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 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 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

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

To explore the effects of vascular endothelial growth factor (VEGF) on the mechanisms of CML pathogenesis, the effect of VEGF on K562 cell apoptosis induced by As(2)O(3) was analyzed through morphologic observation, DNA fragmentation agarose gel electrophoresis and DNA ploidy flow cytometry analysis, and the effect of VEGF on the expression of bcl-X(L), Bax and caspase-3 in K562 cells was determined by Western blot, meanwhile the expression difference between bcl-X(L) and Bax mRNA in above conditions was detected by RT-PCR. The results showed that after VEGF added, the apoptosis of K562 cells reduced, however, there was no significant changes in cell cycle distribution (P > 0.05). At the same time, following the increasing of the concentration of VEGF, expression of mRNA and protein of bcl-X(L) was up-regulated and the expression of Bax protein was down-regulated in K562 cells, and the activation of pro-caspase-3 into caspase-3 was inhibited or reduced. It is concluded that VEGF may suppress the apoptosis of K562 cells through its influence on the bcl-X(L)/Bax expression ratio in K562 cells.
Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Blotting, Western ; Chlorides ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics ; bcl-X Protein ; biosynthesis ; genetics

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