AIM: To investigate the process of human bone marrow stromal cells (hBMSCs) differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS: Purified hBMSCs were treated with β-mercaptoethanol (β-ME) for 1 day and retinoic acid (RA) for 3 days, followed by growth factor (10 μg/L bFGF or 20 μg/L NGF) for another 3 days. Immunofluorescence was performed to detect the expression of nestin (a neural precursor cells marker), Tuj1 (a premature neuronal marker), and neurofilament (NF, a mature neuronal marker) at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS: Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells (34.41%±1.27%) and Tuj1-positive cells (27.79%±1.27%) were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA (1.33), as well as by β-ME/RA/GF (1.77), was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells (37.59%±1.52%). CONCLUSION: After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.

Objective To observe how exogenous estrogen influences the distribution and the expression of NOS positive neurons in the supraoptic nuclei of hypothalamus in the overiectomized female rats. Methods The 2-3-month-old female rats(n=40) were selected as the healthy and nulli-copulatory experimental animals. Rats were divided into following groups: normal control group(n=10), ovariectomized control group(n=10), and two experimental groups that have been injected with estrogen for post-operative 40 days(n=10) and for post-operative 70 days(n=10). Finally, all the animals were infused and the brains were removed. Immunohistochemical (SABC) method was adopted to count the number of NOS poitive neurons and observed the NOS poitive neuronal morphology under the light microscope. The image analysis system was used to test the average gray value of immunoreactivity in NOS positive neurons. Results In the ovariectomized control group, the density of NOS positive neurons in supraoptic nucleus was significantly increased and their shapes were bigger than that of the normal control group(P<0.05). The density and the form of the NOS positive neurons in supraoptic nucleons had no apparent difference between the estrogen for post-operative 40 days group and the ovariectomizeed control group(P>0.05).In the group after estrogen-injection 2 months compared with the normal control group, and the ovariectomized control group, both of the NOS positive neurons' density and the size become significantly decreased, and the staining of cells was lesser in the group injected with estrogen for post-operation 70 days. Conclusion The present results suggest that exogenous estrogen may influence the distribution and the expression of NOS positive neurons in supraoptic nucleus of hypothalamus of ovariectomized female rats.

Objective: To study the mechanisms of cold exposure mediated ileum mechanical barrier injury in mice. Methods: Twenty mice were randomly divided into the control and cold exposure groups. Both the control and cold exposure groups were placed in the climate room with (24±2)℃ and 40% humidity. The mice in the cold exposure group were moved to the climate room at (4±2)℃ every day for 3 hours for three consecutive weeks. Three weeks later, the ileum tissues of mice were collected. Changes in ileum tissue structure were observed by hematoxylin-eosin staining and Masson staining. The related protein expression levels of the tight junction, inflammatory cytokines, and the NF-κB pathway were detected by Western blot. Results: Compared with the control group, the circular muscle layer of the ileum in cold exposed mice became thin, a large number of inflammatory cells infiltrated, the length of villi became short, the depth of recess was increased, and tissue fibrosis appeared. The expression levels of ideal tight junction-associated proteins in cold exposed mice were decreased significantly (P＜0.05), while the protein expression levels of IL-1β, IL-6 and phosphorescent p65 were increased significantly (P＜0.05). Conclusion: Cold exposure can damage the tight junction of the mouse ileum, destroy the integrity of the mechanical barrier and activate the NF-κB signaling pathway to promote the occurrence of the inflammatory response.
Animals ; Cytokines/metabolism* ; Ileum/metabolism* ; Intestinal Mucosa ; Mice ; NF-kappa B/metabolism* ; Tight Junctions/metabolism*

To evaluate immune efficacy of the recombinant Lactobacillus casei, we constructed pLA-Newcastle disease virus (NDV)-F/L. casei and obtained the expression products. PCR amplified the NDV F gene carrying part of the major epitopes. The target gene was inserted to the shuttle plasmid pLA, and then transformed into Escherichia coli BL21 (DE3) in order to screen positive recombinant plasmid. The positive recombinant plasmid was transformed into L. casei by electroporation to construct pLA-NDV-F/L. casei. The positive strains were identified by PCR. The reactivity of the recombinant bacteria was identified by Western blotting and the protein expression was detected by indirect immunofluorescence, flow cytometry and laser confocal microscopy. The 14-day-old chickens in each group were vaccinated by oral plus nose drops. The pLA-NDV-F/L. casei twice immunization group and three times immunization group, the commercial vaccine group, the pLA/L. casei group, the unchallenge PBS and the challenge PBS group were established. IgG in serum and sIgA in the lavage fluid of intestinal, nasal and lung were detected by ELISA. The protection rate of chickens was evaluated. The results showed that 94.10% of the recombinant bacteria expressed the F protein. The recombinant protein was highly expressed on the surface of L. casei with a protein size of 62 kDa, which specifically bound to anti-NDV serum. The levels of anti-F IgG and sIgA antibodies in each test group were significantly higher than those in the control groups. The duration of antibody in the pLA-NDV-F/L. casei three-time immunization group lasted 28 days longer than that in the twice immunized group, and there was no significant difference between antibody peak values. The attack protection rates in each group of immunized pLA-NDV-F/L. casei three times, twice, attenuated vaccine, pLA/L. casei and PBS were 80%, 80%, 90%, 0% and 0%, respectively. Therefore, the antigenic protein of NDV F was successfully expressed by L. casei expression system, which has of reactogenicity and immunogenicity, and could induce protective immune responses in chickens.
Animals ; Antibodies, Viral ; Chickens ; Immunization ; Lactobacillus casei ; Newcastle disease virus ; Vaccines, Attenuated ; Viral Vaccines

Objective:To investigate the mutative rate and spectrum of common hereditary deafness genes in Chinese. Methods:Heel blood samples from 2545 infants born from January to October, 2018, were collected, and screened with microarray chip. Results:There were 119 children with mutation of deafness gene, including 60 cases (2.36%) with GJB2 mutation, male/female = 1∶1 (30/30); 48 (1.88%) with SLC26A4 mutation, male/female nearly 1∶1 (26/22); five (0.20%) with mutation of mitochondrial 12S rRNA gene; five (0.20%) with GJB3 mutation; one (0.04%) with heterozygosis in GJB2 235 and SLC26A4 IVS7-2 mutation. Other more, mutations of 1174A > T, 1229C > T and 15+5G>A of SLC26A4 were found in one child, respectively. Conclusion:The distribution of deafness gene loci has been investigated, which can be reference for prevention and control of hereditary deafness in Chinese.

