Objective To evaluate the role of Toll-like receptor 2/nuclear factor-κB(TLR2/NF-κB)signaling pathway pretreatment in ventilator-induced lung injury(VILI). Methods Thirty male Sprague-Dawley(SD)rats were randomly divided into three groups by using random number scale,with 10 rats in each group. Group A:rats were given 200μL of TLR2 monoclonal antibodies(TLR2mAb,10μg/kg)by slow instillation through tracheal catheter, and then ventilated with a high tidal volume(VT)of 40 mL/kg. Group B:ventilated with a normal VT of 8 mL/kg. Group C:rats were tracheally instilled with 10 μg/kg of TLR2mAb devoid of biologic activity,and then ventilated with a high VT of 40 mL/kg. The rats were mechanically ventilated for 4 hours,the lung wet to dry weight ratio(W/D)was calculated. The changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosorbent assay(ELISA)was performed to determine the concentration of interleukins(IL-1β,IL-6)and tumor necrosis factor-α(TNF-α)in serum and brconchoalveolar lavage fluid(BALF). Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)was used to assess the mRNA expressions of TLR2, NF-κB and myeloid differentiation factor 88(MyD88)in lung tissue. Results No obvious pathological changes in lungs were found in group A and group B,and no obvious damages to ultra-microstructure were found in lung macrophages, typeⅠepithelial cell and typeⅡepithelial cell. In group C,pathological changes were observed,including pulmonary alveoli fusion,alveoli septum thickening,inflammatory cells infiltration,and damages to ultrastructure of lung macrophage,damage to cell membrane of typeⅠepithelial cells and typeⅡepithelial cells,vacuoles in cytoplasm, damage to organelle,and even pyknosis and perinuclear cistern thickening. Compared with group C,W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and B〔W/D ratio:1.151±0.026,1.128±0.048 vs. 1.403±0.062;concentration of IL-1βin serum(ng/L):37.05±5.61, 34.52±4.31 vs. 51.45±8.18;concentration of IL-6 in serum(ng/L):53.65±5.16,55.77±5.62 vs. 89.96±7.08;concentration of TNF-αin serum(ng/L):71.93±13.29,67.36±11.42 vs. 96.20±11.60;concentration of IL-1βin BALF(ng/L):56.48±6.16,54.44±7.26 vs. 99.77±8.41;concentration of IL-6 in BALF(ng/L):172.44±21.26, 163.47±18.70 vs. 216.22±23.90;concentration of TNF-α in BALF(ng/L):235.81±42.75,231.72±40.38 vs. 374.85±69.61,all P<0.01〕,but there were no significant differences between group A and group B(all P>0.05). The mRNA expressions of TLR2,MyD88,and NF-κB were significantly decreased in group A and group B compared with those in group C〔TLR2 mRNA(2-ΔΔCt):1.021±0.287,0.938±0.196 vs. 3.862±0.871;MyD88 mRNA (2-ΔΔCt):1.235±0.277,1.300±0.306 vs. 3.618±1.107;NF-κB mRNA(2-ΔΔCt):0.519±0.036,1.043±0.170 vs. 20.280±9.466,P<0.05 or P<0.01〕,but there was no significant difference among the parameters mentioned above between group A and B(all P>0.05). Conclusion To some extent,pre-intervention with TLR2mAb to block the TLR2/NF-κB signal pathway can inhibit the release of pro-inflammatory factors,and regulate the VILI.

