The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Quercetin exhibits neuroprotective effect by reduc-ing amyloid precursor protein expression and oxida-tive stress in Neuro2a/APPswe cell model / 中国药理学与毒理学杂志

OBJECTIVE The abnormal amyloid-β(Aβ)and oxidative stress assiociated with the progression of Alzheimer disease(AD).Quercetin has been reported to possess antioxidant and anti-inflammatory properties in neurodegenerative disorders.In this present study,we designed to characterize the mechanisms by which quer-cetin exerts neuroprotective effects in murine neuroblas-toma N2a cells stably expressing human Swedish mutant amyloid precursor protein(N2a/APP).METHODS N2a/APP cells were treated with quercetin at concentrations of 10,20 and 50 μ mol·L-1 for 24 h.Cell viability was examined with CCK-8 assays.The protein levels of ERK1/2 and Akt were detected by Western blotting.Intra-cellular reactive oxygen species(ROS)was detected by a fluorescent probe 2,7-dichlorofluorescein diacetate(DCFH-DA).The mitochondrial membrane potential(Δψ m)in N2a/APP cells was detected by using JC-1 staining method.Immunofluorescence was used to detect the generation of 8-hydroxy-2′-deoxyguanosine(8-OHdG)and 4-hydroxynonenal(4-HNE).RESULTS Quercetin attenuated the enhancement of p-ERK1/2,reductions of p-Akt,and decreased levels of APP expression.More-over,quercetin alleviated loss of mitochondria membrane potential(MMP)since it attenuates these oxidative stress,as reflected in the levels of ROS,4-HNE and 8-OHdG,was elevated in N2a/APP cells and these effects were again ameliorated by quercetin.CONCLUSION Neuroprotection by quercetin in N2a/APP cells involves normalizing the impaired mitochondrial function and reducing oxidative stress via inactivation of the ERK1/2 and activation of the Akt pathways.

AGO and RDRP genes are involved in the stress response of Aspergillus flavus / 安徽医科大学学报

Objective @# To explore the role of Argonaute ( Ago) gene and RNA⁃Dependent RNA Polymerase (RDRP) gene of Aspergillus flavus in the growth and development about the RNAi mechanism . @*Methods @# A. flavus Ago1 , Ago2 , RDRP1 , RDRP3 gene mutant strains were constructed by homologous recombination . The growth and development of the mutant strains were observed on potato dextrose agar(PDA) + uracil uridine (UU) medium inoculated with 3 μl 106 CFU/mL spores . 200 , 400 μg cell wall pressure agent conidored ( CR) , 0. 8 mol/L , 1 . 6 mol/L osmotic pressure agent NaCl , 2 mmol/L , 4 mmol/L oxidative pressure agent hydrogen peroxide (H2 O2 ) and 0. 01% , 0. 02% genomic damage agent methyl mesylate (MMS) were added to the Yeast extract Glucose Minimum (YGM) + UU medium to analyze the stress response of the mutant strains . @*Results @#A. flavus mutant strains about ΔAgo1 , ΔAgo2 , ΔRDRP1 , ΔRDRP3 were successfully constructed and its growth and development were normal . The ΔAgo1 and ΔAgo2 strains reduced the stress effects on cell wall and osmotic pressure compared to the control . Ago1 gene deletion reduced the effect of H2 O2 , and conversely RDRP3 gene deletion increased the inhibition of H2 O2 . The Ago2 and RDRP1 strains reduced the effect on genetic damage agent . In addition , ΔRDRP1 increased the effect of osmotic stress . @*Conclusion @# The Ago1 , Ago2 , RDRP1 and RDRP3 genes of A. flavus are not in⁃ volved in the regulation of growth rate and asexual reproduction and can participate in the regulating of the host stress response to the environment .

Analysis of DNA methylation in peripheral blood of patients with coal-burning-borne endemic fluorosis / 中华地方病学杂志

Objective:To explore the DNA methylation patterns and methylation differential genes of patients with coal-burning-borne endemic fluorosis, and to provide a basis for study of the pathogenesis of fluoride-induced body injury.Methods:A case-control study was conducted in Shuicheng County, Liupanshui, Guizhou Province, ten patients with severe fluorosis were selected as the fluorosis group in Douqing Township, where people burning high fluorine coal in open range all year round; and ten people without fluorosis phenotype were selected as the control group in Huaga Township, where firewood was the main fuel. Peripheral blood samples were collected from the two groups of people. Reduced representation bisulfite sequencing (RRBS) technique was used to detect the whole genome DNA methylation pattern ( n = 4) and DNA differentially methylated region (DMR), the DMR differential degree (log 2Ratio) and KEGG pathway enrichment analysis were used to screen the methylation differential genes, and real-time PCR was used to verify the mRNA expression levels of the candidate methylation differential genes( n = 10). Results:The methylation pattern analysis results showed that the methylation levels of all C bases in the genome DNA of the fluorosis group and the control group were (61.53 ± 0.59)% and (62.48 ± 1.53)%, respectively; among them, the methylated levels at CG sites were (63.75 ± 0.65)%, (64.36 ± 1.01)%, at CHG sites were (13.79 ± 0.72)%, (16.69 ± 4.06)%, and at CHH sites were (25.12 ± 1.72)%, (29.77 ± 3.97)%. Compared with the control group, patients in the fluorosis group had 1 000 DMR distributed on different autosomes; and the chromosome 19 was the most with 104 segments. There were 978 DMR-related genes, including 265 hypermethylation genes and 713 hypomethylation genes; KEGG pathway enrichment analysis showed that methylation differential genes were mainly involved in cell metabolism, cancers, and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and other signaling pathways; combined with the differential degree of DMR, the hypermethylated succinate dehydrogenase complex flavoprotein subunit A pseudogene 3 (SDHAP3, log 2Ratio = 3.487) and hypomethylated nuclear factor κB inhibitor kinase regulatory subunit γ (IKBKG, log 2Ratio =-4.436) were selected as the candidate genes. There were statistically significant differences in the mRNA expression levels of SDHAP3 (0.54 ± 0.08, 1.00 ± 0.00) and IKBKG (1.32 ± 0.39, 1.00 ± 0.00) between fluorosis group and control group ( F = 22.94, 15.09, P < 0.01 or < 0.05). Conclusion:There are a large number of methylation differential genes in the genomes of patients with coal-burning-borne endemic fluorosis and controls, the hypermethylated SDHAP3 and hypomethylated IKBKG may be involved in fluoride induced body injury.

Protective effect of exogenous H2S on oxygen-glucose deprivation and reoxygenation-induced cell injury of SH-SY5Y cells / 西安交通大学学报(医学版)

【Objective】 To explore the protective effects of exogenous hydrogen sulfide （H2S） on oxygen glucose deprivation and re-oxygenation （OGD/R）-induced SH-SY5Y cell injury. 【Methods】 OGD/R model was established in SH-SY5Y cells. Based on interferences， SH-SY5Y cells were divided into control group （no interference）， OGD/R model group， and treatment groups （OGD/R model treated with different concentrations of NaHS （H2S donor）. The cells were cultured in low glucose medium without fetal bovine serum under 37 ℃， 93% N2 and hypoxia （2% O2） for 12 h， then in complete medium under normoxia （5% O2 ） for 24 h. CCK-8 was used to detect cell viability， and flow cytometry was used for assaying the level of cellular apoptosis. Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins including glucose regulatory protein 78 （GRP78）， CCAAT/enhancer-binding homologous protein （CHOP）， NF-κB P65 and COX-2. 【Results】 The cell viability was lower in OGD/R model group than in the control group （P<0.05）. NaHS treatment at a concentration of 0-0.5 mmol/L increased cell viability in OGD/R model group in a dose-dependent manner （P<0.05）. In contrast， NaHS （C>0.5 mmol/L） decreased cell viability of OGD/R （P<0.05）. The apoptosis level of early stage in OGD/R model group was significantly higher than that in control group （P<0.05）. NaHS treatment at concentrations of 0.2 and 0.4 mmol/L lowered the apoptosis level of early stage in OGD/R model group （P<0.05）. Western blot showed that the protein expression levels of GRP78， CHOP， NF-κB p65， and COX-2 were significantly higher in OGD/R model group than those in control group （P<0.05）. When compared with OGD/R model group， the expression levels of GRP78， CHOP， NF-κBp65 and COX-2 were decreased by NaHS at concentrations of 0.2， 0.4 and 4 mmol/L （P<0.05）， but there was no statistically significant difference among the treatment groups （P>0.05）. 【Conclusion】 NaHS can rescue cell viability of OGD/R-induced cell injury. It may be achieved by inhibiting the activation of NF-κBp65 and COX-2 proteins induced by endoplasmic reticulum stress to reduce the level of cell apoptosis.

A temperature-inducible Targetron system for efficient gene inactivation in Escherichia coli / 生物工程学报

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.

The study of postoperative delirium in predicting dementia in elderly patients with femoral neck fracture / 重庆医学

Objective To study the postoperative delirium in predicting dementia in elderly patients with femoral neck frac‐ture and provide prevention advises for postoperative delirium .Methods 120 elderly patients with femoral neck fracture were in‐cluded .All patients were tested normal by Clinical Dementia Rating (CDR) preoperative .Basic imformation and postoperative deliri‐um in predicting dementia were recored in detail .After one year of follow up ,all patients were tested by CDR again and divided into dementia group and without dementia group .Results There were 40 patients (33 .3% ) with delirium postoperative with dementia 1 week after operation;there were 16 patients got 0 .5 -3 .0 CDR score after one year follow up (40 .0% ) .There were 80 patients (66 .7% ) did not experience delirium postoperative 1 week after operation ,and 4 patients (5 .0% ) with dementia got CDR score higher than zero after one year folloew up;the difference was statistically significant (P<0 .05) .Single factor analyse showed that there were close correlation between age ,introverted ,level of education < 6 years ,diabetes mellitus ,delirium ,LDL‐C level and de‐mentia (P<0 .05) .Multiple factors showed that age ,diabetes history and delirium were the independent risk factors of dementia in elderly patients with femoral neck fracture (P<0 .05) .ROC curve showed that the AUC area of postoperative delirium in predic‐ting dementia in elderly patients with femoral neck fracture was 0 .878 .Conclusion In elderly patients without the history of de‐mentia ,age ,diabetes history and delirium after hip fracture surgery are the major predictor of dementia within half years .

Study on fingerprints correlated with pharmacodynamic of constituents in Herba Erigerontis against neurotoxicity induced by beta-amyloid peptide / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the neuroprotective effects of the constituents in Herba Erigerontis on neuroblastoma SH-SY5Y cells, and find out its possible material foundation in treating Alzheimer's disease (AD).</p><p><b>METHOD</b>Different combinations of the three constituents in Herba Erigerontis were prepared according to the orthogonality experiment, and the indexes (MTT reduction assay, lipid peroxidation and expressions of nAChR alpha7 protein)were observed upon the SH-SY5Y cells followed by treatment of these combinations and beta-amyloid peptide (AP). The pharmacology data thus obtained and peak data in UPLC fingerprint were analyzed through ANOVA and correlationship by SPSS to give the information of active possible material foundation.</p><p><b>RESULT</b>Constituents B and C showed clear activity and peaks of 4, 7-12 did positive correlationship according to the correlation of fingerprints and pharmacology.</p><p><b>CONCLUSION</b>This study makes a valid approach for deducing the active constituents even the exact compounds against neurotoxicity induced by Abeta by correlation of fingerprints and pharmacology.</p>

Protection of Tianshen Yizhi Recipe against low expression of nicotinic receptor and neurotoxicity induced by beta-amyloid peptide / 中西医结合学报

OBJECTIVE: To investigate the inhibition effects of Tianshen Yizhi Recipe (TSYZR), a compound traditional Chinese herbal medicine, on decreased expression of nicotinic acetylcholine receptor (nAChR) and the neurotoxicity as well as lipid peroxidation induced by beta-amyloid peptide (Abeta) in human SH-SY5Y neuroblastoma cells. METHODS: The SH-SY5Y cells were treated by a certain concentration of TSYZR, and then exposed to Abeta(25-35). Methyl thiazolyl tetrazolium reduction assay was carried out to understand the influences of the drugs on cellular viability. Expressions of nAChR subunits (alpha3 and alpha7) at protein and mRNA levels were detected by Western-blotting and reverse transcription polymerase chain reaction, respectively. Lipid peroxidation was measured by thiobarbituric acid to observe the capacity of antioxidant of the drugs. RESULTS: TSYZR at a safe concentration could increase alpha7 protein in the cells, inhibit decreased expressions of alpha3 and alpha7 nAChR subunit proteins, prevent lower expression of alpha7 mRNA in SH-SY5Y cells induced by Abeta, reduce the neurotoxicity and lipid peroxidation resulting from Abeta, but had no significant effect on the lower expression of alpha3 mRNA. CONCLUSIONS: TSYZR can up-regulate the expression of alpha7 nAChR subunit protein and prevent decreased expressions of nAChRs and neurotoxicity as well as lipid peroxidation induced by Abeta. This drug may play an important therapeutic role in treatment of Alzheimer disease.

