Objective To study the influence of pregnant rats' prenatal chronic stress (PS) on learning and memory of their offspring rats and its possible molecular mechanisms.Methods Pregnant females were individually restrained for 45 min 3 times a day during pregnancy from day 14 to day 21.Control pregnant females were left undisturbed in their home cages.The rat offsprings were randomly assigned to PS group or control group.Males and females were kept for the study separately.The learning and memory of the developing rat offspring in the Morris water maze were examined.The basal levels of corticosterone (COR) and adreno-cortico-tropic-hormone (ACTH) were analyzed by using radioimmunoassay.The Golgi-Cox impregnation technique was used to compare density and morphology of the CA1 hippocampal dendritic spines.Results The escape latency (EL) to find the platform in the control group was significantly less than that in the PS group in female rat offspring (F =4.533,P ＜ 0.05),and the difference was statistically significant on the 5th day (t =2.788,P ＜ 0.01).EL to find the platform in the control group was significantly less than that in the PS group in male rat offspring (F =6.101,P ＜0.05),and the difference was statistically significant on the second day (t =3.051,P ＜ 0.01).In the space exploration experiments of the water maze,the retention time observed for the control group and the PS group in the goal quadrant was similar(P ＞ 0.05).The basal levels of the serum COR in the PS group were higher than those in the control group of female rat offspring(t =3.658,P ＜ 0.01) and the basal levels of the serum ACTH in the PS group were higher than those in the control group of male rat offsprings(t =2.319,P ＜ 0.05).A simplified pattern was observed in the CA1 hippocampal dendritic spines in the PS group,showing a less extent of dendritic arborization and the density was significantly lower than that in the control group(t =-3.072,P ＜ 0.01).Conclusions Altered function of the hypothalamic-pituitary-adrenal axis in the offspring mediates the cognitive alterations observed following prenatal stress should to be associated with the lower density and simplified pattern of CA1 dendritic spines.

Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines.

Objective To observe the effect of different thyroid hormone level on the expression of synaptotagmin Ⅰ(Syt Ⅰ) in adult rat hippocampus. Methods All 28 adult male SD rats were assigned randomly into hypothyroid, hyperthyroid and control group, hypothyroid group was established by daily intraperitoneal injections with propylthiou raci(PTU, 10.0 mg/kg body weight) for 6 weeks and hyperthyroid group with L-Thyroxine (L-T4, 0.5 mg/kg body weight) for 3 weeks. Radioimmunity method was used to assay the levels of serum T3 and T4, immunohistochemical S-P technology to assay the levels of Syt Ⅰ protein in hippoeampus CA1, CA3 and dentate gyrus (DG). The layers analyzed in the different subfields include the polymorphic cell layer(the stratum oriens, SO), pyramidal cell layer(PCL), stratum radiatum (SR), lacunosum-molecular layer (SLM) in CA1 and CA3, granular cell layer(GL) and molecular layer(ML) in DG. Results The levels of serum T3 and T4[(0.34±0.12), (41.03± 11.37)nmol/L]in the hypothyroid rats were significantly lower than those in the control group[(0.65±0.15), (55.20±10.68)nmol/L, P < 0.01 or < 0.05], and the positive granule of Syt Ⅰ was significantly lower in PCL and SR of CA1 and CA3, GL of DG. The average optical value responsible for Syt Ⅰ immunoreactivity was obviously reduced in SO(0.048±0.007), PCL(0.299±0.035), SR(0.042±0.007), SLM(0.038±0.006) of CA1, PCL(0.085± 0.019), SR(0.040±0.011), SLM (0.038±0.006) of CA3, GL (0.076±0.019) of DG than normal controls (0.068± 0.014, 0.376±0.053, 0.053±0.008,0.056±0.009,0.118±0.026,0.052±0.010,0.053±0.009,0.099±0.015; P< 0.01 or < 0.05). Serum T3 and T4 levels [(1.43±0.30), (157.18±19.95)nmol/L]of hyperthyroid rats were significantly higher than those of control group(P < 0.01). The value was reduced in PCL(0.322±0.050), SR(0.039±0.006), SLM (0.042±0.006) of CA1, PCL(0.098±0.034), SR(0.046±0.013), SLM(0.046±0.010) of CA3 and GL(0.085± 0.024), ML (0.042±0.009) of DG (P < 0.05 or < 0.01). Conclusion Adult-onset of hypothyroidism and hyperthyroidism can reversibly decrease the expression of Syt Ⅰ in CA1, CA3 and DG regions of hippocampus.

Signal transducer and activator of transcription (STAT) 3 is a critical transcription factor for cell proliferation and survival. It is activated within cells by many cytokines to mediate immune and inflammatory responses to injury. Inflammatory bowel disease (IBD), represented by Crohn′s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disease of the intestinal tract. STAT3 has been shown to be abnormally activated in IBD colon tissues by many pro-inflammatory cytokines, leading to disruption of the intestinal mucosal barrier and excessive innate immune and Th17 responses. The persistent chronic inflammation eventually leads to intestinal fibrosis and stenosis. In addition to immune responses, STAT3 is also involved in intestinal fibrosis in IBD by promoting the transcription of fibrosis-related genes. Colitis-associated cancer (CAC) is a particularly aggressive subtype of colorectal cancer and is associated with chronic inflammation-induced IBD. STAT3 has also been associated with CAC initiation and development. STAT3 is overactivated in tumors, which leads to suppression of the anti-tumor activity of immune cells and promotion of cancer cell proliferation, tumor angiogenesis, invasion, and migration. In the present article, we summarize the role of STAT3 in IBD and CAC and the research progress of the related drugs developed for UC and CAC treatment.