The clinical characteristics, laboratory tests, treatment and prognostic characteristics of a case of neurexin-3α antibody-mediated autoimmune encephalitis,who was admitted in Xi′an International Medical Center Hospital on January 4, 2020 were summaried. The patient was a 56-year-old male with pre-symptoms of infection before his illness. The main manifestations were rapid progress of cognitive dysfunction, mental and behavioral abnormalities, refractory epilepsy, and status epilepticus, dystonia, accompanied by autonomic nerve involvement such as increased heart rate, breathing, and sweating, suspected of central hypoventilation, clinical manifestations were similar to N-methyl-D-aspartate-receptor (NMDAR) encephalitis and magnetic resonance imaging suggested bilateral hippocampal, temporal, and insular high signals, cerebrospinal fluid examination and other laboratory tests were normal. After treatment with methylprednisolone and intravenous immunogloblin, he died a few days after being discharged. The clinical manifestations of neurexin-3α antibody-mediated autoimmune encephalitis were similar to NMDAR encephalitis and severe, and neurexin-3α antibodies need to be further tested to confirm the diagnosis when NMDAR antibody was negative.

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

OBJECTIVETo investigate osteogenesis and integration of osteointergrated dental implants with marrow stromal osteoblast and cancellous bone matrix compound artificial bone (MCCAB) when embedded subcutaneously.

METHODSOsteointergrated dental implants (3 mm in diameter) were inserted into cancellous bone matrix (CBM) columns (5 mm in diameter). Marrow stromal osteoblast (MSO) were cultured and expanded in the column and on the surface. The osteointergrated dental implants loaded MSO-Alginate-CBM compound was formatted. This compound was then implanted subcutaneously in nude mice, and the osteointergrated dental implants loaded Alginate-CBM compounds were implanted as control. The compound was in the mice for 4 to 8 weeks and then harvested and assessed by means of gross observation, X-ray examination, histologic observation and computerized histomorphometry for evaluation of bone formation.

RESULTSThe osteogenesis of the osteointergrated dental implants loaded MSO-Alginate-CBM compound was better than that of the the osteointergrated dental implants loaded Alginate-CBM compound. Both intramembranous and cartilaginous osteogenesis was seen but the former was predominant. A large amount of new bone formed around the implant and integrated well with the implant. In the control, only slight cartilage osteogenesis was seen and no integration was found.

CONCLUSIONSThe results suggest that the new bone forms in the scaffolds and on the surface of the implant, and integration between the implant and artificial bone also occurs when they are implanted in the nude mice.

Animals ; Bone Matrix ; transplantation ; Bone Substitutes ; Cells, Cultured ; Dental Implantation, Endosseous ; methods ; Dental Implants ; Mice ; Mice, Nude ; Osseointegration ; physiology ; Osteoblasts ; transplantation ; Osteogenesis ; physiology ; Rabbits ; Tissue Engineering

An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Amino Acid Sequence ; Animals ; Anthrax Vaccines ; immunology ; Antigens, Bacterial ; chemistry ; genetics ; immunology ; toxicity ; Bacterial Toxins ; chemistry ; genetics ; immunology ; toxicity ; Base Sequence ; Mice ; Molecular Sequence Data ; Plasmids ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism.

METHODSTwo-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10-min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions.

RESULTSHistological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region (P < 0.01 vs 10-min ischemia group). Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates (P < 0.01 vs 10-min ischemia group).

CONCLUSIONIschemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Animals ; Brain Ischemia ; pathology ; Cell Count ; methods ; Cell Death ; Disease Models, Animal ; Gene Expression Regulation ; physiology ; HSP70 Heat-Shock Proteins ; metabolism ; Hippocampus ; blood supply ; metabolism ; pathology ; Ischemic Preconditioning ; Male ; Neurons ; metabolism ; Proteins ; metabolism ; Rats ; Rats, Wistar ; Time Factors

OBJECTIVETo study the expression of Aurora-B in human glioma tissue and its significance.

METHODSThe total RNA was extracted from 41 human glioma tissues and 11 normal brain tissues by Trizol reagent. After reverse transcription of the total RNA into cDNAs, Aurora-B mRNA expressions in these samples were detected by quantitative real-time PCR. The protein expression in these samples was detected using immunohistochemical staining.

RESULTSAurora-B mRNA and protein expressions were significantly increased in glioma tissues as compared with those in normal brain tissues.

CONCLUSIONAurora-B mRNA and protein show markedly higher expressions in glioma tissue, suggesting that Aurora-B may be one of the malignant biomarkers in the pathogenesis and progression of human glioma.