5, 10-methylenetetrahydrofolate reductase polymorphism in three nationalities of Guizhou in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) among the Han, Buyi and Miao populations in Guizhou and to provide genetic data for establishment of the genetic polymorphism bank of Guizhou Minorities.</p><p><b>METHODS</b>The technique of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype and allele frequencies at two mononucleotide sites (677 and 1298) of MTHFR among the Han population in Libo county, the Buyi population in Libo county and the Miao population in Leishan county.</p><p><b>RESULTS</b>At the site of 677, the T allele frequencies were found to be 22.8%, 16.1%, 10.6%, for the Han, Buyi, Miao populations respectively. At the site of 1298, the C allele frequencies were 28.9%, 39.1%, 48.7% for the Han, Buyi, Miao populations respectively. The frequencies for the combined heterozygote of 677CT/1298AC were 16.66%, 22.7%, 11.1% for the three populations respectively. Moreover, one case with combined homozygote of 677TT/1298CC was seen in the Miao population.</p><p><b>CONCLUSION</b>The polymorphisms of the two mononucleotide sites (677 and 1298) of MTHFR are diverse in different populations. The C allele frequencies at the site of MTHFR 1298 of the Miao population in Leishan county and the Buyi population in Libo county are high, and the C allele frequency in the Miao population is higher than those hitherto reported in literature.</p>

Preparation of heart valve scaffold and cell seeding / 生物医学工程学杂志

To prepare scaffolds for heart valve tissue engineering, porcine heart valves were treated with varied concentrations of trypsin for 32, 56, 80 and 104 h or followed with DNase. And then the structure of acellular valves was observed under light microscope, scanning and transmission electron microscope. Porcine endothelial cells, human endothelial cells, and canine myofibroblasts were reseeded onto the acellularized porcine heart valve scaffolds once a day for 3 days. The valves were analyzed by immunohistochemical staining and electron microscopy. Results show that all endothelial cells and the majority of interstitial cells were removed from the heart valves after digestion with trypsin for 104 h, and the collagen fiber structure remains intact, but the space between collagen fibers increased slightly. Incubation with trypsin for 80 h and then with DNase almost removed all cells, and the collagen fiber structure and the space between the fibers remain intact. After reseeding, human endothelial cells almost fully cover the valve scaffold surface as shown by H-E staining and platelet endothelial cell adhesion molecules (PECAM-1) staining. Xenogeneic porcine endothelial cells also adhered to and grew on the scaffolds. As shown by H-E staining and actin staining, canine myofibroblasts not only adhered to the surface of valve scaffold but also migrated to the inner part of matrix after one week culture. These results suggest that the digestion of porcine heart valves with trypsin combining with DNase is a suitable method to remove cells. The acellular porcine heart valve scaffolds have a quite favorable biocompatibility with human and porcine endothelial cells as well as canine myofibroblasts.

Study on association of apolipoprotein J gene polymorphism with type 2 diabetes mellitus / 中国实用内科杂志

Objective To investigate the association between apolipoprotein J(ApoJ)gene polymorphism and type 2 diabetes mellitus(T2DM).Methods The exons 3、4、7、8 of ApoJ gene were screened by polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)in 61 type 2 DM patients and 60 healthy control subjects of Chinese population.Abnormal bands were sequenced.Results The deletion/insertion polymorphism site in exon 7 of the ApoJ gene on two subject groups had significant difference (P0.05).Conclusion ApoJ of exon 7 deletion/insertion polymorphism is one of the genetic marker of 2 DM and the ApoJ polymorphism may be associated with 2 DM.