Objective To investigate the efficacy of small interfering RNA against Pseudomonos aeruginosa expressing MexA-MexB-OprM multidrug efflux pump in vivo.Methods Two short hairpin (sh)RNA expression vectors targeting the MexB gene,and negative controls,were designed,synthesized,and electrotransformed into the P.aeruginosa strain PAO1.The in vivo therapeutic efficacy of the MexB small interfering (si)RNAs was determined by infecting a murine model of chronic P.aeruginosa lung infection (1 × 107 CFU/ml).The mice were killed on day 3,5 and 7 after infection with the Pseudomonas aeruginosa strains.Results In the murine infection model,treatment with MexB-siRNAs led to significantly reduced bacteria burden of the bellows by day 5 and 7 post-infection,and reduced the P.aeruginosa-induced pathological changes.In addition,MexB-siRNA2 treatment enhanced neutrophil recruitment and production of inflammatory cytokines (IL-1β,IL-12) in the early infection stage (day 3) (P＜0.05),both of which decreased by day 7.Conclusion MexB-siRNA could inhibit both mRNA expression and the activity of P.aeruginosa in vitro.siRNA was effective in reducing the bacterial load in a murine model of chronic lung infection.Targeting of MexB with siRNA appears to be a novel strategy for treating P.aeruginosa infections.

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.

ObjectiveTo investigate the alteration of MexB protein expression by plasmid containing siRNA template strand was transformed into Pseudomonas aeruginosa.Methods siRNA molecules specifically against mexB were designed and ligated into pGPU6/GFP/Neo vector to construct the recombinant plasmid pGPU6/GFP/Neo-siRNA.MexB gene was cloned into expression vector pET22b+ to construct plasmid pET22b+/mexB,the recombinant expression plasmid was transformed into E.coli BL21 (DE3)plysS and protein MexB was induced to express,then purified protein MexB was used to prepare specific antibodies in rabbits.The plasmids with siRNA molecules specifically against mexB were transformed into wild type strain,clinical multiresistant strain and mexB overexpression strain of Pseudomonas aeruginosa by electroporation respectively,and the changes of the expression of MexB were detected in 8,12,24 h respectively by Western blot.ResultspGPU6/GFP/Neo-siRNA was constructed successfully.Protein MexB was expressed successfully and the rabbit polyclonal antibodies against MexB was prepared well.The gene silence of mexB by siRNA molecules was effective in the three strains of Pseudomonas aeruginosa,but it depended on the silencing time.ConclusionThe expression of MexB was reduced in 8 h and 12 h,but in 24 h,the expression was unchanged.

ObjectiveTo explore the effects of hepatitis B virus X protein (HBx) on the expression and promoter methylation of the p16 tumor suppressor gene, and to investigate the epigenetic role of HBx in the development and progression of hepatitis B virus (HBV)-associated hepatocellular carcinomas (HCC). MethodsExperiments were performed in the human hepatoblastoma cell line HepG2, HepG2 cells expressing green fluorescent protein (HepG2/GFP), and HepG2 cells stably expressing GFP-HBx fusion protein (HepG2/GFP-HBx). Western blot was used to determine the expression levels of the p16 protein in HepG2 cells, HepG2/GFP cells, and HepG2/GFP-HBx cells. HepG2/GFP-HBx cells were treated with a universal inhibitor of DNA methyltransferase (DNMT), 5-aza-2'-deoxycytidine (5-aza-2'-dC). Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation of the p16 tumor suppressor gene in HepG2 cells, HepG2/GFP cells, and HepG2/GFP-HBx cells treated with or without 5-aza-2′-dC. Multiple-group comparison was made by analysis of variance. ResultsAccording to the results of Western blot, HepG2/GFP-HBx cells had a significantly lower expression level of the p16 protein than HepG2 cells and HepG2/GFP cells (P=0.0007; P=00014); there was no significant difference in the expression level of the p16 protein between HepG2/GFP and HepG2 cells (P＞0.05). The MSP assay revealed partial CpG methylation in the p16 promoter region in HepG2/GFP-HBx cells. No promoter methylation was detected in HepG2 cells or HepG2/GFP cells. Non-methylation in the p16 promoter region was restored in HepG2/GFP-HBx cells treated with 5-aza-2′-dC. ConclusionIn the hepatoblastoma cell line, HBx down-regulates the expression of the p16 tumor suppressor gene by inducing methylation in its promoter region. The DNMT inhibitor, 5-aza-2′-dC, restores non-methylation in the p16 promoter region. The reversible modification provides new insights for the treatment and prevention of HBV-associated HCC.

