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With the implementation of Regulation (European Union [EU]) 2017/746 on in vitro diagnostic medical devices (IVDR), from May 26, 2022, onwards, the development and use of diagnostic tests will be governed by a vastly expanded and upgraded EU regulatory framework. We provide here an overview of the amended transition timelines, the role of notified bodies, EU reference laboratories, expert panels, and the Medical Device Coordination Group (MDCG). We also describe the implications of the IVDR for innovative laboratory medicine by explaining the exemption for in-house devices (IH-IVDs). Two key challenges faced by the academic diagnostic sector are: (1) the stipulation on equivalence of tests (article 5.5d), which poses a new condition for the use of IH-IVDs and (2) the gray area between CE marked in vitro diagnostics (CE-IVDs), modified CE-IVDs, Research Use Only (RUO) tests, and IH-IVDs. Furthermore, the results of a questionnaire on current diagnostic practice conducted by European medical societies collaborating in the BioMed Alliance indicate widespread use of IH-IVDs in diagnostic laboratories across Europe and emphasize the need for support and guidance to comply with the IVDR. Diagnostic equivalents of the European Reference Networks (ERNs) for rare diseases could help ensure affordable and equal access to specialized diagnostics across the EU. Concerted action by clinical and laboratory disciplines, regulators, industry, and patient organizations is needed to support the efficient and effective implementation of the IVDR in a way that preserves innovation and safeguards the quality, safety, and accessibility of innovative diagnostics.

Summary Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation-sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer- to non-bilayer-forming lipids, thus contributing to protein and membrane stabilization.

Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes represents a challenging task because of its low abundance. DAG can be measured by direct infusion mass spectrometry (MS) on a quadrupole time-of-flight mass spectrometer after purification from the crude plant lipid extract via solid-phase extraction on silica columns. Different internal standards are employed to compensate for the dependence of the MS and MS/MS signals on the chain length and the presence of double bonds in the acyl moieties. Thus, using a combination of single MS and MS/MS experiments, quantitative results for the different molecular species of DAGs from Arabidopsis can be obtained.

Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes represents a challenging task because of its low abundance. DAG can be measured by direct infusion mass spectrometry (MS) on a quadrupole time-of-flight mass spectrometer after purification from the crude plant lipid extract via solid-phase extraction on silica columns. Different internal standards are employed to compensate for the dependence of the MS and MS/MS signals on the chain length and the presence of double bonds in the acyl moieties. Thus, using a combination of single MS and MS/MS experiments, quantitative results for the different molecular species of DAGs from Arabidopsis can be obtained.

Suberin is a highly persistent cell wall polymer, predominantly composed of long-chain hydroxylated fatty acids. Apoplastic suberin depositions occur in internal and peripheral dermal tissues where they generate lipophilic barriers preventing uncontrolled flow of water, gases, and ions. In addition, suberization provides resistance to environmental stress conditions. Despite this physiological importance the knowledge about suberin formation has increased slowly for decades. Lately, the chemical characterization of suberin in Arabidopsis enabled the proposal of genes required for suberin biosynthesis such as β-ketoacyl-CoA synthases (KCS) for fatty acid elongation and cytochrome P450 oxygenases (CYP) for fatty acid hydroxylation. Advantaged by the Arabidopsis molecular genetic resources the in silico expression pattern of candidate genes, concerted with the tissue-specific distribution of suberin in Arabidopsis, led to the identification of suberin involved genes including KCS2, CYP86A1, and CYP86B1. The isolation of mutants with a modified suberin composition facilitated physiological studies revealing that the strong reduction in suberin in cyp86a1 mutants results in increased root water and solute permeabilities. The enhanced suberin 1 mutant, characterized by twofold increased root suberin content, has increased water-use efficiency and is affected in mineral ion uptake and transport. In this review the most recent findings on the biosynthesis and physiological importance of suberin in Arabidopsis are summarized and discussed.

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Natural habitats are often characterized by a low availability of phosphate. In plants and many bacteria, phosphate deficiency causes different physiological responses, including the replacement of phosphoglycerolipids in the membranes with nonphosphorous lipids. We describe here a processive glycosyltransferase (Pgt) in Mesorhizobium loti ( Rhizobiales ) involved in the synthesis of di- and triglycosyldiacylglycerols (DGlycD and TGlycD) during phosphate deprivation. Cells of the corresponding Δ pgt deletion mutant are deficient in DGlycD and TGlycD. Additional Pgt-independent lipids accumulate in Mesorhizobium after phosphate starvation, including diacylglyceryl trimethylhomoserine (DGTS) and ornithine lipid (OL). The accumulation of the nonphosphorous lipids during phosphate deprivation leads to the reduction of phosphoglycerolipids from 90 to 50%. Nodulation experiments of Mesorhizobium wild type and the Δ pgt mutant with its host plant, Lotus japonicus , revealed that DGlycD and TGlycD are not essential for nodulation under phosphate-replete or -deficient conditions. Lipid measurements showed that the Pgt-independent lipids including OL and DGTS accumulate to higher proportions in the Δ pgt mutant and therefore might functionally replace DGlycD and TGlycD during phosphate deprivation.