الملخص
@#Objective To construct a yeast two-hybrid recombinant bait plasmid of human programmed cell death ligand 1(PD-L1)immunoglobulin variable region(IgV)domain gene,detect its expression in yeast and detect the cytotoxicity and self-activation of PD-L1 IgV protein as well as the interaction between PD-L1 IgV and human thioredoxin(hTrx).Methods Human PD-L1 was analyzed by bioinformatics method,and primers were designed to amplify PD-L1 IgV domain based on the coding region of PD-L1 gene registered in NCBI GenBank database. PCR amplification was carried out with pENTERPD-L1 plasmid as template,and then cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait plasmid and pGBKT7 empty vector were transformed into Y2HGold yeast cells respectively,and the PD-L1 IgV gene and its expression were detected by PCR and Western blot;Meanwhile,the protein toxicity and self-activation of PD-L1 IgV were detected,and the interaction between PD-L1 IgV and hTrx was detected by drip plate method.Results The bioinformatics analysis results of PD-L1 were consistent with related reports. The recombinant bait plasmid pGBKT7-PD-L1 IgV was correctly constructed,and Y2HGold positive clone was obtained,in which PD-L1 IgV was stably expressed. The empty vector pGBKT7 and recombinant bait plasmid pGBKT7-PD-L1 IgV grew well on SD/-Trp and SD/-Trp/X-α-Gal plates with the same colony size and number and white colony,but they did not grow on SD/-Trp/X-α-Gal/AbA plates,which indicated that PD-L1 IgV protein had no toxicity and no self-activation effect on yeast. The results of drip plates test showed that all experimental groups grew well on SD/-Trp/-Leu plate,while only positive control group grew on SD/-Trp/-Leu/X-α-Gal/AbA plate and showed blue color,which indicated that bait protein PD-L1 IgV and hTrx did not self-activate,and there was no interaction between them.Conclusion Recombinant human PD-L1 IgV bait plasmid was successfully constructed. PD-L1 IgV protein showed no toxicity and self-activation effect on yeast cells,and there was no interaction between PD-L1 IgV and hTrx. Subsequently,hTrx can be used to construct a peptide aptamer library,from which peptide aptamers that specifically bind to PD-L1 IgV can be screened.
الملخص
Objective In this study, a strain of colistin and tigecycline-resistant bacteria isolated in 2009 was analyzed, and the structure of drug-resistant plasmid and genetic environment were discussed, so as to provide basis for the prevention and control of multidrug-resistant bacteria. Methods A strain (GZ12244) with positive mcr and tet(M) was obtained by screening colistin and tigecycline resistance genes. Vitek-2 was used for strain identification, and the drug sensitivity test was carried out by broth dilution method. The molecular typing, drug resistance genes, insertion sequences, plasmid structure and genetic background were analyzed by genome-wide sequencing and bioinformatics. Results Strain GZ12244 is Klebsiella pneumoniae, which is resistant to colistin B, tigecycline, cefuroxime and tetracycline, and carries a variety of drug-resistant related genes such as mcr-1 and tet(M), and some of the drug-resistant genes with antibiotic efflux and antibiotic target change have amino acid substitution mutations. Mcr-1 and tet(M) coexist in a plasmid, and mcr-1 flanked by two insertion sequences ISApl1. There are insertion sequences such as IS15, IS1D and ISEc63 in the upstream and downstream of tet(M) gene. Conclusion Klebsiella pneumoniae GZ12244 is a multidrug-resistant strain. The drug-resistant gene exists in plasmid, and the mobile elements in upstream and downstream may spread the drug-resistant gene.
الملخص
AIM:To construct a recombinant adenovirus vector carrying the mouse ubiquitin-like with plant homeodomain and RING finger domains 1(Uhrf1)gene,validate the expression of Uhrf1 in neonatal mouse cardiomyo-cytes and explored its role in hydrogen peroxide(H2O2)-induced DNA damage.METHODS:The mouse Uhrf1 gene cod-ing sequence was amplified by polymerase chain reaction(PCR),digested,and inserted into the pADM-CMV-C-FH vec-tor to create the recombinant adenoviral plasmid ADM-Uhrf1.Following transfection into HEK293T cells,we generated re-combinant adenoviral particles,amplified,purified,and determined the titer.Neonatal mouse cardiomyocytes were infect-ed at an multiplicity of infection(MOI)of 50,UHRF1 protein expression was validated via Western blot and immunofluo-rescence staining.H2O2-induced DNA damage was explored along with adenovirus-mediated Uhrf1 overexpression to inves-tigate its role in DNA damage repair.RESULTS:ADM-Uhrf1 virus titer,determined by capsid immunofluorescence as-say,was 1.8×1013 pfu/L.Western blot confirmed a significant increase in UHRF1 protein expression(P＜0.05),with im-munofluorescence indicating predominant nuclear localization.Uhrf1 overexpression effectively inhibited the expression of the DNA damage marker,phosphorylated H2AX protein(γH2AX)(P＜0.01).CONCLUSION:We successfully con-structed a recombinant adenoviral vector carrying the mouse Uhrf1 gene,facilitating Uhrf1 overexpression in neonatal mouse cardiomyocytes.Furthermore,this overexpression effectively alleviated DNA damage in cardiomyocytes.
الملخص
Objective @#To construct lentiviral vector of p62 gene over expression , and stably express p62 gene in human monocytic leukemia cells 1 (THP 1) , and to provide a way to study the role of p62 gene at the cellular lev el .@*Methods @#The p62 gene fragment was amplified by polymerase chain reaction (PCR) , and the amplified product was ligated to the linearized pcDNA3 1 Flag PCDH10 lentiviral vector. After identifying with PCR , the PCR product was cotransfected with the packaging plasmid into human embryonic kidney cells 293 (HEK 293T) . THP 1 cells were infected with recombinant lentivirus . Positive cell clones were screened by ampicillin . Western blot and real time fluorescence quantitative polymerase chain reaction (RT qPCR) were used to detect THP 1 cell lines with high p62 expression ( overexpression group) and THP 1 cell lines transfected with empty plasmid without p62 gene ( control group) . The expression levels of TNF α, IL 1βand Cxcl1 after K. p. infection were detected by RT qPCR .@*Results @#The p62 gene fragment was successfully obtained by PCR and ligated to pcDNA3 1 Flag PCDH10 vector. PCR confirmed that p62 pcDNA3 1 Flag PCDH10 recombinant plasmid was constructed successfully. Am picillin resistant cell lines were selected after lentiviral infection of THP 1 cells . The results of Western blot analysis showed that the THP 1 cells with drug sieve survival increased the expression of P62 protein compared with the con trol cells (P < 0.001) , and RT qPCR analysis showed that the relative mRNA expression of p62 increased (P < 0.001) . THP 1 cells with high expression of P62 were successfully constructed . The levels of TNF-α、IL-1βand C xcl1 from THP 1 cells with high expression of P62 significantly increased after infection with K. p. (P < 0.01) . @*Conclusion @#P62 pcDNA3 1 Flag PCDH10 vector and THP 1 cells with high expression of P62 can be successful ly constructed by three plasmid packaging system , which provides a basis for the study of p62 .
الملخص
Quinolone antibiotics have been commonly used to treat cases of multiple antibiotic resistance. Unfortunately, quinolone antibiotics have so much been resisted by infectious bacterial agents. This study aimed to evaluate the susceptibility of some clinical isolates of E. coli to some commonly used quinolone antibiotics and the determination of the plasmid-encoded quinolone resistance genes. Our results showed the plasmid quinolone-resistance genes in the following prevalence: qnr genes: qnr S (71.4 %); qnr B (15.4 %); qnr S and B (12.1 %); aac (6) lb-cr (4 %); Efflux genes: oqxA (7.7 %); oqxB (25.3 %); qepA (12.1 %); oqxA and oqxB (5.5 %). We conclude that there is a high frequency of Plasmid-mediated quinolone resistance genes in Escherichia coli isolates from clinical samples in South-Eastern Nigeria. These could be responsible for the high incidence of quinolone resistance reported in Enugu. There is a need for whole-genome sequencing to map out all resistance genes.
الملخص
@#Abstract: Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance. However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure (A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection (LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.
الملخص
The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter Pldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×102-8.93×102 CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with Pldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.