Objective To explore the molecular mechanisms of hepatic stimulator substance (HSS) gene knockout in promoting the development and progression of nonalcoholic steatohepatitis (NASH).Methods NASH model mice (n=20) with HSS wild-type (HSS+/+) or HSS gene knockout (HSS-/-) were constructed using modified choline-deficient diet (CD-diet),untreated C57BL6-HSS-/-and C57BL6-HSS+/+ mice (n=20) were considered as control.Ten mice of each group were killed at month 1 and 2,respectively.The levels of triglyceride (TG) and total cholesterol (TC) in liver were measured using ELISA method.Histopathology and collagen deposition in liver tissue were observed using HE staining and Masson staining,respectively.Lipid content in liver tissue was observed and calculated using oil red O staining.The levels of mRNA and proteins of peroxisome proliferators activated receptor gama coactivator 1 alpha (PGC-1α),mitochondrial transcription factor A (TFAM),transcription factor-E2 related factor α (Nrf2),[-loop,dynamin-related protein 1 (Drp1),mitochondrial fission 1 protein (Fis1),mitofusins 1 (Mfn1),autophagy related gene 3 (Atg3) in liver tissue were detected using Real-time PCR and Western blot,respectively.Content of malonaldehyde (MDA),cyclooxygenase Ⅳ (COX Ⅳ) and adenosine tirphosphate (ATP) were measured using kits,and the activity of respiratory chain complex Ⅴ and cytochrome C oxidase in liver tissue were measured using spectrophotometry.the comparison between groups was done by t test.Results The levels of HSS mRNA and protein in mice-HSS-/-were 0.154± 0.04 and 0.08± 0.01,respectively,which were both significantly lower than those in mice-HSS+/+ (0.952 ± 0.08 and 1.362±-0.130,respectively),and t he differences had statistical significance (t =10.244 and 10.375,respectively,both P＜0.05).One month and 2 months after NASH modeled,TC contents in mice-HSS-/ were (248.6±21.5) μmol/g and (217.4±18.0) μmol/g,respectively,which were both remarkably higher than those in mice-HSS+/+ [(153.5 ± 11.2) μmol/g and (140.8 ±7.5) μmol/g,respectively],and the differences had statistical significance (t=15.270 and 10.524,respectively,both P＜0.05).The results form HE staining,oil red O staining and Masson staining indicated that fat deposition,collage deposition and inflammation in liver tissues of mice-HSS-/-were severer than those in mice-HSS+/+.One month after NASH modeled,protein levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=10.705,24.072,9.892 and 17.540,respectively,all P＜ 0.05).Two months after NASH modeled,protein levels of Drp1,Fis1,Mfn1and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=125.378,15.926,34.330 and 13.437,respectively,all P＜0.05).One month after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,the differences had statistical significance (t=36.337,40.825,33.508 and 28.104,respectively,all P＜0.05).Two months after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=35.210,42.375,27.753 and 20.560,respectively,all P＜0.05).The protein levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were lower than those in liver of C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=20.548,31.036,19.445 and 10.974,respectively;two months:t=9.887,13.330,22.375 and 18.903,respectively,all P＜0.05).The mRNA levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were all lower than those in C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=9.087,12.582,21.451 and 7.774,respectively;two months:t=23.758,17.924,9.924 and 15.209,respectively,all P＜0.05).One month and 2 months after NASH modeled,the levels of ATP mRNA in liver of C57BL6-HSS / group were both significantly lower than those in C57BL6-HSS+/+,and the differences had statistical significance 0=43.775 and 28.375,respectively,both P＜0.05);the levels of COXⅣ mRNA in liver of C57BL6-HSS / group were 0.142 ± 0.06 and 0.068± 0.001,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.255± 0.08 and 0.172 ±0.06,respectively),and the differences had statistical significance (t=28.337 and 19.782,respectively,both P＜0.05);the levels of MDA mRNA in liver of C57BL6-HSS-/-group were 0.973 ±0.112 and 1.253±0.054,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.366±0.02 and 0.872±0.05,respectively),and the differences had statistical significance (t=8.357 and 6.582,respectively,both P＜0.05).Conclusion Deletion of HSS accelerates NASH progression via inhibiting mitochondrial fusion,which leads to dysfunction of mitochondrial respiratory chain and inhibition of fatty acid oxidation.

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.
Animals ; Bacterial Outer Membrane Proteins ; metabolism ; Lung ; microbiology ; Membrane Transport Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Pseudomonas Infections ; metabolism ; microbiology ; Pseudomonas aeruginosa ; metabolism

Objective:To investigate the clinical characteristics of patients with aspergillus spondylitis, and to provide reference for timely diagnosis and treatment.Methods:The clinical manifestations, imaging performance, laboratory examination results, diagnosis and treatment outcomes of six patients with confirmed aspergillus spondylitis in Department of Infectious Diseases, Henan Provincial People′s Hospital during April 30, 2015 and May 1, 2020 were retrospectively analyzed.Results:The main manifestations of six patients were fever and neck pain or low back pain. The time from the onset of clinical manifestations to diagnosis was more than two months to 14 months. Spine magnetic resonance imaging (MRI) showed long T1 and T2 signals on vertebral body, high pressure lipid signal, obvious enhanced scan enhancement, and paravertebral abscess formation might be presented. Among the six patients, C-reactive protein increased in four patients, erythrocyte sedimentation rate increased in five patients, β-D-glucan test (G test) increased in three patients, galactomannan antigen test (GM test) increased in four patients. Six patients with aspergillus spondylitis were all confirmed by biopsy of diseased tissue for fungal smear, tissue culture or metagenomics next generation sequencing. After treatment with voriconazole or itraconazole, five patients recovered and one patient was still under treatment.Conclusions:The clinical manifestations and imaging examination of patients with aspergillus spondylitis are nonspecific. Peripheral blood G test and GM test need to be combined for diagnosis. The diagnosis depends on tissue puncture pathology examination, and the metagenomics next generation sequencing is needed if necessary.

Objective:To investigate the efficacy of metagenomic next generation sequencing (mNGS) in the etiological diagnosis of patients with spinal infection, so as to provide reference for timely diagnosis and treatment.Methods:A total of 40 patients with suspected spinal infection admitted to the Department of Infectious Diseases in Henan Provincial People′s Hospital from January 2020 to July 2022 were included. The results of tissue culture, histopathological examination and tissue mNGS detection were analyzed retrospectively. According to the clinical diagnose, the patients were divided into the spinal infection group (28 cases) and the non-spinal infection group (12 cases). The positive rate, sensitivity and specificity of mNGS and tissue culture in the pathogen detection of patients with spinal infection were compared. McNemar test was used for statistical analysis.Results:There were 23 males and 17 females in 40 patients. The positive rate of mNGS was higher than that of tissue culture (75.0%(30/40) vs 12.5%(5/40)), and the difference was statistically significant ( χ2=0.08, P<0.001). Based on clinical diagnostic criteria, the sensitivity of mNGS in the diagnosis of spinal infection was higher than that of tissue culture (82.1% vs 17.9%), with a statistically significant difference ( χ2=0.02, P<0.001), while the specificity compared to the tissue culture (33.3% vs 100.0%), the difference was not statistically significant ( P>0.05). Conclusions:mNGS has a high pathogen detection rate and sensitivity in the etiological diagnosis of patients with spinal infection, which could provide clinical guidance for the diagnosis and treatment of patients with spinal infection.