OBJECTIVETo investigate the relationship between subtelomeric rearrangements and idiopathic mental retardation (MR).

METHODSThirty unrelated patients were recruited using strict selection criteria. Patients were screened by multiplex ligation-dependent probe amplification (MLPA) for subtelomeric imbalance.

RESULTSFive subtelomeric deletions/duplications were identified. They were: 4p deletion, 21p duplication, 10p duplication combined with 4p deletion, 15p duplication, and 9p deletion combined with 3p duplication. These subtelomeric rearrangements were previously unidentified by conventional technique.

CONCLUSIONChildren with unexplained mental retardation are related with subtelomeric rearrangements. MLPA is a rapid and an effective technique for detecting genetic abnormalities in patients with idiopathic MR.

Child ; Chromosome Aberrations ; Female ; Gene Deletion ; Gene Duplication ; Humans ; Intellectual Disability ; diagnosis ; genetics ; Ligase Chain Reaction ; methods ; Male

OBJECTIVETo study the association between tumor necrosis factor-α (TNF-α) G-308A polymorphisms and genetic susceptibility to spontaneous preterm birth (SPTB).

METHODSThe case group enrolled 753 SPTB infants and the control group included 681 term infants. TNF-α G-308A polymorphisms were genotyped using Sequenom MassARRAY®SNP.

RESULTSThe frequencies of the allele (G and A) in the case and control groups were not significantly different (P=0.35). The frequencies of the genotypes (GG, GA and AA) in the case and control groups were not significantly different (P=0.64). The logistic regression analysis found that TNF-α G-308A was not associated with genetic susceptibility to SPTB (OR=0.85; 95%CI: 0.61-1.19; P=0.35).

CONCLUSIONSThere is no association between the polymorphisms of TNF-α G-308A and the genetic susceptibility to SPTB.

Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Infant, Newborn ; Male ; Polymorphism, Single Nucleotide ; Premature Birth ; genetics ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVETo provide mutation analysis and prenatal diagnosis for a family affected with congenital factor VII(FVII) deficiency.

METHODSDNA was extracted from peripheral blood samples from the proband and his parents. All exons and flanking sequence of the FVII gene were amplified with PCR and subjected to direct sequencing. Prenatal diagnosis was performed by amniocentesis.

RESULTSA homozygous mutation (NM_000131.3) c.572-1G>A was identified in the proband. Both parents of the fetus were carriers of the mutation.

CONCLUSIONA method for molecular diagnosis of congenital factor VII deficiency was established and successfully applied for an affected family.

Factor VII Deficiency ; genetics ; Humans ; Infant, Newborn ; Male ; Mutation ; Prenatal Diagnosis

Objective: To investigate the accuracy and reliability of lung ultrasound in diagnosis of pulmonary hemorrhage of the newborn infants. Method: From January 2014 to May 2016, 142 neonates from the Army General Hospital of the Chinese PLA were enrolled in the study. They were divided into two groups: a study group of 42 neonates, who were diagnosed with pulmonary hemorrhage according to their medical history, clinical manifestations and chest X-ray findings, and a control group of 100 neonates with no lung disease. All subjects underwent bedside lung ultrasound in a quiet state in a supine, lateral or prone posture, performed by a single experienced physician. The ultrasound findings were compared between the two groups.Fisher′s exact test was uesd for comparison between two groups. Result: The lung ultrasound main findings associated with pulmonary hemorrhage included: (1) Shred sign: which was seen in 40 patients(95%). (2) Lung consolidation with air bronchograms: which were seen in 35 patients(83%). (3) Pleural effusion: which was seen in 34 infants(81%), pleurocentesis confirmed that the fluid was really bleeding.(4)Atelectasis: which was seen in 14 cases(33%). (5) Pleural line abnormalities and disappearing A-lines with an incidence of 100%. (6) Alveolar-interstitial syndrome: 5 patients(12%)had the main manifestations of alveolar-interstitial syndrome. The above signs were not seen in normal controls (all P<0.01) and the normal lung mainly manifested with clear pleural line and A-lines under ultrasound. Conclusion Lung ultrasonography is accurate and reliable for diagnosing pulmonary hemorrhage, which is suitable for routine application for the diagnosis of pulmonary hemorrhage in the neonatal intensive care unit.

Drug-induced liver injury (DILI) with extrahepatic adverse drug reaction (EHADR) is the injury of extrahepatic organs which is caused by the same drug inducing liver injury and may occur simultaneously or successively with DILI, such as the skin, gastrointestinal tract, blood system, cardiovascular system, and bone and joint system, and it should be taken seriously in clinical practice. DILI with EHADR is not rare, and its pathogenesis may be associated with various factors including the physicochemical properties, distribution, metabolism, excretion, biological activity, and immunological properties of drugs, host genetic background, and underlying diseases. The diagnosis and treatment of DILI with EHADR is more challenging than that of DILI alone.