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Acinetobacter — Microbiology
Published in E. Bergogne-Bénézin, M.L. Joly-Guillou, K.J. Towner, Acinetobacter, 2020
Characterisation of microorganisms by ribotyping has appeared useful in a number of reports (Owen, 1989; Grimont and Grimont, 1986). The method as used for Acinetobacter has been described in detail by Gerner-Smidt (1992). Briefly, it comprises purification of DNA, digestion by restriction enzymes, separation of resulting fragments by agarose gel electrophoresis, and transfer to nylon membrane by vacuo- blotting. Selective banding patterns are visualised by hybridisation with a non-radioactive digoxigenin-ll-dUTP-labelled probe of cDNA from rRNA of E. coli (see Appendix VIII). By this method Gerner-Smidt (1992) investigated 70 strains that were identified as either A. calcoaceticus, A. baumannii or the unnamed genomic species 3 and 13TU by DNA-DNA hybridisation. Patterns generated by restriction with EcoRI, Clal or Sall were specific for each genomic group and are thus useful for identification. Reproducibility was excellent, and the discriminatory power for typing by combined use of the three enzymes was high, with 52 different patterns in 70 unrelated strains.
Fucosidosis
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
The gene is composed of eight exons spanning 23 kb [31]. Three patterns of mRNA were found [32] in Italian patients: two lacked mRNA; one had reduced amounts of an RNA with a cDNA by hybridization of a pattern indicating loss of a restriction site; and three had mRNA that was normal in size and content. Among mutations defined, a deletion of two exons [10] resulted in marked reduction of cross-reacting material (CRM) and absence of enzyme activity; a C-to-T transition leading to a TAA stop-codon, p.Q422X, deleting the carboxyl end of the enzyme [16, 33, 34]. This mutation causes loss of an EcoRI restriction site that is useful for molecular diagnosis [11]. A C-to-A change in exon 6 led to a stop codon p.W382X [12]. Among other mutations, most have been missense, not deletions or other major changes in gene structure [10–13, 35, 36]. Nonsense mutations were common. A homozygous nonsense mutation c1295G>A,p.W423X was found in exon 8 in a patient with unusual phenotype [23\ (v.s.). A patient with a missense mutation, p.L405R, was 46 years old despite less than 1 percent of control enzyme activity and no CRM [37]. Most of the mutations reported have led to virtual absence of activity of the enzyme, and this has been independent of the variability seen clinically.
Genetics
Published in Rachel U Sidwell, Mike A Thomson, Concise Paediatrics, 2020
Rachel U Sidwell, Mike A Thomson
These are varying Length fragments of DNA that are used to detect DNA polymorphisms in individuals. Bacterial enzymes known as restriction enzymes, e.g. EcoRl, cut the DNA at recognized sequences known as restriction sites. If a polymorphism or mutation exists within the DNA sequence cut by the restriction enzyme, the DNA will not be cut at this point and the DNA Length will vary from the expected Length and may be detected by gel electrophoresis. Once a variation is detected, DNA sequencing of the DNA region will determine the precise gene change.
Immunogenicity and protection efficacy of enhanced fitness recombinant Salmonella Typhi monovalent and bivalent vaccine strains against acute toxoplasmosis
Published in Pathogens and Global Health, 2021
Fei-Kean Loh, Sheila Nathan, Sek-Chuen Chow, Chee-Mun Fang
Coding sequences of SAG1 (NCBI GenBank: S76248.1, nucleotides 311–1321) and GRA2 (NCBI GenBank: L01753.1, nucleotides 987–1223) were codon-optimized using Jcat JAVA Codon Adaptation Tool (Technical University of Braunschweig, DE). The genes were synthesized with NheI at 5ʹ and AvrII at 3ʹ of the sequence (GenScript, USA), which were then restricted and inserted into the NheI site of expression plasmid pSEC10. Successfully obtained clones were DNA sequenced (1st BASE, MYR) and designated as pSEC-SAG1 and pSEC-GRA2 (Figure 1a). Subsequently, PompC-clyA-gene cassette was cleaved as EcoRI-AvrII fragment from recombinant pSEC-gene plasmid and inserted into pSMART-gua-aph-FRT cleaved at MfeI and NheI sites (Figure 1(a-b)). The recombinant integration plasmids were DNA sequenced (1st BASE, MYR). Linear fragment flanked by EcoRI sites was restricted for integration into guaBA locus using λ Red recombination method [19] (Figure 1c). Briefly, the linear fragments were transformed into CVD910 expressing λ red proteins from pKD46 [19]. Removal of kanamycin resistance gene was accomplished using flippase recombinase encoded by pCP20 [20]. The resulting monovalent strains were termed CVD910-SAG1 and CVD910-GRA2. The original guaBA locus promoter (PguaBA) was preserved. Finally, recombinant plasmid pSEC-SAG1 was transformed into CVD910-GRA2 to form the bivalent CVD910-GS strain (Figure 1d). The positive transformants were selected through PCR screening. CVD910-SAG1 was omitted for further construction due to poor fitness.
