Occurrence of Ribotypes in Germany and Their Association with Different Host Plants and Viruses

The plasmodiophorid Polymyxa graminis transmits plant viruses to cereal crops such as wheat, rye, barley and triticale. Soil samples from different locations and cereal host plants were analyzed for the presence of P. graminis ribotypes I and II, and tested for the occurrence of soil-borne viruses. P. graminis sequences mainly from fields in Germany used for virus resistance trials, but also from a site each in Poland and Denmark were obtained and deposited in the European Nucleotide Archive. The interactions between the components of the pathogen complex – vector ribotype and virus – and the host are discussed

Viruses in winter barley – is the production of this crop in Germany threatened by another soil-borne virus?

Der Wintergerstenanbau wird in Deutschland durch eine Reihe insekten- und pilzübertragbarer Viren bedroht. Die Situation kann sich zukünftig durch ein weiteres bodenbürtiges Virus verschärfen, das aus Gerstenpflanzen, die gegenüber den Erregern der Gelbmosaikvirose resistent waren, isoliert und auf diese rückübertragen werden konnte. In symptomtragenden Pflanzen waren stäbchenförmige Viruspartikeln mit ca. 180 und 300 nm Länge und einem Durchmesser von ca. 20 nm vorhanden, die sich mit goldmarkierten Furovirus-spezifischen Anti­körpern dekorieren ließen. Die serologischen Tests und vorläufigen molekularen Analysen weisen auf eine enge Verwandtschaft des neuen Gerstenvirus mit Isolaten des Soil-borne cereal mosaic virus bzw. Soil-borne wheat mosaic virus hin, die aber bisher in Deutschland nicht an Wintergerste sondern nur in Roggen, Triticale und Winter­weizen gefunden wurden. Weitere Untersuchungen müssen zeigen, welche geographische Verbreitung und wirtschaftliche Bedeutung dieses neue Virus für den Getreideanbau in Deutschland besitzt. Außerdem muss seine genaue Identität zu ähnlichen Furoviren, die in Frankreich bzw. Japan aus Gerste isoliert wurden, aufgeklärt werden.    The production of winter barley in Germany is threatened by a number of insect- and fungus- transmitted viruses. The situation is now worsened by the discovery of a new soil-borne virus. It was isolated from barley varieties possessing resistance to viruses of the barley yellow mosaic disease complex. The new virus has two sizes of rod-shaped virions that measure about 180 nm and 300 nm in length and 20 nm in diameter and can be decorated by immuno­gold labeling using selected furovirus-specific antibodies. Based on results of serological tests and preliminary mole­cular analysis, the new barley virus was closely related to isolates of Soil-borne cereal mosaic virus or Soil-borne wheat mosaic virus, two viruses hitherto only isolated in Germany from rye, triticale and winter wheat but never from winter barley. Further investigations will focus on the geographic distribution and relative importance of the new furovirus in Germany and on its identity to similar furoviruses isolated from barley in France and Japan.   &nbsp

Computational Immunohistochemistry: Recipes for Standardization of Immunostaining

Cancer diagnosis and personalized cancer treatment are heavily based on the visual assessment of immunohistochemically-stained tissue specimens. The precision of this assessment depends critically on the quality of immunostaining, which is governed by a number of parameters used in the staining process. Tuning of the staining-process parameters is mostly based on pathologists' qualitative assessment, which incurs inter- and intra-observer variability. The lack of standardization in staining across pathology labs leads to poor reproducibility and consequently to uncertainty in diagnosis and treatment selection. In this paper, we propose a methodology to address this issue through a quantitative evaluation of the staining quality by using visual computing and machine learning techniques on immunohistochemically-stained tissue images. This enables a statistical analysis of the sensitivity of the staining quality to the process parameters and thereby provides an optimal operating range for obtaining high-quality immunostains. We evaluate the proposed methodology on HER2-stained breast cancer tissues and demonstrate its use to define guidelines to optimize and standardize immunostaining

Predominant modifier of extreme liver cancer susceptibility in C57BR/cdJ female mice localized to 6 Mb on chromosome 17

Sex hormones influence the susceptibility of inbred mice to liver cancer. C57BR/cdJ (BR) females are extremely susceptible to spontaneous and chemically induced liver tumors, in part due to a lack of protection against hepatocarcinogenesis normally offered by ovarian hormones. BR males are also moderately susceptible, and the susceptibility of both sexes of BR mice to liver tumors induced with N,N-diethylnitrosamine relative to the resistant C57BL/6J (B6) strain is caused by two loci designated Hcf1 and Hcf2 (hepatocarcinogenesis in females) located on chromosomes 17 and 1, respectively. The Hcf1 locus on chromosome 17 is the predominant modifier of liver cancer in BR mice. To validate the existence of this locus and investigate its potential interaction with Hcf2, congenic mice for each region were generated. Homozygosity for the B6.BR(D17Mit164-D17Mit2) region resulted in a 4-fold increase in liver tumor multiplicity in females and a 4.5-fold increase in males compared with B6 controls. A series of 16 recombinants covering the entire congenic region was developed to further narrow the area containing Hcf1. Susceptible heterozygous recombinants demonstrated a 3- to 7-fold effect in females and a 1.5- to 2-fold effect in males compared with B6 siblings. The effect in susceptible lines completely recapitulated the susceptibility of heterozygous full-length chromosome 17 congenics and furthermore narrowed the location of the Hcf1 locus to a single region of the chromosome from 30.05 to 35.83 Mb

Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations

Evolution of Retinoid and Steroid Signaling: Vertebrate Diversification from an Amphioxus Perspective

Although the physiological relevance of retinoids and steroids in vertebrates is very well established, the origin and evolution of the genetic machineries implicated in their metabolic pathways is still very poorly understood. We investigated the evolution of these genetic networks by conducting an exhaustive survey of components of the retinoid and steroid pathways in the genome of the invertebrate chordate amphioxus (Branchiostoma floridae). Due to its phylogenetic position at the base of chordates, amphioxus is a very useful model to identify and study chordate versus vertebrate innovations, both on a morphological and a genomic level. We have characterized more than 220 amphioxus genes evolutionarily related to vertebrate components of the retinoid and steroid pathways and found that, globally, amphioxus has orthologs of most of the vertebrate components of these two pathways, with some very important exceptions. For example, we failed to identify a vertebrate-like machinery for retinoid storage, transport, and delivery in amphioxus and were also unable to characterize components of the adrenal steroid pathway in this invertebrate chordate. The absence of these genes from the amphioxus genome suggests that both an elaboration and a refinement of the retinoid and steroid pathways took place at the base of the vertebrate lineage. In stark contrast, we also identified massive amplifications in some amphioxus gene families, most extensively in the short-chain dehydrogenase/reductase superfamily, which, based on phylogenetic and genomic linkage analyses, were likely the result of duplications specific to the amphioxus lineage. In sum, this detailed characterization of genes implicated in retinoid and steroid signaling in amphioxus allows us not only to reconstruct an outline of these pathways in the ancestral chordate but also to discuss functional innovations in retinoid homeostasis and steroid-dependent regulation in both cephalochordate and vertebrate evolution

Antigen-antibody binding kinetics for quantitative molecular diagnostics

The binding of antibody and antigen constitutes the basis of immunoassays. In this thesis, we aim to explore this binding through three perspectives: a) improving the kinetics of the reaction by increasing mass transport, b) developing a novel method to study the kinetics of binding, and c) using immunoassays for quantifying surface antigen on tumors to uncover tumor heterogeneity. Nowadays, immunoassays are mostly performed on surfaces, which facilitates washing steps and exchanging reagents. A key limitation of such assays is the formation of a layer depleted of analyte, which reduces the reaction rate and thus decreases the overall sensitivity of the test. In the first part of this thesis, we explore the most common current strategies to reduce the depletion layer, i.e., the use of shakers. We show that while shakers are easy to use, they often offer unpredictable results. Such results can be greatly improved through the use of microfluidics, which offers the possibility to control mass transport through convective flows, improving signal while reducing total assay time. Nevertheless, microfluidics has not been adapted to the most common substrates used for immunoassays, microtiter plates. Thus, we propose a new microfluidic concept for controlling the mass transport in microtiter plate wells, which through the use of different kinetic zones allows the exploration of samples with high dynamic ranges in a single test. We then develop a method to evaluate the kinetics of antibody binding using fluorescence lifetime imaging microscopy. Such a method allows a characterization of kinetic constants by comparing the fraction of bound to unbound antibodies on a variety of substrates. We demonstrate its use in breast cancer derived cell blocks and in cancer tissues. Finally, we explore the heterogeneity in cancer, i.e., the different phenotypical profiles that cells in cancer show. Studying tumor heterogeneity is critical to understand cancer evolution in a patient and thus be able to perform accurate patient stratification. We develop a method easily implementable in pathology labs that uses the kinetics of binding of antibodies through immunohistochemical stains to quantify the presence of antigens on different regions of a sample. By performing this localized quantification, we can also explore the heterogeneity present on the tissue. We further investigate heterogeneity using a workflow involving the local tissue lysis and antibody microarray analysis. This method shows a higher capacity of multiplexing, which allowed us to investigate the relative variations of 13 proteins and their inter and intra-tumoral heterogeneity, highlighting the presence of multiple phenotypical variants in a single patient. Thus, in this thesis, we show several use scenarios of antibody-antigen binding kinetics, which highlight the potential of this simple binding to provide us with powerful techniques

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening

Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases