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This paper describes the cloning of a gene (pcp) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removes N-terminal pyroglutamic acid residues from polypeptides. This gene was isolated from Streptococcus pyogenes by construction of a gene library with a bacteriophage λ-derived cosmid-Escherichia coli host system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215-amino-acid product of Mr 23 135 consistent with the 26 kDa polypeptide obtained from in vivo overexpression in E. coli. Southern hybridization confirmed that pcp is a single-copy gene on the S. pyogenes chromosome. 5′ and 3′ endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of the pcp gene product from S. pyogenes are discussed and compared with that from Bacillus subtilis. The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new class of peptidase. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is a One Health pathogen found in humans, animals, and the environment, with food representing a potential transmission route. One Health studies are often limited to a single country or selected reservoirs and ribotypes. This study provides a varied and accessible collection of C. difficile isolates and sequencing data derived from human, animal, and food sources across 13 European countries. A total of 441 strains (human hospital- and community-associated cases n  = 280, animal n  = 96, food n  = 65) were analysed by ribotyping, toxinotyping and whole-genome sequencing (WGS). We detected 83 sequence types (STs), with ST11 ( n  = 80 isolates) and ST1 ( n  = 54 isolates) being the most represented. Several STs included strains originating from all source combinations. Further genomic analysis confirmed close genetic relatedness in some of the STs. Additionally, the genomic analysis identified 10 strains from cryptic clades (C-I to C-III) and 4 of them were mono-toxigenic possessing only a variant form of tcd A gene. Amongst 106 ribotypes, 10 were shared between all 3 sources and 68 were source-specific. Some ribotypes were only found at the intersection of human and food source (RT023, RT027), or between human and animal source (RT009, RT045, RT046). C. difficile ribotypes and STs in Europe were diverse. In this collection, some ribotypes showed potential association with food or animal transmission routes. C. difficile strains from divergent clades CI-III, currently emerging in the human population, were rare and mostly food-associated. Trial registration: ClinicalTrials.gov identifier: NCT03503474.

BackgroundThere is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI.AimTo compare data on the populations with CDI in hospitals vs the community across 12 European countries.MethodsFor this point-prevalence study (July-November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin.ResultsThe CDI positivity rate was 4.4% (country range: 0-16.2) in hospital samples, and 1.3% (country range: 0-2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9-35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2-4.3), p = 0.01; OR: 2.0 (1.1-3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe).ConclusionThese findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI.

The ability to realize simultaneous genotyping of multiple single-nucleotide polymorphisms or mutations is valuable in DNA samples from complex multigenic pathologies such as cancer. In this way, the complexity (number of hybridization units per chip) of the developed MICAM DNA chip, and the orientation of the grafted pyrrole oligonucleotides, make it particularly well adapted to the analysis of single-nucleotide polymorphisms/mutations in multiple potential tumoral markers. The proposed genotyping methodology is based on solid-phase minisequencing, where oligonucleotides are designed to anneal immediately upstream of the polymorphism sites, and labeled dideoxynucleotides are used as substrates for polymerase extension. The developed assay was applied to the analysis of the TP53 codon 72 polymorphism on DNA from cell lines and human colorectal samples.

Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping.

With the increasing availability of genetic information and its relationship to human diseases, there is a growing need in the medical diagnostic field for technologies that can proceed to the parallel genotyping of multiple markers. In this paper, we report the development of a new flexible microarray-based method that aims to be inexpensive, accurate, and adapted to routine analysis. The construction of the MICAM (MICrosystem for Analysis in Medicine) DNA chip is based on the controlled electro-synthesis of a conducting polymer film bearing oligonucleotide probes on gold electrodes. First, accessible 3'OH-ends of grafted probes are directly used to conduct single template-dependent nucleotide extension reactions with fluorescence-labeled chain terminators. Then, the fluorescence of incorporated dideoxynucleotides on controls and probes of interest are recorded to assess base calling. Here, we present the development of the methodology to assign the genotype of TP53 (tumor protein p53) codon 72 polymorphism and its application to analysis of genomic DNA from cell lines and from human colorectal samples. The genotyping results obtained by mini-sequencing on the polypyrrole DNA chip were 100% concordant with data obtained by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Moreover, the developed probe array assay has been successfully applied to the detection of TP53 loss of heterozygosity.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

A multiplexed assay based on the codetection of nucleic acids and antibodies in human serum infected by human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus was proposed. The combined immuno- and oligosorbent array (CombOLISA) microarray is prepared in 96-well standard microplates by spotting (1). nucleic probes specific for a virus genome, (2). viral proteins for the capture of serum antibodies, and (3). nonspecific proteins for verifying specificity. Experimental assay conditions were optimized so that both DNA hybridization and immunological reactions can be achieved simultaneously in the same well and buffer and all at the same temperature. A generic detection system based on the precipitation of an insoluble colorimetric substrate in the presence of enzyme-labeled antibodies or streptavidin was proposed. The optical density of each spot was correlated to the corresponding analyte concentration. The influence of critical parameters on CombOLISA performance such as serum concentration was studied. Calibration curves and sensitivity thresholds were established for each parameter. Serial dilutions of serum were correlated to results obtained with validated immunoassay platforms such as a microplate enzyme-linked immunosorbent assay or the VIDAS automat. Also, several HIV- and HBV-infected serum samples were tested independently by CombOLISA and VIDAS. Coefficients of variation for genomic and proteomic parameters vs spot density were below 15%.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) is an exopeptidase commonly called PYRase, which hydrolytically removes the pGlu-proteins. pGlu also known as pyrrolidone carboxylic acid may occur naturally by an enzymatic procedure or may occur as an artifact in proteins or peptides. The enzymatic synthesis of pGlu suggests that this residue may have important biological and physiological functions. Several studies are consistent with this supposition. PYRase has been found in a variety of bacteria, and in plant, animal, and human tissues. For over two decades, biochemical and enzymatic properties of PYRase have been investigated. At least two classes of PYRase have been characterized. The first one includes the bacterial and animal type I PYRases and the second one the animal type II and serum PYRases. Enzymes from these two classes present differences in their molecular weight and in their enzymatic properties. Recently, the genes of PYRases from four bacteria have been cloned and characterized, allowing the study of the primary structure of these enzymes, and their over-expression in heterelogous organisms. Comparison of the primary structure of these enzymes revealed striking homologies. Type I PYRases and bacterial PYRases are generally soluble enzymes, whereas type II PYRases are membrane-bound enzymes. PYRase II appears to play as important a physiological role as other neuropeptide degrading enzymes. However, the role of type I and bacterial PYRases remains unclear. The primary application of PYRase has been its utilization for some protein or peptide sequencing. Development of chromogenic substrates for this enzyme has allowed its use in bacterial diagnosis.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N-terminal pyroglutamyl group from some peptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp). This gene is present in a single copy in the genome of Bacillus subtilis as indicated by Southern blot hybridization analysis. The pcp transcripts were analyzed in Escherichia coli by Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcp gene has been over-expressed in E. coli, allowing the identification and partial characterization of Pcp protein.

The occurrence of beta-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli, coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and amplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes for E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei), independent of the beta-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 x 10(4) bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.