Objective To construct a novel enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) recombinant baculovirus. Methods The target gene(EGFP and GDNF) was cloned into baculovirus transfer vector pFastBacDual, pFB-EGFP-GDNF was constructed and restriction enzyme analysis was conducted. pFB-EGFP-GDNF was transposited with baculovirus shuttle vector (Bacmid) into DH10Bac competent cells, and recombination baculovirus vector Bacmid-EGFP-GDNF was constructed. The plasmid was extracted and PCR was performed for identification. Bacmid-EGFP-GDNF was transfected with Sf9 insect cell package virus by liposomal transfection method. Immunofluorescent staining was employed to detect the expression of EGFP and GDNF protein in St9 cells. Results The target gene fragment was correctly cloned into pFastBaeDual vector, and recombinant Bacmid was constructed. Bacmid-EGFP-GDNF was successfully transfected, and higher virus titer was obtained. The coexpression of GDNF and EGFP protein in Sf9 cells was identified by immunofluorescent staining. Conclusion The recombinant baculovirus Bacmid-EGFP-GDNF can be successfully constructed, and the protein of EGFP and GDNF is coexpressed in St9 cells, which paves a way for the research of GDNF gene therapy.

OBJECTIVETo explore the effects of different anesthesia Methods on the cellular immune function and mean arterial pressure, heart rate in patients with abdominal tumor in the peri-operational period.

METHODSPatients were anesthetized by general anesthesia (GA), combined acupuncture-general anesthesia (AGA), combined epidural-general anesthesia (EGA), and combined acupuncture-epidural-general anesthesia (AEA), respectively. Changes of T lymphocyte subsets and hemodynamics were observed at different time points before and after treatment.

RESULTSPatients' cellular immune function after GA was lowered, which could be alleviated by combination of GA with acupuncture anesthesia (AA) or with epidural anesthesia. Combined acupuncture-drug anesthesia showed a stabilizing effect on hemodynamics in peri-operational period to some extent.

CONCLUSIONEGA and AGA are good anesthesia choice in clinical practice, which would be favorable for early recovery of immune function in patients with abdominal tumor after operation.

Acupuncture Analgesia ; Adult ; Aged ; Aged, 80 and over ; Anesthesia, Epidural ; Anesthesia, General ; Electroacupuncture ; Female ; Hemodynamics ; Humans ; Male ; Middle Aged ; Stomach Neoplasms ; immunology ; physiopathology ; surgery ; T-Lymphocyte Subsets ; immunology

Antibody Formation ; China ; epidemiology ; Female ; Hepatitis B ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Vaccines ; immunology ; Humans ; Infant, Newborn ; Male

OBJECTIVEAdrenocortical carcinoma (ACC) is a rare but highly malignant tumor, and its diagnosis is mostly delayed and prognosis is poor. We report estrogen receptor (ER) expression in this tumor and our clinical experiences with 17 ACC cases.

METHODSThe data of the 17 patients (9 females and 8 males, age range from 16 to 69 years, mean age of 42.6 years) with ACC were reviewed, and symptoms, diagnostic procedures, treatment, and results of follow-up were evaluated. Immunohistochemistry was used to detect ER expression in tumor samples from the 17 patients.

RESULTSAt the time of diagnosis, 4 tumors were classified as Stage I, 4 as Stage II, 3 as Stage III, and 6 as Stage IV. Eight patients demonstrated positive nuclear immunostaining of ER. The prognosis of patients with ER positive was significantly better (P<0.05) than that of patients with ER negative, with 1- and 5-year survival rates at 86% and 60% for ER-positive patients, and 38% and 0% for ER-negative patients, respectively.

CONCLUSIONER-positivity may be one of the factors associated with a worse prognosis of ACC.

