Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol)

New sensors benchmark report on Kompsat-3

The following document has been drawn up as a follow up to the Quality Control Record L [i] on the commissioning phase of the Kompsat-3 imagery, planned benchmarking tests as well as the methodology used in the tests. Benchmarking is necessary to be performed in order to estimate the usability of the imagery collected by particular sensor in The Common Agricultural Policy (CAP) image acquisition Campaign. The main requirement that should be fulfilled concerns the planimetric accuracy of the orthoimagery which should not exceed particular thresholds given in VHR Specifications [iii]. The methodologies used in the benchmarking tests were performed based on Guidelines for Best Practice and Quality Checking of Ortho Imagery [ii]. However, in addition the tests were performed according to alternative methodology, described in [i], which differs from the standard one, the GCPs selection/measurement phase i.e. image to image correlation techniques are used.JRC.H.6-Digital Earth and Reference Dat

New sensors benchmark report on WorldView-4 - Geometric benchmarking over Maussane test site for CAP purposes

Imagery collected by recently launched WorldView-4 satellite can be potentially used in The Common Agricultural Policy (CAP) image acquisition Campaign. The qualification and certificate is conducted by performing benchmarking tests, i.e. it has to be checked whether planimetric accuracy of produced orthoimagery does not exceed certain values regulated by JRC. Therefore, benchmarking tests were carried out on three WorldView-4 imagery collected in March and April 2017. This report describes in details how the tests were performed i.e. auxiliary data used, methodology and workflow as well as outcome from the Internal Quality Control. However, to make the tests objective, the orthoimagery have been handed to JRC for External Quality Control which is a base for certification of the sensorJRC.D.5-Food Securit

New sensors benchmark report on WorldView-3

Imagery collected by recently launched WorldView-3 satellite can be potentially used in The Common Agricultural Policy (CAP) image acquisition Campaign. The qualification and certificate is conducted by performing benchmarking tests namely, it has to be checked whether planimetric accuracy of produced orthoimagery does not exceed certain values regulated by JRC. Therefore, benchmarking tests were carried out on two WorldView-3 imagery acquired in October and November 2014. This report describes in detail how the tests were performed i.e. auxiliary data used, methodology and workflow as well as outcome from the Internal Quality Control. However, to make the tests objective, the orthoimagery was handed to JRC for External Quality Control which is a base for certification of the sensor. Such external QC has been performed by the JRC and included in Chapter 7.JRC.H.6-Digital Earth and Reference Dat

Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

<p>Abstract</p> <p>Background</p> <p>The opportunistic food-borne gram-positive pathogen <it>Listeria monocytogenes </it>can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σ^B, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σ^Bexpression during growth of <it>L. monocytogenes </it>EGD-e. Whole genome-based transcriptional profiling was used to identify σ^B-dependent genes at different growth phases.</p> <p>Results</p> <p>We detected 105 σ^B-positively regulated genes and 111 genes which appeared to be under negative control of σ^Band validated 36 σ^B-positively regulated genes <it>in vivo </it>using a reporter gene fusion system.</p> <p>Conclusion</p> <p>Genes comprising the σ^Bregulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σ^B-positively regulated genes of <it>L. monocytogenes </it>are absent in <it>L. innocua</it>. A total of 75 σ^B-positively regulated listerial genes had homologs in <it>B. subtilis</it>, but only 33 have been previously described as being σ^B-regulated in <it>B. subtilis </it>even though both species share a highly conserved σ^B-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.</p

Spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort.

BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. FUNDING: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario

Dopaminergic Neurodegeneration in the Mouse Is Associated with Decrease of Viscoelasticity of Substantia Nigra Tissue.

The biomechanical properties of brain tissue are altered by histopathological changes due to neurodegenerative diseases like Parkinson's disease (PD). Such alterations can be measured by magnetic resonance elastography (MRE) as a non-invasive technique to determine viscoelastic parameters of the brain. Until now, the correlation between histopathological mechanisms and observed alterations in tissue viscoelasticity in neurodegenerative diseases is still not completely understood. Thus, the objective of this study was to evaluate (1) the validity of MRE to detect viscoelastic changes in small and specific brain regions: the substantia nigra (SN), midbrain and hippocampus in a mouse model of PD, and (2) if the induced dopaminergic neurodegeneration and inflammation in the SN is reflected by local changes in viscoelasticity. Therefore, MRE measurements of the SN, midbrain and hippocampus were performed in adult female mice before and at five time points after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin hydrochloride (MPTP) treatment specifically lesioning dopaminergic neurons in the SN. At each time point, additional mice were utilized for histological analysis of the SN. After treatment cessation, we observed opposed viscoelastic changes in the midbrain, hippocampus and SN with the midbrain showing a gradual rise and the hippocampus a distinct transient increase of viscous and elastic parameters, while viscosity and-to a lesser extent-elasticity in the SN decreased over time. The decrease in viscosity and elasticity in the SN was paralleled by a reduced number of neurons due to the MPTP-induced neurodegeneration. In conclusion, MRE is highly sensitive to detect local viscoelastic changes in specific and even small brain regions. Moreover, we confirmed that neuronal cells likely constitute the backbone of the adult brain mainly accounting for its viscoelasticity. Therefore, MRE could be established as a new potential instrument for clinical evaluation and diagnostics of neurodegenerative diseases

A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.

Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein

Enhanced Adult Neurogenesis Increases Brain Stiffness: <i>In Vivo</i> Magnetic Resonance Elastography in a Mouse Model of Dopamine Depletion

<div><p>The mechanical network of the brain is a major contributor to neural health and has been recognized by in vivo magnetic resonance elastography (MRE) to be highly responsive to diseases. However, until now only brain softening was observed and no mechanism was known that reverses the common decrement of neural elasticity during aging or disease. We used MRE in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) mouse model for dopaminergic neurodegeneration as observed in Parkinson’s disease (PD) to study the mechanical response of the brain on adult hippocampal neurogenesis as a robust correlate of neuronal plasticity in healthy and injured brain. We observed a steep transient rise in elasticity within the hippocampal region of up to over 50% six days after MPTP treatment correlating with increased neuronal density in the dentate gyrus, which could not be detected in healthy controls. Our results provide the first indication that new neurons reactively generated following neurodegeneration substantially contribute to the mechanical scaffold of the brain. Diagnostic neuroimaging may thus target on regions of the brain displaying symptomatically elevated elasticity values for the detection of neuronal plasticity following neurodegeneration.</p></div