Modulation of ABCG2 surface expression by Rab5 and Rab21 to overcome multidrug resistance in cancer cells
Published in Xenobiotica, 2020
Mammalian expression vector pEGFP-C2 (Clontech), was used to construct N- terminally tagged ABCG2 (EGFP-ABCG2) fusion plasmid. The plasmid pSIN4-EF2-ABCG2-IRES-Neo containing full length human ABCG2 coding region was a gift from Ren-he Xu (Addgene plasmid # 25983) (Zeng et al., 2009). Fast digest EcoRI and BamHI (Thermo Fisher Scientific) were used to digest the plasmid and full-length ABCG2 fragment was ligated into EcoRI/BamHI digested pEGFP-C2 vector, followed by the transformation into E. coli (DH5-α cells). Positive transformants were sequence verified. GFP tagged expression construct of inactive Rab5A mutant, pEGFP-Rab5A-S34N (Addgene plasmid # 28045) and constitutively active Rab5A mutant pEGFP-Rab5A-Q79L (Addgene plasmid # 28046) were gift from Qing Zhong (Sun et al., 2010).
Tumor-targeting oncolytic virus elicits potent immunotherapeutic vaccine responses to tumor antigens
Published in OncoImmunology, 2020
Yong Luo, Chaolong Lin, Yidi Zou, Fei Ju, Wenfeng Ren, Yanhua Lin, Yale Wang, Xiaoxuan Huang, Huiling Liu, Zeng Yu, Pingguo Liu, Guowei Tan, Quan Yuan, Jun Zhang, Chenghao Huang, Ningshao Xia
HSV-1 strain KOS virus was purchased from ATCC and propagated in U-2 OS cells. The viral titer was determined in U-2 OS cells as previously described.26 For the construction of the donor plasmids, different strategies were used. To generate the 27p plasmid, the 5ʹ and 3ʹ regions flanking the genomic regions of core ICP27 promoter (from nt113422 to nt113590) were amplified with the primer pair #1 and #2, and then sequentially cloned into BamHI and PstI digested PUC57 vector with a linker containing the PmeI and SpeI sites. To generate the 27p-hTERT plasmid, the region for core ICP27 promoter in 27p was replaced with DNA fragments containing the core hTERT promoter,27 using the restriction enzymes PmeI and SpeI. To generate the fICP0 plasmid, the 5ʹ and 3ʹ regions flanking the genomic regions of ICP0 were amplified with the primer pair #3 and cloned into HindIII and EcoRI digested PUC57 vector. To generate the fICP0-GFP plasmid, the ICP0 region of picp0 was replaced with DNA fragments encoding eGFP using the restriction enzymes NcoI and SalI. To generate the PF0 plasmid, the DNA fragment covered 478 bp upstream of the ICP34.5 initiation codon to 409 bp downstream of the ICP0 stop codon was amplified with the primer pair #4 and ligated into PstI and SacI digested pMD18-T vector. The d34.5/0GFP plasmid was derived from PF0 that the ICP34.5 and ICP0 coding region was replaced by a DNA fragment encoding eGFP at the NcoI and SalI site. All primers used in this study are listed in Table S5.