This study was aimed at understanding the serotypes,virulence,drug resistance,and genetics of the pathogenic genes from 29 strains of foodborne Listeria monocytogenes in Guizhou Province in 2022.The minimum inhibitory concentration(MIC)values against eight antibiotics were determined with the microbroth dilution method,and whole genome sequencing was performed on 29 L.monocytogenes strains isolated from food microbiology and foodborne disease surveillance efforts in the province in 2022.The genome sequences were assembled,and bioinformatics analyses were conducted to examine genealogy,serogroups,sequence analysis(ST type),clonal groups(CC type),resistance genes,and virulence genes.A total of 29 strains of L.monocytogenes had resistance to zero to eight antibiotics,and carried six resistance genes.All strains of L.monocytogenes carried fosX,mprF,Lin,and norB.The 29 strains of L.monocytogenes belonged to 2 lineages(Ⅰ and Ⅱ):17 strains belonged to lineage Ⅱ,which was the dominant strain,and 12 strains belonged to lineage Ⅰ.The strains were classified into 13 ST types,among which ST8 was dominant,accounting for 31.03％(9/29 strains),and was followed by ST619 and ST121,accounting for three strains each.The strains were di-vided into four serogroups,with 15 strains in serogroups 1/2a and 3a;11 strains in serogroups 1/2b,3b,and 7;2 strains in serogroups 1/2c and 3c;and 1 strain in serogroups 4b,4d,and 4e.The strains were divided into 12 CC types,and one unsub-divided CC type,ST2348,among which CC8 was dominant,accounting for 27.59％(nine strains).A total of 25 virulence genes were found,which belonged to three virulence islands(LIPI-1,LIPI-2,and LIPI-3)and 23 CL types,including one or two strains each,and three CL types including two strains each.Foodborne L.monocytogenes in Guizhou Province has a low level of drug resistance,carries a high number of virulence genes,and shows genetic diversity in serogroups and molecular phe-notypes.These findings should strengthen the continuous surveillance efforts for Listeria monocytogenes.

OBJECTIVETo evaluate the clinical effect of Xuefu Zhuyu Decoction (XFZYD) on the incidence of complications of rib fracture in patients with blunt chest injury.

METHODSOne hundred and twenty patients with rib fracture stratified according to the AIS scale in three layers (1-3) were equally assigned to two groups, the treated group and the control group, all received conventional treatment, but XFZYD was administered to patients in the treated group additionally. The incidence of complications in patients, including atelectasis, pleural effusion, pulmonary contusion, pleurocentesis and closed thoracic drainage, were observed.

RESULTSThe incidence of pleural effusion in patients of AIS-1 and -2 in the treated group was 20% and 45% respectively, which was remarkable lower than that in the control group (55% and 85%) respectively (P < 0.05); in the treated group, 10% patients of AIS-3, for whom close thoracic drainage was applied, while in the control group, the percentage reached 60%, showing significant difference between groups (P < 0.05).

CONCLUSIONXFZYD could reduce the incidence of pleural effusion in patients with blunt chest injured rib fracture of AIS-1 or -2, and reduce the utilization of close thoracic drainage in those of AIS-3, so it is good for clinical practice.

Adult ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Pleural Effusion ; etiology ; prevention & control ; Rib Fractures ; complications ; drug therapy ; Thoracic Injuries ; complications ; Wounds, Nonpenetrating ; complications

OBJECTIVETo identify metastasis-associated genes in tongue carcinoma and to better understand the mechanism underlying tongue carcinoma metastasis. To compared mRNA expression profiles of two tongue carcinoma cell strains with high and low metastatic potentials using microarray technology.

METHODSTca8113 and Tb cells were used as model systems to study the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes were prepared from the mRNA samples of Tca8113 and Tb cells by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double dots of 1 152 human genes, and scanned at two wave lengths. Differential expression genes from the above two cell lines were analyzed using computer. Then six of the different expression genes were further validated by RT-PCR technique.

RESULTSIn the 1 152 clones of known genes and expressed sequence tags that were analyzed, 37 showed significantly different (minimum 2 folds) expression levels in two cell lines. Among the 37 genes, 15 were up regulated (with ratio more than 2) and 22 down regulated (with ratio less than 1/2). The results of RT-PCR analysis were coincident with those of microarray assay.

CONCLUSIONSome of these genes are known to be involved in human tumor antigen, immune surveillance, adhesion, cell signaling pathway and growth control. It is suggested that the microarray in combination with a relevant analysis facilitates rapid and simultaneous identification of multiple genes of interests and in this study it provided a profound clue to screen candidate targets for early diagnosis and intervention.

Carcinoma ; Gene Expression Profiling ; Humans ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; Tongue Neoplasms

OBJECTIVETo construct the plasmid containing short hairpin RNA (shRNA) of TGF-beta1 expression vector.

METHODSShort chain oligonucleotide was designed according to the TGF-beta1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-beta1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-beta1 expression vector was transfected into salivary gland mucoepidermoid carcinoma (Ms) cells by Lipofectomine TM 2000, and its effect on TGF-beta1 expression was observed by RT-PCR and immunohistochemistry.

RESULTSSuccessful construction was identified by enzyme cutting and the constructed plasmid was called pWH1-TGF-beta1. The shRNA and it inhibited the TGF-beta1 mRNA and protein expression effectively.

CONCLUSIONThe constructed TGF-beta1 shRNA expression vector can block the TGF-beta1 expression in salivary gland mucoepidermoid carcinoma cells.

Genetic Vectors ; Humans ; Immunohistochemistry ; Plasmids ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Transforming Growth Factor beta1

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology