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Background: Glycosaminoglycans (GAGs), including hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS) are essential components of the bone and cartilage tissues. CS isolated from the cartilage tissue of various animals has found application in pharmaceuticals, cosmetics and food industries. In the first part of the present work, three methods were used and compared to extract and purify glycosaminoglycans (GAGs) from the cartilage powder of a local cartilaginous marine species «Scyliorhinus canicula». One of these GAGs, chondroitin sulfate (CS), will be exploited for the development of an anti-osteoarthritis generic at the request of a collaborative pharmaceutical industry. Thus this active ingredient must meet the requirements and tests described by the European Pharmacopoeia (Ph. Eur.). These tests are treated in the second part of this work. Results: Among the three methods that have been applied in the present work, in order to optimize the best process for GAGs preparation, enzymatic hydrolysis with papain followed by deproteinisation using trichloroacetic acid (TCA) was found the best one. The separation of the extracted GAGs using agarose gel electrophoresis, and the identification of bands by Fourier Transform Infrared (FT-IR) Spectroscopy, revealed that the cartilage GAGs of « Scyliorhinus canicula» are exclusively chondroitin sulfate (CS) and dermatane sulfate (DS), with proportions of 12.889 and 87.111% respectively, and that CS is of type C. The extraction technique with papain provides a product with GAGs content of around 90%. The TCA deproteinisation yielded the lowest level of protein (2.8%) in the extracted GAGs, less than 3%, which is the standard required by the European Pharmacopoeia (Ph. Eur.). Cetylpyridinium chloride (CPC) assay suggests that the titration technique, although is introduced by the Ph. Eur. for the determination of CS content, is not an accurate method, and that the values obtained by the optimized and validated HPLC method, described in this work, are more exact. Conclusion: The extracted and purified active ingredient is perfectly conform to the tests described by the Ph. Eur. The results suggest that the co-product of Scyliorhinus canicula would be a perfect source of molecules of pharmacological interest, obtained by a simple and non-agressive process. [ABSTRACT FROM AUTHOR]

Objectives: NDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals.Methods: During a 4 year survey (January 2013-December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed.Results: Molecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types.Conclusions: This study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans. [ABSTRACT FROM AUTHOR]

Objectives: To discover the Escherichia coli STs and associated resistance mechanisms in the community in Islamabad, Pakistan by analysis of E. coli isolates in sewage.Methods: One hundred and ten E. coli were isolated from sewage across the city of Islamabad without antibiotic bias and confirmed as E. coli by MALDI-TOF MS. Isolates were characterized by fumC/fimH (CH) typing and core-genome MLST. Resistance mechanisms, virulence genes, phylotypes and plasmid incompatibility types were determined in a subset of isolates by in silico analysis. The genomic position of blaCTX-M-15 was determined using S1-PFGE, probing and Nanopore MinION sequencing.Results and conclusions: The most prevalent STs were ST394, ST10 and ST648, accounting for 39% of all isolates collected and were found at many sites across Islamabad. Carbapenemase genes were absent and only a single isolate of ST131 was found. The most prevalent resistance mechanisms were qnrS1 and blaCTX-M-15, with blaCTX-M-15 penetrating many STs and found in 31% of all collected isolates. However, the majority of the successful STs were blaCTX-M-15 negative indicating that resistance is not the main driver of prevalence. Twenty-three percent of blaCTX-M-15 genes were chromosomally encoded and large ISEcp1-mediated insertions included qnrS1 and several plasmid genes. In all chromosomally encoded isolates no plasmid copies of blaCTX-M-15 were found. The most prevalent ST (ST394) contained many enteroaggregative E. coli virulence genes and the fimH30 variant allele previously linked to the success of ST131. [ABSTRACT FROM AUTHOR]

Phylogeographical research has revealed several paradigm patterns of postglacial range expansion from the Mediterranean peninsulas to more northern parts of Europe. These range expansions have consequences for the genetic constitution of populations. Although many studies have been performed in mainland Europe, the colonization history of the British Isles is relatively poorly studied; the genetic consequences of the last glacial readvances and the climate optimum conditions, as well as the implications of the recent climatic conditions on the population genetic structures, are little understood. Therefore, we selected the common blue butterfly Polyommatus icarus as a model species for understanding more generally the colonization patterns of the British Isles and the genetic dynamics on these islands. Allozyme analyses of this butterfly show a rather high genetic diversity over continental Europe without major genetic differentiation. The situation on the British Isles is completely different. Here, populations show a much lower genetic diversity compared to mainland Europe. The genetic constitution is well differentiated from that observed on the European mainland, and the genetic differentiation among populations in Britain is stronger than at the European scale. These results support the hypothesis that a relatively cold-tolerant species such as the common blue could have colonized the British Isles early during the late glacial period and survived the last glacial readvances in small refugia in the South. The retraction of this species in small isolated populations could have caused the genetic impoverishment found. The subsequent forest climax during the climate optimum possibly restricted further expansion of this early succession species to small pockets all over the British Isles, resulting in the genetic patchwork that is still observed. Additionally, the relatively cool and rainy conditions one these islands might have caused bottlenecks, possibly enforcing these genetic patterns. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ••, ••-••. [ABSTRACT FROM AUTHOR]

