How standardization of the pre-analytical phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics.

Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced

The Cell Membrane of a Novel Rhizobium phaseoli Strain Is the Crucial Target for Aluminium Toxicity and Tolerance

Soils with low pH and high aluminium (Al) contamination restrict common bean production, mainly due to adverse effects on rhizobia. We isolated a novel rhizobium strain, B3, from Kenyan soil which is more tolerant to Al stress than the widely used commercial strain CIAT899. B3 was resistant to 50 µM Al and recovered from 100 µM Al stress, while CIAT899 did not. Calcein labeling showed that less Al binds to the B3 membranes and less ATP and mScarlet-1 protein, a cytoplasmic marker, leaked out of B3 than CIAT899 cells in Al-containing media. Expression profiles showed that the primary targets of Al are genes involved in membrane biogenesis, metal ions binding and transport, carbohydrate, and amino acid metabolism and transport. The identified differentially expressed genes suggested that the intracellular γ-aminobutyric acid (GABA), glutathione (GSH), and amino acid levels, as well as the amount of the extracellular exopolysaccharide (EPS), might change during Al stress. Altered EPS levels could also influence biofilm formation. Therefore, these parameters were investigated in more detail. The GABA levels, extracellular EPS production, and biofilm formation increased, while GSH and amino acid level decreased. In conclusion, our comparative analysis identified genes that respond to Al stress in R. phaseoli . It appears that a large portion of the identified genes code for proteins stabilizing the plasma membrane. These genes might be helpful for future studies investigating the molecular basis of Al tolerance and the characterization of candidate rhizobial isolates that perform better in Al-contaminated soils than commercial strains

CORK1, A LRR-Malectin Receptor Kinase, Is Required for Cellooligomer-Induced Responses in Arabidopsis thaliana

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical and chemical distortions, pH changes, and breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers that induce Ca 2+ -dependent CWI responses. We show that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis and are preferentially activated by cellotriose (CT). CORK1 is required for cellooligomer-induced cytoplasmic Ca 2+ elevation, reactive oxygen species (ROS) production, mitogen-associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes, including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that CORK1 is required for CWI and immune responses activated by cellulose breakdown products

Beneficial and pathogenic Arabidopsis root-interacting fungi differently affect auxin levels and responsive genes during early infection

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within three hours after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within one day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content

Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins.

Plants interact with a wide variety of fungi in a mutualistic, parasitic or neutral way. The associations formed depend on the exchange of nutrients and signalling molecules between the partners. This includes a diverse set of protein classes involved in defence, nutrient uptake or establishing a symbiotic relationship. Here, we have analysed the secretomes of the mutualistic, root-endophytic fungus Piriformospora indica and Arabidopsis thaliana when cultivated alone or in a co-culture. More than one hundred proteins were identified as differentially secreted, including proteins associated with growth, development, abiotic and biotic stress response and mucilage. While some of the proteins have been associated before to be involved in plant-microbial interaction, other proteins are newly described in this context. One plant protein found in the co-culture is PLAT1 (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase). PLAT1 has not been associated with plant-fungal-interaction and is known to play a role in abiotic stress responses. In colonised roots PLAT1 shows an altered gene expression in a stage specific manner and plat1 knock-out plants are colonised stronger. It co-localises with Brassicaceae-specific endoplasmic reticulum bodies (ER-bodies) which are involved in the formation of the defence compound scopolin. We observed degraded ER-bodies in infected Arabidopsis roots and a change in the scopolin level in response to the presence of the fungus

Alternaria Brassicae Induces Systemic Jasmonate Responses in Arabidopsis Which Travel to Neighboring Plants via a Piriformsopora Indica Hyphal Network and Activate Abscisic Acid Responses

Stress information received by a particular local plant tissue is transferred to other tissues and neighboring plants, but how the information travels is not well understood. Application of Alternaria Brassicae spores to Arabidopsis leaves or roots stimulates local accumulation of jasmonic acid (JA), the expression of JA-responsive genes, as well as of NITRATE TRANSPORTER (NRT)2.5 and REDOX RESPONSIVE TRANSCRIPTION FACTOR1 (RRTF1). Infection information is systemically spread over the entire seedling and propagates radially from infected to non-infected leaves, axially from leaves to roots, and vice versa. The local and systemic NRT2.5 responses are reduced in the jar1 mutant, and the RRTF1 response in the rbohD mutant. Information about A. brassicae infection travels slowly to uninfected neighboring plants via a Piriformospora Indica hyphal network, where NRT2.5 and RRTF1 are up-regulated. The systemic A. brassicae-induced JA response in infected plants is converted to an abscisic acid (ABA) response in the neighboring plant where ABA and ABA-responsive genes are induced. We propose that the local threat information induced by A. brassicae infection is spread over the entire plant and transferred to neighboring plants via a P. indica hyphal network. The JA-specific response is converted to a general ABA-mediated stress response in the neighboring plant