 74 TB pleurisy cases and 45 non-TB pleurisy cases were screened randomly from June 2017 to July 2018 in Dalian Tuberculosis Hospital. Value of IL-27, ADA and TB-Ab in blood and pleural fluid of the two groups of cases was detected, and diagnostic value of these biomarkers in TB pleurisy was compared. Results The value of IL-27, ADA and TB-Ab in blood and pleural effusion of patients with TB pleurisy were higher than those of control group (P < 0.05). ROC curve analysis showed that areas under the blood IL-27, ADA and TB-Ab curves were 0.820, 0.744 and 0.589 (P<0.05) respectively, while those under the pleural effusion curves were 0.921, 0.876 and 0.708 (P<0.05) respectively. The area under the curve of IL-27 and ADA ROC curve was 0.921 (P<0.05), but 95% CI was higher than that of pleural effusion ADA (0.804-0.930) and IL-27(0.857-0.962). Conclusions Detection of IL-27 and ADA in pleural effusion is of great value in the diagnosis of TB pleurisy. The combined detection of IL-27 and ADA in pleural effusion would improve the diagnostic value.

Objective To explore the prognostic values of telomerase reverse transcriptase promoter (TERTp) mutation and 1p/19q co-deletion in newly-diagnosed O6-methylguanine-DNA methyltransferase (MGMT) promoter un-methylated/isocitrate dehydrogenase (IDH) wild-type glioblastoma multiform (GBM). Methods A total of 82 patients pathologically newly-diagnosed MGMT promoter un-methylated/IDH wild-type GBM, admitted to our hospitals from March 2016 to November 2018, were included in this study. TERTp mutations (TERTp wild-type and TERTp mutation [C228 mutation and C250 mutation]) in GBM specimens were detected by PCR sequencing, 1p/19q co-deletion in GBM specimens was detected by fluorescence in situ hybridization (FISH), and clinical data, adverse reactions and prognoses of patients with different molecular typing were compared. Results There were 33 patients in the TERTp wild type group with mean age of 48 years, and 49 patients in the TERTp mutation group with mean age of 59 years; the difference of age was significant (P<0.05); there were no statistical differences in gender distribution, Karnofsky performance status (KPS) scores, tumor sites and surgical resection degrees between the two groups (P>0.05). There were 8 patients with 1p/19q co-deletion and 74 patients without 1p/19q co-deletion; no significant differences in above clinical parameters were noted between the two groups. There were no statistically significant differences in the incidences of bone marrow suppression, digestive tract response and fatigue, disease progression rate, or survival rate between patients from TERTp wild type group and TERTp mutation group, and between patients with 1p/19q co-deletion and patients without 1p/19q co-deletion (P>0.05). No significant differences in above clinical parameters, disease progression rate, and survival rate were noted between patients with C228 mutation and C250 mutation (P>0.05). Conclusion TERTp typing and 1p/19q co-deletion status do not have prognostic value in newly-diagnosed MGMT un-methylated/IDH wild-type GBM patients; patients with TERTp mutations have older age than wild-type patients; patients with C250 mutation trend to have higher survival rate than those with C228 mutation.

The American Association for the Study of Liver Diseases (AASLD) updated and published the Practice Guidance for the Diagnosis and Management of Nonalcoholic Fatty Liver Disease (NAFLD) in July 2017, which provides recommendations for the accurate diagnosis, treatment, and effective prevention of NAFLD. Related metabolic diseases should be considered during the initial evaluation of patients suspected of NAFLD. Noninvasive diagnostic techniques including transient elastography, magnetic resonance elastography, and serum biochemical models should be used to evaluate the development and progression of liver fibrosis in patients with NAFLD. Clinical liver pathology report should clearly differentiate between nonalcoholic fatty liver (NAFL), NAFL with inflammation, and nonalcoholic steatohepatitis (NASH) and identify the presence or absence of liver fibrosis and its degree. Early medication for NAFLD can only be used in patients with pathologically confirmed NASH and liver fibrosis, and it is not recommended to use pioglitazone and vitamin E as the first-line drugs for patients with NASH which has not been proven by biopsy or non-diabetic NASH patients. Foregut bariatric surgery can be considered for obese patients with NAFLD/NASH who meet related indications. It is emphasized that the risk factors for cardiovascular disease should be eliminated for NAFLD patients. Statins can be used for the treatment of dyslipidemia in patients with NAFLD/NASH, but they cannot be used in patients with decompensated liver cirrhosis. Routine screening or hepatocellular carcinoma surveillance is not recommended for NASH patients without liver cirrhosis. Cardiovascular disease should be taken seriously during liver transplantation evaluation. There is still no adequate clinical evidence for the treatment of NAFLD in children and adolescents, and intensive lifestyle intervention is recommended as the first-line therapy for such patients.

Objective To investigate the clinical effect of cisplatin combined with cisplatin and radiotherapy in the treatment of glioblastomas patients receiving surgery.Methods 68 cases of glioblastomas patients with surgery were divided into control group (n=36) and treatment group (n=32).The control group was treated with temozolomide and radiotherapy;the treatment group was treated with cisplatin combined with temozolomide and radiotherapy.Short-term efficacy,adverse reactions,and survival time were documented during following up in both groups.Results The efficiency in the treatment group (87.5％) was significantly higher than that in the control group (66.67％),P＜0.05.There were no significant difference of adverse reaction including bone marrow inhibition,digestive tract symptoms,and psychiatric symptoms among the two groups,P＞0.05.The medium survival time of the treatment group (26.0 month) was significantly longer than that of the control group (17.5 month),P ＜0.05.There was no significant difference of 1-year survival rate between the treatment group (81.25％) with control group (63.89％),P＞0.05;however,2-and 3-year survival rate in treatment group (52.13％,31.25％) were significantly higher than that in the control group (27.78％,11.11％),P＜0.05.1-year progression free survival (PFS) rate of treatment group (68.75％) was significantly higher that of control group (41.67％),P＜0.05.Conclusion Cisplatin combined with temozolomide and radiotherapy can significantly improve the treatment efficiency without leading to higher rate of adverse reactions and significantly improve the 2-,3-year survival rate as well as 1-year progression free survival (PFS) rate in patients with glioblastomas,it is worth being promoted in clinical application.