الموضوعات
Lacticaseibacillus , Lacticaseibacillus paracasei , Plasmids/genetics , Genetic Vectors/genetics , Lactobacillus/genetics , Escherichia coli/geneticsالملخص
Objective:To investigate the distribution of integrons and plasmid-mediated quinolone resistance (PMQR) genes in clinical isolates of Klebsiella aerogenes and to analyze the relationship between integrons and bacterial resistance to antimicrobial agents. Methods:Ninety-one Klebsiella aerogenes strains isolated from clinical samples in the Fengxian District Central Hospital from November 2015 to March 2021 were used in this study. Class 1 and class 2 integron-integrase genes ( intI1 and intI2) and PMQR genes were screened by PCR. The types of promoters and gene cassette arrays of variable regions were determined by sequencing. Besides, the relationship between integrons and antimicrobial resistance was analyzed. Results:The resistance rate of the 91 Klebsiella aerogenes isolates to aztreonam was more than 40.00% and the resistance rates to other commonly used antimicrobial agents were less than 35.00%. Among the 91 isolates, 30 carried the intI1 gene, while none of them carried the intI2 gene. Seven class 1 integron gene cassette arrays of variable regions were detected and the gene cassette array of aac(6′)-11 C- ΔereA2- IS1247- aac3- arr- ΔereA2 was detected in Klebsiella aerogenes. PcH1 with weak activity was the predominant variable region promoter of class 1 integrons. The detection rates of intI1-positive and intI1-negative isolates in ICU, neurosurgery and other clinical departments were statistically different ( P<0.05). The resistance rate of intI1-positive isolates to some commonly used antibiotics was significantly higher than that of intI1-negative isolates ( P<0.05). qnrS gene was the prevalent PMQR gene. The detection rates of integrons and PMQR genes in Klebsiella aerogenes isolates was low except for the strains isolated in 2016. Conclusions:Antimicrobial resistance in Klebsiella aerogenes was closely related to integrons. The distribution of integrons in Klebsiella aerogenes strains isolated from different clinical departments was different, and the monitoring of drug-resistant strains should be strengthened in ICU and neurosurgery. The resistance to quinolones in Klebsiella aerogenes strains in this region was mainly related to qnrS gene.
الملخص
Aims@#This study was carried out to evaluate the antibiotic susceptibility pattern and plasmid profiling of selected multi-drug resistant bacteria isolated from ear, nose and throat samples collected from two different hospitals in Cross River State.@*Methodology and results @#One hundred two samples of wounds (53), throat (7), ear (11) and sputum (31) were collected from ambulatory patients in two hospitals in Cross River State. Standard microbiological analysis methods were used to examine the samples. The Kirby-Bauer disc diffusion method was used to conduct susceptibility studies. Zippy plasmid miniprep kits were used to extract the plasmid DNA. Ethidium bromide was used for plasmid curing. SSPS was used to analyze replicate data. Results suggest that 71.6% of the samples analyzed were positive for bacterial growth and 28.4% were negative. Bacterial species identified were Staphylococcus aureus, Proteus mirabilis, Klebsiella sp, Escherichia coli, Providencia sp., Pseudomonas aeruginosa, Enterobacter sp., Streptococcus pyogenes and Citrobacter sp. Over 60% of the isolates were resistant to antibiotics used, with augmentin having the highest while graxone recorded the least resistance rate. The plasmid analysis of the multi-drug resistant bacteria revealed 2-4 plasmids per bacterial cell with sizes ranging from 2.0-42.1 kb. The result of the plasmid curing showed that ethidium bromide is a potent plasmid curing agent as none of the genes was amplified. @*Conclusion, significance and impact of study @#This study revealed that the prevalence of antibiotic resistance was alarmingly high in isolated bacteria. It also revealed that the resistance is plasmid-mediated. Therefore, it is important that the rational use of antibiotics be emphasized among the public.
الملخص
Objective To construct a fluorescent expression cell model of botulinum toxin type B light chain(BoNT/B,BLC),and evaluate the effect of small molecule compounds with this model.Methods The BLC gene was inserted into the fluorescent expression vector pEGFP-N1 to construct a recombinant plasmid before being transfected into the neural cell line Neuro-2a cells for expression.The fluorescence expression level of BLC-EGFP protein in the cells was observed under a fluorescence microscope,and the enzyme digestion activity and stability of BLC-EGFP in the cells were detected by Western blotting.Furthermore,the model was used to evaluate the effect of SRC kinase inhibitor KX2-391 on the intracellular stability of BLC-EGFP protein.Results The recombinant expression plasmid pEGFP-N1-BLC was constructed.It was found that the expression level of BCL-EGFP protein in Neuro-2a cells gradually increased over time,and that the intracellular substrate vesicle-associated membrane protein-2(VAMP-2)was cleaved after plasmid transfection.CHX was added to terminate protein synthesis after the plasmid was transfected for 12 h,and the intracellular level of BLC-EGFP did not change significantly within 72 h.Twenty-four hours of treatment with KX2-391 could significantly promote the intracellular degradation of BLC-EGFP protein.Conclusions A cell model of fluorescent expression of botulinum toxin type B light chain has been established,which provides a technical reserve for the subsequent study onthe intracellular persistence mechanism and intracellular antidote screening of botulinum toxin type B light chain.
الملخص
@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.
الملخص
Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.
الموضوعات
Plasmids/genetics , DNA/genetics , Nucleic Acids , Genetic Techniques , Magnetic Phenomenaالملخص
Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
الموضوعات
Animals , Foot-and-Mouth Disease Virus/genetics , Capsid Proteins , Viral Proteins/metabolism , Foot-and-Mouth Disease/prevention & control , Tetracyclines/metabolism , Viral Vaccines , Antibodies, Viral , Mammals/metabolismالملخص
Objective @# Angiotensin Ⅱ Type 2 receptor (AT2R) is a major receptor of angiotensin Ⅱ , which has a protective effect on damage to various tissues and organs．In this study，an overexpression plasmid of AT2R was con- structed to explore the effect of overexpression of AT2R on lipopolysaccharide ( LPS ) -induced inflammatory re- sponse in mouse hepatocytes (AML12 cells) ．@*Methods @#Mice tissues and organs were used as samples to amplify the gene of interest (AT2R) fragments containing EcoR Ⅰ and Hind Ⅲ restriction sites，and then the final product was obtained by digestion and ligation．The final positive clones were sequenced and identified．The pCMV-Flag-N- AT2R plasmid was transfected into HEK 293T cells，and the expression of Flag protein was detected by the Western blot method after 24 h．AT2R expression on AML12 cells was observed by Western blot and laser confocal microsco- py．AML12 cells were treated differently and divided into control group，LPS treatment group，pCMV-Flag-N-AT2R group and pCMV-Flag-N-AT2R + LPS group，and cell viability was detected by CCK8．Western blot detected PCNA proteins and observed cell proliferation ; Cytoinflammatory factor levels were detected by qPCR ; Western blot detec- ted the expression level of nuclear transcription factor NF-кB (p65) in cells. @*Results @#The identification and se- quencing results of EcoR I and Hind III double restriction showed that pCMV-Flag-N-AT2R plasmid was successful- ly constructed，and the detection results of the Western blot method showed successful expression of AT2R protein. Laser confocal observed that there were AT2R receptors on AML12 cells，and AT2R recombinant plasmids could be expressed on AML12; Compared with the control group，the viability and proliferation ability of LPS-treated AML12 cells were weakened，while the levels of IL-6 and TNF-α increased，and the expression level of nuclear transcription factor NF-кB (p65) increased．Compared with the LPS group，the viability and proliferation of cells in AML12 cells treated with pCMV-Flag-N-AT2R and LPS were enhanced，while the levels of IL-6 and TNF-α decreased．The ex- pression of the nuclear transcription factor NF-кB ( p65 ) decreased．@*Conclusion @# AT2R overexpression plasmids were successfully constructed and successfully expressed on AML12 cells，and AT2R could inhibit the inflammatory response of LPS-induced AML12 cells.
الملخص
Las bacterias son capaces de desarrollar mecanismos de resistencia a los antimicrobianos, aquellos adquiridos y transmisibles son los más significativos debido al potencial de diseminación. La aparición de Salmonella enterica con resistencia a C3aG, quinolonas y a colistina representa una amenaza progresiva. El objetivo fue determinar la resistencia a los antimicrobianos y la presencia de los mecanismos de resistencia plasmídicos a quinolonas, ß-lactámicos y colistina en aislados de Salmonella provenientes de la vigilancia integrada de enteropatógenos. Fueron estudiadas 501 cepas de Salmonella spp. colectadas entre los años 2020 y 2021, por la red de enteropatógenos del Laboratorio Central de Salud Pública. Se investigó la resistencia a las C3aG, quinolonas y colistina, en aislamientos de humanos, alimentos, animales de consumo y ambiente. Las cepas estudiadas exhibieron resistencia a tetraciclina (32,5%), ácido nalidíxico (29%), ampicilina (13,2%), nitrofurantoína (11,6%), C3aG (7,2%), cotrimoxazol (5,8%), ciprofloxacina (2,2%). El 18% (90/501) presentaron resistencia trasferible por plásmidos, fueron detectados 111 genes (71 cepas con un gen, 17 cepas dos genes y 2 cepas tres genes diferentes). Qnr B: 41,1% (37/90), mcr-1: 38,9% (35/90), CMY: 23,3% (21/90), CTX-M: 16,7% (15/90) y Qnr S: 3,3% (3/90). Heidelberg fue el serovar predominante en muestras de pollo y el mayor portador de genes de resistencia de tipo CMY y mcr-1. La detección de genes en alimentos y animales de consumo, que pueden transmitirse fácilmente al ser humano es motivo de alerta y resalta la importancia de continuar fortaleciendo la vigilancia multisectorial y multidisciplinaria.
Bacteria can develop antimicrobial resistance mechanisms, those acquired and transmissible being the most significant due to the potential for dissemination. The emergence of Salmonella enterica with resistance to third-generation cephalosporins, quinolones, and colistin represents a progressive threat. The objective was to determine antimicrobial resistance and the presence of plasmid resistance mechanisms to quinolones, ß-lactams, and colistin in Salmonella isolates from integrated surveillance of enteropathogens. Five hundred and one strains of Salmonella spp. collected between 2020 and 2021 were studied by the enteropathogen network of the Laboratorio Central de Salud Publica (Central Public Health Laboratory). Research was conducted on the resistance to third-generation cephalosporins, quinolones, and colistin, isolated from humans, foodstuffs, animals for consumption, and the environment. The strains studied exhibited resistance to tetracycline (32.5%), nalidixic acid (29%), ampicillin (13.2%), nitrofurantoin (11.6%), third-generation cephalosporins (7.2%), cotrimoxazole (5.8%), and ciprofloxacin (2.2%). Eighteen percent (90/501) presented plasmid-transferable resistance, 111 genes were detected (71 strains with one gene, 17 strains with two genes, and 2 strains with three different genes). Qnr B: 41.1% (37/90), mcr-1: 38.9% (35/90), CMY: 23.3% (21/90), CTX-M: 16.7% (15/90), and Qnr S: 3.3% (3/90). Heidelberg was the predominant serovar in chicken samples and the largest carrier of CMY and mcr-1 resistance genes. The detection of genes in foodstuffs and animals for consumption, which can be easily transmitted to humans, is a cause for alarm and highlights the importance of continuing to strengthen multisectoral and multidisciplinary surveillance.
الملخص
Abstract In the last decade Achromobacter spp. has been associated with chronic colonizationin patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequentspecies recovered within this genus, other species such as A. ruhlandii have also been reportedin these patients. Descriptions of mobile elements are scarce in Achromobacter and none ofthem have been originated in A. ruhlandii. The aim of this study was to report the full char-acterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates.Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patientwith CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequencedby Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plas-mid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotidesubstitution translated into one amino acid change among them. These plasmids have a class 1integron containing the aac-(6)-Ib gene, a mercury resistance operon region and the relE/stbEtoxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 fromPseudomonas aeruginosa. This is the first full description and characterization of a plasmid fromA. ruhlandii which was maintained over time.
Resumen Durante la última década, Achromobacter spp. han sido asociadas con la colonización crónica en pacientes con fibrosis quística. Si bien Achromobacter xylosoxidans es la especie más frecuentemente recuperada, otras especies como Achromobacter ruhlandii también fueron reportadas en nuestra región. Sin embargo, pocos reportes se han centrado en la descripción de elementos móviles, y ninguno de ellos los documenta en A. ruhlandii. El objetivo de este estudio fue reportar la caracterización completa de un plásmido conservado en 4 aislamientos clonalmente relacionados de A. ruhlandii. Se recuperaron 9 aislamientos de A. ruhlandii entre 2013 y 2015 de un único paciente con fibrosis quística proveniente de un hospital pediátrico de Buenos Aires, Argentina. Se realizó la secuenciación completa del genoma de los 4 aislamientos seleccionados según el perfil de resistencia antibiótica en un equipo Illumina MiSeq. Estos fueron anotados mediante RAST y curados manualmente. Se detectó la presencia de un solo plásmido de 34.096 pb y 50CDS en los 4 aislamientos, observándose únicamente un cambio nucleotídico traducido en un cambio aminoacídico en un aislamiento. Los plásmidos ensamblados se caracterizaron por presentar un integrón de clase 1 que contenía el gen aac-(6')-Ib, un operón de resistencia a mercurio y el sistema de toxina-antitoxina relE/stbE. Cabe destacar que estos plásmidos poseen un 79% de similitud y un 99% de identidad con el plásmido pmatvim-7 de Pseudomonas aeruginosa. Esta es la primera descripción y caracterización completa de un plásmido proveniente de A. ruhlandii.
الملخص
Drug-resistant bacteria have become a serious threat to human health. Polymyxin has shown strong bactericidal activity to some Gram-negative and Gram-positive bacteria that are resistant to antibiotics， and has become a last-resort treatment option against a variety of multi-drug resistant bacteria. However， due to the abuse of polymyxin in animal breeding， the drug resistance rate of polymyxin in human population has significantly increased. In order to further understand the mechanism of polymyxin resistance， and to take measures to reduce the incidence of polymyxin resistance in the population， this paper reviewed the progress in research of the antibacterial mechanism of polymyxin， the prevalence of polymyxin resistance in the population， the mechanism of polymyxin resistance， and its transmission mode.
الملخص
Drug-resistant bacteria have become a serious threat to human health. Polymyxin has shown strong bactericidal activity to some Gram-negative and Gram-positive bacteria that are resistant to antibiotics， and has become a last-resort treatment option against a variety of multi-drug resistant bacteria. However， due to the abuse of polymyxin in animal breeding， the drug resistance rate of polymyxin in human population has significantly increased. In order to further understand the mechanism of polymyxin resistance， and to take measures to reduce the incidence of polymyxin resistance in the population， this paper reviewed the progress in research of the antibacterial mechanism of polymyxin， the prevalence of polymyxin resistance in the population， the mechanism of polymyxin resistance， and its transmission mode.
الملخص
ObjectiveTo study the underlying mechanism of Liuwei Dihuangwan in inhibiting triple-negative breast cancer through mitogen-activated protein kinase kinase kinase 1 （MAPKKK1） and Krüppel-like factor 4 （KLF4）. MethodFour hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days. The model mice of spontaneous tumors were randomly divided into a model group （normal saline）， a paclitaxel group （0.025 g·kg-1·d-1， ip， 21 days）， and high-， medium- and low-dose Liuwei Dihuangwan groups （7.2， 3.6， 1.8 g·kg-1·d-1， ig）. Tumor tissues were separated until the moribund stage. The tumor volume and weight were measured， and the tumor inhibition rate and the survival time of the tumor mice were calculated （after 6 months， tumor-free mice were assigned into the normal group）. SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture， and MAPKKK1 in MDA-MB-231 cells was silenced. The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot. ResultThe in vivo experimental results showed that compared with the conditions of the normal group， the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped （P<0.01）. Compared with the model group， all medication groups showed reduced tumor volume and weight （P<0.05， P<0.01）， increased tumor inhibition rate， prolonged survival time of tumor mice （P<0.05）， and increased protein expression of MAPKKK1 （P<0.01）. Additionally， the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4 （P<0.01）. The in vitro experiments showed that compared with the conditions of the normal group， the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated， and the protein expression of MAPKKK1 in the paclitaxel group and the high- and medium-dose Liuwei Dihuangwan groups， and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased （P<0.01）. After silencing of MAPKKK1， compared with the conditions of the negative plasmid group （unsilenced MAPKKK1）， the fluorescence intensities of MAPKKK1 and KLF4 and the protein expression decreased in the RNAi-27 positive plasmid group （silenced MAPKKK1） （P<0.05， P<0.01）. Compared with the RNAi-27 positive plasmid group， all medication groups had enhanced fluorescence intensities of MAPKKK1 and KLF4 and protein expression to different degrees （P<0.05， P<0.01）. ConclusionLiuwei Dihuangwan can inhibit the growth of triple-negative breast cancer， and the underlying molecular mechanism is related to the up-regulation of MAPKKK1 and activation of KLF4 expression.
الملخص
Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.