Aurora Kinase B ; Aurora Kinases ; Biomarkers, Tumor ; metabolism ; Brain Neoplasms ; enzymology ; pathology ; Female ; Glioma ; metabolism ; pathology ; Humans ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Tumor Cells, Cultured

Objective To summarize the clinical features of 22 probands diagnosed with congenital muscular dystrophy (CMD),and to provide genetic counseling and prenatal diagnosis for 23 fetuses of these pedigrees.Methods Data of 22 CMD patients who were treated in the Pediatric Department of Peking University First Hospital during October 2006 to March 2016 were analyzed.Informed written consents for participation in this study were obtained from the parents or guardians.Prenatal diagnosis was performed using DNA samples extracted from fetal villus cells of 12 cases at 11-13 gestational weeks and amniotic fluid of 11 cases at 18-22 gestational weeks.Direct DNA sequencing by polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) were used to detect CMD-related gene mutations.Linkage analysis of short tandem repeats (STRs) was used to identify maternal blood contamination and biological parents.Results Thirteen out of the 22 probands with CMD were diagnosed with congenital muscular dystrophy type 1 A (MDC1A),and all of them carried compound heterozygous mutations in LAMA2 gene.Prenatal diagnosis of 13 fetuses from these pedigrees found that four fetuses were wild-type,seven were heterozygotes and two carried the same mutations as their proband.Three probands with LMNA-related congenital muscular dystrophy (L-CMD) carried de novo mutations in LMNA gene.In these pedigrees,two fetuses were wild-type and one whose mother was mosaicism carried the same mutations as the proband.One proband with Ullrich congenital muscular dystrophy carried compound heterozygous mutations in COL6A2 gene and the fetus of the same pedigree was wild-type.Five probands were diagnosed with α-dystroglycanopathies.And among them,two cases of muscle-eye-brain disease (MEB) carried compound heterozygous mutations in POMGnT1 gene and the fetuses of the two peidgrees were heterozygotes;one case of congenital muscular dystrophy type 1C (MDC1C) had compound heterozygous mutations in FKRP gene and the fetus carried the same mutations;one patient diagnosed with POMGnT1-related congenital muscular dystrophy with mental retardation (CMD-MR) carried compound heterozygous mutations in POMGnT1 gene,and the fetus was positive for the same mutations;one proband with POMT1-related CMD-MR was positive for compound heterozygous mutations in POMT1 gene and the results of prenatal diagnosis for two fetuses of this pedigree showed that the first fetus had the same mutations as the proband,while the second was heterozygote.Conclusions No effective therapeutic method is available for CMD.Therefore,accurate genetic counseling and prenatal diagnosis are necessary to prevent CMD child from birth.

Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1(from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co-localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to af-fect the formation of SND1 containing stress granules.

Background Since the measurement method establishment of serum ferritin abroad in early period of theseventies, the iron deficiency had been divided into two types: the non-anemia and anemia types. In orderto go step further studies, we must ertablish the bemoglobin targets of the two types. Methods One hurdred and fifty-two children in experimontal group, from 6 to 7 years old, and allcome from Qinghai province. There are 29 children in Xining city, 24 in Guide, 26 in Gongbe, 40 in Gui-nan and 33 in Maduo countics. There are 36 health children aged from 6 to 7 years old in the controlgroup, and all comes from Beijing. The Hb, RBC, HCT, HCTW and FEP wcre determined. Results The three targets correlating with Hb (Hb, MCH and MCHC); correlating with RBC (RBC,HCT and MCV); the two targets correlating with RBC_weight (HCTW and CMCW) and correlating withFEP of RBC(FEP and MCEP) have very significant difference between experimental group and control group. Conclusion The determination values of the 10 targets are not same in children in different districts,and the values of all the target: are increased on different degree along with the increase in altitude of ele-vation. There is very important significance on the studies of iron deficiency and altitude hypoxia to establish the normal values of the 10 targets.