Adolescent ; Adrenal Cortex Neoplasms ; diagnosis ; metabolism ; mortality ; Adrenocortical Carcinoma ; diagnosis ; metabolism ; mortality ; Adult ; Aged ; Biomarkers, Tumor ; analysis ; China ; Female ; Humans ; Incidence ; Male ; Middle Aged ; Neoplasm Proteins ; analysis ; Receptors, Estrogen ; analysis ; Risk Assessment ; methods ; Risk Factors ; Survival Analysis ; Survival Rate ; Young Adult

Abstract: Objective　To construct SARS-CoV-2 receptor binding domain molecular probe for monoclonal memory B cell sorting and obtain RBD specific neutralizing antibodies from peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents by single-cell sorting. Methods　The SARS-CoV-2 RBD sequence was downloaded from GenBank, and the Avi tag and 6-histidine tags were added at the C-terminal. After codon optimization, it was chemically synthesized, cloned into the pDRVI1.0 vector, expressed after transfection of 293F cells, and biotinylated consequently. RBD-specific B cells were sorted out with this probe1 from the PBMCs of convalescents recovered from COVID-19. After B cells were lysed, the variable regions of heavy chain and light chain were amplified, cloned into the antibody expression vector, and transfected into 293F cells to express the antibody. Then the antibody was purified from the supernatant using protein A column and SARS-CoV-2 pseudovirus was used to test their neutralizing activity. Results　RBD-Avi probe was produced and successfully biotinylated sequentially with an efficiency of 30%-50%. Western blot analysis revealed that the biotinylated probe was recognized by the antibodies purified from COVID-19 convalescent plasma. Using this probe, 7 and 16 RBD-specific memory B cells were successfully isolated from the PBMCs of two convalescent individuals, accounting for 0.24% and 0.17% of the total cell population, respectively. After amplifying the variable regions of antibody heavy and light chains from the lysed B cells, 7 and 12 pairs of antibody heavy-light chains were obtained. A total of 16 antibodies were expressed in the convalescent individuals, and most of the purified antibodies showed neutralizing activity against the pseudovirus, with IC50 values of 6 antibodies below 1 μg/mL. The IC50 values of XJ-A9 and SCF-F1 against the wild-type pseudovirus were 0.07 μg/mL and 0.35 μg/mL, respectively. Conclusion　The SARS-CoV-2 RBD molecular probe constructed in this study has good antigenicity, and the isolated antibodies present neutralizing activity against wild-type SARS-CoV-2 pseudovirus.

OBJECTIVETo study the effect and indications of intracranial pressure (ICP) monitoring for frontal lobe contusion patients.

METHODSDuring January 2005-December 2008, 34 cases of frontal lobe contusion received ICP monitoring in our department (monitoring group). Different treatment protocols were adopted according to the results of ICP. Meanwhile 46 cases of same type of head-injured patients who did not undergo ICP monitoring served as control group.

RESULTSWe found that ICP elevated dramatically within 24 hours after head injury if the contusions were located in frontal longitudinal dehiscence, bilateral undersurface of frontal lobe or dispersed in bilateral lobe. After half a year follow-up and on the basis of Glasgow Coma Scale assessment, the monitoring group showed better outcome than the control group with good recovery in 24 cases (70.6%) , moderate disability in 7 cases (20.6%), severe disability in 2 (5.88%) and death in 1 (2.94%). The outcome of control group displayed good condition in 25 cases (54.3%), moderate disabilities in 8 (17.4%), severe disability in 7 (15.2%), and death in 6 (13.0%).

CONCLUSIONSFrontal lobe contusions are vulnerable and complex head injuries, especially when the contusions are located in frontal longitudinal dehiscence, bilateral undersurface of frontal lobe or diffused in bilateral lobes. These patients should undergo ICP monitoring regardless of their consciousness status. If ICP elevates over 25 mm Hg, the craniotomy is mandatory and will markedly reduce the mortality and disability of these patients.

Adolescent ; Adult ; Aged ; Contusions ; physiopathology ; Craniocerebral Trauma ; physiopathology ; Female ; Frontal Lobe ; injuries ; Glasgow Coma Scale ; Humans ; Intracranial Pressure ; Male ; Middle Aged

Adult ; Aged ; Female ; Humans ; Intestine, Small ; surgery ; Lymphangiectasis, Intestinal ; pathology ; surgery ; Male ; Middle Aged

OBJECTIVETo study the feasibility and the characteristics of recombinant baculovirus as spiral ganglion cells (SGC) gene transfer vector.

METHODSAfter the generation of baculovirus- green fluorescent protein( Bac-GFP) according to Bac-to-Bac baculovirus expression system, SGC were infected by Bac-GFP with different multiplicities of infection (MOI) and different concentrations of sodium butyrate. The transfection cell rate and mean fluorescence strength (MFS) were detected by fluorescence microscopy and flow cytometry. Toxicity effects of recombinant baculovirus vectors and sodium butyrate on SGC were determined by spectroscopic measurement of 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTF).

RESULTSBaculovirus was able to infect primary SGC cultures. The dose-response characteristics of Bac-GFP were determined on SGC, and the expression level could be up-regulated by sodium butyrate. Infection with Bac-GFP in the absence or presence of sodium butyrate (< or =10 mmol/L) was considered to be non-cytotoxic to primary SGC. GFP had been expressed in SGC at 6 h post-infection and the highest numbers of cells expressing GFP were observed at approximately 48 h post-infection.

CONCLUSIONSBaculovirus is a novel and promising tool for gene transferring into the cochlear nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.

Animals ; Baculoviridae ; genetics ; Cells, Cultured ; Gene Transfer Techniques ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology ; Transfection

OBJECTIVETo investigate the clinical features and treatment strategy of dissymmetric bilateral frontal contusion, and to summarize our experience in treating these patients by minimally invasive surgery.

METHODSOver the past 3 years, we have treated a total of 31 patients with dissymmetric bilateral frontal contusion using endoscopy-assisted unilateral cerebral falx incision. Other 30 patients treated by routine bilateral approaches within the same period were taken as control.

RESULTSSeventeen cases (54.8%) in the unilateral operation group survived and were in good condition, 8 (25.8%) had moderate disability, 4 (12.9%) had severe disability, 1 (3.2%) was in vegetative state, and 1 (3.2%) died. Compared with the control group, the Glasgow Outcome Scale score was not significantly different in the unilateral operation group, but the operation time, blood transfusion volume, the length of hospital stay, the incidences of mental disorder and olfactory nerve injury were greatly reduced in the unilateral operation group.

CONCLUSIONSEndoscopy-assisted unilateral cerebral falx incision can shorten the operation time, reduce surgical trauma and complications in treatment of patients with dissymmetric bilateral frontal contusion. It can obviously diminish the chance of delayed intracerebral hematoma and subsequently minimize the incidences of subfalcial and centrencephalic herniation.

Brain Injuries ; Contusions ; Dura Mater ; Endoscopy ; Humans ; Treatment Outcome

OBJECTIVETo detect the duplication or deletion of peripheral myelin protein 22(PMP22) gene in Chinese patients with Charcot-Marie-Tooth disease(CMT) or hereditary neuropathy with liability to pressure palsies(HNPP) using real-time quantitative polymerase chain reaction.

METHODSDuplications or deletions of PMP22 gene were detected in 113 CMT cases, 4 HNPP cases and 50 normal controls by using real-time quantitative PCR.

RESULTSThirty-six of 113 CMT cases had the PMP22 duplication, 4 HNPP cases had the PMP22 deletion. No duplication or deletion was found in 50 normal controls.

CONCLUSIONThe PMP22 duplication rate in Chinese patients with CMT is 31.9%(36/113). PMP22 deletion is the common cause of HNPP.

Adult ; Charcot-Marie-Tooth Disease ; genetics ; Female ; Gene Duplication ; Humans ; Male ; Myelin Proteins ; genetics ; Polymerase Chain Reaction ; methods ; Sequence Deletion ; Young Adult