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes. [ABSTRACT FROM AUTHOR]

Purpose: Humulus lupulus (H. lupulus), more commonly known as hop, is a member of the Cannabaceae family with male and female flowers on separate plants. It is native in Europe including Lithuania, Asia and North America. Hop has been recognized as a medicinal plant for centuries, nevertheless different medicinal activities Of hop are currently investigated and discovered. An important class of hop compounds is the hop acids, which are classified as α-acids and β-acids. Different varieties of hops vary in amount and composition of hop acids. Methods: Simple capillary zone electrophoresis method has been optimized and applied for the analysis of hop acids in hop cone extracts. Results: With this method the analysis takes ca. 10 min. Repeatability for migration times and peak areas expressed as relative standard deviation were up to 0.21% and 5.96%, respectively. Conclusions: Comparative results of capillary zone electrophoretic analysis of extracts of different hop varieties and conductometric titration, as a standard method for determination of α-acids, are presented. Both methods provide consistent results, however capillary zone electrophoresis is capable of separating co, form of humulones from other forms. [ABSTRACT FROM AUTHOR]

Aim Climatic changes and fluctuations in the past have strongly influenced the distribution of animal and plant species. Such fluctuations are also reflected in the patterns of genetic diversity on both local and global scales. The genetic pattern of the pearly heath butterfly, Coenonympha arcania, was used to evaluate the genetic differentiation of isolated (in north-western Europe), peripheral (in north-eastern Europe) and central (in southern Europe) populations in the context of post-glacial distributional changes of the species. Location Europe (Sweden, Germany, the Baltic states, Italy, Slovenia, Hungary, Romania, Bulgaria). Thus, samples were collected from large parts of the species’ distribution representing the three categories mentioned above. Methods We analysed 18 loci of 569 individuals from 28 populations by allozyme electrophoresis. We used both individual-based and population-based analyses, including F-statistics, various clustering methods and Markov chain Monte Carlo simulations. Results All loci, except Fum, were polymorphic. The mean FST for all samples was 0.18. The mean genetic distance among populations was 0.046. Two major genetic lineages were distinguished. Populations from the centre of the distributional range in southern Europe and the northern periphery of the distributional range differed significantly in their level of genetic variability. The central populations of south-eastern Europe showed high levels of genetic diversity and no differentiation among populations. Main conclusions Most probably the two major genetic lineages evolved during glacial isolation in two disjunct Mediterranean refugia. The lack of genetic differentiation across south-eastern Europe implies a continuous Würm ice age distribution in this area, thus supporting the functional existence of steppe forests throughout this region. The peripheral-isolated populations in Sweden seem to have suffered from one or more severe bottlenecks, resulting in substantial genetic impoverishment. The peripheral-connected eastern Baltic populations, on the other hand, are affected by post-glacial and possibly recurrent gene flow from more central parts of the distribution. [ABSTRACT FROM AUTHOR]

In total, 96 carbapenem-resistant isolates of Acinetobacter baumannii were obtained from 25 hospitals in 17 European countries. Imipenem MICs ranged from <4 to 128 mg/L on retesting by Etest, with MICs ≥16 mg/L being associated with the carriage of genes encoding at least one other class D carbapenemase in addition to the intrinsic OXA-51-like enzyme. Molecular typing results obtained by random amplified polymorphic DNA analysis, followed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested chromosomal DNA, were highly congruent, with 17 different PFGE types being delineated at a cut-off similarity level of 85%. With few exceptions, multiple isolates from a single hospital belonged to the same PFGE type. Seven sequence groups were identified among the 96 A. baumannii isolates, with the majority of isolates ( n = 81) belonging to the previously defined sequence groups 1 and 2, which each included eight PFGE types. These two multinational lineages included the previously defined European clones II and I, respectively, but the problem of resistant A. baumannii in Europe appeared not to be confined solely to these two European clones. Rather, two broader lineages of carbapenem-resistant A. baumannii now seem to be spreading throughout Europe. [ABSTRACT FROM AUTHOR]

Background: The main components of the spatial genetic structure of the populations are neighbourhood size and isolation by distance. These may be inferred from the allele frequencies across a series of populations within a region. Here, the spatial population structure of Proclossiana eunomia was investigated in two mountainous areas of southern Europe (Asturias, Spain and Pyrenees, France) and in two areas of intermediate elevation (Morvan, France and Ardennes, Belgium). Results: A total of eight polymorphic loci were scored by allozyme electrophoresis, revealing a higher polymorphism in the populations of southern Europe than in those of central Europe. Isolation by distance effect was much stronger in the two mountain ranges (Pyrenees and Asturias) than in the two areas of lower elevation (Ardennes and Morvan). By contrast, the neighbourhood size estimates were smaller in the Ardennes and in the Morvan than in the two high mountain areas, indicating more common movements between neighbouring patches in the mountains than in plains. Conclusion: Short and long dispersal events are two phenomena with distinct consequences in the population genetics of natural populations. The differences in level of population differentiation within each the four regions may be explained by change in dispersal in lowland recently fragmented landscapes: on average, butterflies disperse to a shorter distance but the few ones which disperse long distance do so more efficiently. Habitat fragmentation has evolutionary consequences exceeding by far the selection of dispersal related traits: the balance between local specialisation and gene flow would be perturbed, which would modify the extent to which populations are adapted to heterogeneous environments. [ABSTRACT FROM AUTHOR]

Abstract: Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres. [Copyright &y& Elsevier]

The phylogeography of ‘southern’ species is relatively well studied in Europe. However, there are few data about ‘northern’ species, and so we studied the population genetic structure of the arctic-alpine distributed burnet moth Zygaena exulans as an exemplar. The allozymes of 209 individuals from seven populations (two from the Pyrenees, five from the Alps) were studied by electrophoresis. All 15 analysed loci were polymorphic. The mean genetic diversities were moderately high ( A: 1.99; He: 11.5; P: 65%). Mean genetic diversities were significantly higher in the Alps than in the Pyrenees in all cases. FST was 5.4% and FIS was 10%. Genetic distances were generally low with a mean of 0.022 between large populations. About 62% of the variance between populations was between the Alps and the Pyrenees. The two samples from the Pyrenees had no significant differentiation, whereas significant differentiation was detected between the populations from the Alps ( FST = 2.8%, P = 0.02). Zygaena exulans had a continuous distribution between the Alps and the Pyrenees during the last ice age. Most probably, the species was not present in Iberia, and the samples from the Pyrenees are derived from the southern edge of the glacial distribution area and thus became genetically impoverished. Post-glacial isolation in Alps and Pyrenees has resulted in a weak genetic differentiation between these two disjunct high mountain systems. [ABSTRACT FROM AUTHOR]

Objective-To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. Sample Population-27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. Procedure-Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (POR) assay, 16S rANA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. Results-Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. Conclusions and Clinical Relevance-This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms. [Copyright &y& Elsevier]

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies. [ABSTRACT FROM AUTHOR]

Background The pollens from Parietaria Judaica and Parietaria officinalis are a major cause of pollinosis in Europe. Par o I (13.5 kDa) and Par j I (12 kDa), the major allergens from these species, are highly crossreactive. Methods We have immunoscreened a P. judaica pollen cDNA expression library with a rabbit antiserum specific for Par j I and with a serum pool from allergic patients. An immunopositive clone containing a 26 bp insert was further characterized. The insert sequence was determined and the beta-galactosidase fusion protein was partially purified by electroelution from sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)gels. Results This fusion protein specifically and extensively inhibited Par o I and Par o I binding of a rabbit antiserum and of a serum pool obtained from allergic patients. The antifusion-protein antiserum obtained in a rabbit (anti 6a) specifically precipitated radioiodinated purified Par o I in the double antibody radioimmunoassay (DARIA) and competed with antibodies of sera from allergic patients for the binding to Parietaria pollen extract allergens by enzyme linked immunosorbent assay (ELISA). We investigated the prevalence of antibody response towards the 6a epitope in patients naturally sensitized to Parietaria. The presence of 6a specific IgE antibodies was assessed in the sera of 33 patients using inhibition assays. All sera had antibodies with this specificity: the extensive percentage of inhibition reached suggested that they dominated individual ab response. Conclusion In conclusion, the antibody response induced by natural exposure to the pollen of Parietaria appears to be higly focused on a single linear antigenic determinant of the major allergens which may play a relevant role in the development of clinical allergy. This report is, to our knowledge, the first description of a dominant linear epitope of a major allergen. [ABSTRACT FROM AUTHOR]

The article presents clinical recommendations for diagnosis, treatment and follow-up of multiple myeloma from the European Society for Medical Oncology (ESMO). The incidence of the disease in Europe is 6.0/100,000/year. The tests in which diagnosis should be based include detection and evaluation of the monoclonal component by serum and urine protein electrophoresis. Information is also presented on staging and risk assessment and treatment.