Micromechanical testing at high strain rates and varying temperatures of 3D-printed polymer structures

Following recent developments, in-situ nanoindenter systems can now perform tests at much faster speeds, enabling us to accomplish micro-compression experiments using strain rates in the range of 103 s-1 for micron-sized specimens. Thus, it has become possible to study deformation processes occurring in the very small scales of all types of materials. Here, we focus on the effect that dynamic testing speeds have on the mechanical behaviour of 3D-printed micron-sized polymer structures. We look at the influence of temperature and compare results obtained from compression and tensile tests. Please click Additional Files below to see the full abstract

Population genomics of Francisella tularensis subsp. holarctica and its implication on the eco-epidemiology of tularemia in Switzerland

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of; Francisella tularensis; subsp.; holarctica; , the causative agent of tularemia in Switzerland. A total of 59; Fth; isolates were obtained from castor bean ticks (; Ixodes ricinus); , animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the; Fth; population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting; Fth; to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks

Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay.; To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed.; MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2 × 10(2) to 8 × 10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample.; Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

BACKGROUND Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis. RESULTS To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms "Membrane" and "Protein Secretion." CONCLUSION In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis

Nanopore sequencing improves the draft genome of the human pathogenic amoeba Naegleria fowleri.

Naegleria fowleri is an environmental protist found in soil and warm freshwater sources worldwide and is known for its ability to infect humans and causing a rapid and mostly fatal primary amoebic meningoencephalitis. When contaminated water enters the nose, the facultative parasite follows the olfactory nerve and enters the brain by crossing the cribriform plate where it causes tissue damage and haemorrhagic necrosis. Although N. fowleri has been studied for several years, the mechanisms of pathogenicity are still poorly understood. Furthermore, there is a lack of knowledge on the genomic level and the current reference assembly is limited in contiguity. To improve the draft genome and to investigate pathogenicity factors, we sequenced the genome of N. fowleri using Oxford Nanopore Technology (ONT). Assembly and polishing of the long reads resulted in a high-quality draft genome whose N50 is 18 times higher than the previously published genome. The prediction of potentially secreted proteins revealed a large proportion of enzymes with a hydrolysing function, which could play an important role during the pathogenesis and account for the destructive nature of primary amoebic meningoencephalitis. The improved genome provides the basis for further investigation unravelling the biology and the pathogenic potential of N. fowleri

Anthrax Spore Detection by a Luminex Assay Based on Monoclonal Antibodies That Recognize Anthrose-Containing Oligosaccharides▿

The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-reactive B. cereus strains, correlating with the results obtained in direct spore ELISA. The Luminex assay (detection limit 103 to 104 spores per ml) was much more sensitive than the corresponding sandwich ELISA. Although not strictly specific for B. anthracis spores, the developed Luminex assay represents a useful first-line screening tool for the detection of B. anthracis spores

Ein nicht alltägliches türkisches Souvenir

Schlussfolgerungen für die Praxis Bei entsprechender Exposition in einem Endemiegebiet und typischer Klinik mit schmerzlosem, zentral nekrotischem, kutanen Ulkus sollte an einen kutanen Anthrax gedacht werden. Bei Einsendung einer Probe in das Primärlabor sollte unbedingt über die Verdachtsdiagnose informiert werden, damit die entsprechenden Biosicherheitsvorgaben eingehalten und eine Gefährdung von Personal und Umwelt ausgeschlossen werden können. Nach Abnahme eines Wundabstrichs bzw. einer Biopsie sollte unverzüglich eine antibiotische Therapie mit Ciprofloxacin oder Clindamycin für sieben Tage eingeleitet werden. Schon der Verdachtsfall ist in der Schweiz innerhalb von zwei Stunden meldepflichtig

Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri.

BACKGROUND The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media. RESULTS As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles. CONCLUSION This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis