Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth.

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Maturation strategy influences expression levels and cofactor occupancy in Fe-S proteins.

Iron-sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe-S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe-4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron-sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered "SufFeScient" derivative of BL21(DE3). Our results show that co-expression of a Fe-S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe-S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe-S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe-S cluster maturation and points out the pitfalls of chemical reconstitution.

Integration of transcriptomic and metabolomic analyses provides insights into response mechanisms to nitrogen and phosphorus deficiencies in soybean.

Nitrogen (N) and phosphorus (P) are two essential plant macronutrients that can limit plant growth by different mechanisms. We aimed to shed light on how soybean respond to low nitrogen (LN), low phosphorus (LP) and their combined deficiency (LNP). Generally, these conditions triggered changes in gene expression of the same processes, including cell wall organization, defense response, response to oxidative stress, and photosynthesis, however, response was different in each condition. A typical primary response to LN and LP was detected also in soybean, i.e., the enhanced uptake of N and P, respectively, by upregulation of genes for the corresponding transporters. The regulation of genes involved in cell wall organization showed that in LP roots tended to produce more casparian strip, in LN more secondary wall biosynthesis occurred, and in LNP reduction in expression of genes involved in secondary wall production accompanied by cell wall loosening was observed. Flavonoid biosynthesis also showed distinct pattern of regulation in different conditions: more anthocyanin production in LP, and more isoflavonoid production in LN and LNP, which we confirmed also on the metabolite level. Interestingly, in soybean the nutrient deficiencies reduced defense response by lowering expression of genes involved in defense response, suggesting a role of N and P nutrition in plant disease resistance. In conclusion, we provide detailed information on how LN, LP, and LNP affect different processes in soybean roots on the molecular and physiological levels.

Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases.

Exploding seed pods evolved in the Arabidopsis relative Cardamine hirsuta via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11, and 17, that precisely colocalize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell-wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

BACKGROUND: The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. METHODS: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. RESULTS: All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. CONCLUSIONS: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

Cisplatin Protein Binding Partners and Their Relevance for Platinum Drug Sensitivity.

Cisplatin is a widely used drug in the treatment of various solid tumors, such as ovarian cancer. However, while the acquired resistance significantly limits the success of therapy, some tumors, such as colorectal cancer, are intrinsically insensitive to cisplatin. Only a small amount of intracellular platinum binds to the target-genomic DNA. The fate of the remaining drug is largely obscure. This work aimed to identify the cytosolic protein binding partners of cisplatin in ovarian and colorectal cancer cells and to evaluate their relevance for cell sensitivity to cisplatin and oxaliplatin. Using the fluorescent cisplatin analog BODIPY-cisplatin, two-dimensional gel electrophoresis, and mass spectrometry, we identified the protein binding partners in A2780 and cisplatin-resistant A2780cis ovarian carcinoma, as well as in HCT-8 and oxaliplatin-resistant HCT-8ox colorectal cell lines. Vimentin, only identified in ovarian cancer cells; growth factor receptor-bound protein 2, only identified in colorectal cancer cells; and glutathione-S-transferase π, identified in all four cell lines, were further investigated. The effect of pharmacological inhibition and siRNA-mediated knockdown on cytotoxicity was studied to assess the relevance of these binding partners. The silencing of glutathione-S-transferase π significantly sensitized intrinsically resistant HCT-8 and HCT-8ox cells to cisplatin, suggesting a possible involvement of the protein in the resistance of colorectal cancer cells to the drug. The inhibition of vimentin with FiVe1 resulted in a significant sensitization of A2780 and A2780cis cells to cisplatin, revealing new possibilities for improving the chemosensitivity of ovarian cancer cells.

A Novel Method for Identification and Quantification of Sulfated Flavonoids in Plants by Neutral Loss Scan Mass Spectrometry.

Sulfur is present in plants in a large range of essential primary metabolites, as well as in numerous natural products. Many of these secondary metabolites contain sulfur in the oxidized form of organic sulfate. However, except of glucosinolates, very little is known about other classes of such sulfated metabolites, mainly because of lack of specific and quantitative analytical methods. We developed an LC-MS method to analyze sulfated flavonoids, a group of sulfated secondary metabolites prominent, e.g., in plants of the genus Flaveria. The method uses a linear gradient of methanol/formic acid in water on a Restek Raptor C18 Core-Shell column for separation of the compounds. The sulfated flavonoids are detected by mass spectrometry (MS) in a negative mode, using a neutral loss of 80 Da after a collision induced dissociation. With this method we were also able to quantify the sulfated flavonoids. We could detect all (mono)sulfated flavonoids described before in Flaveria plus a number of new ones, such as isorhamnetin-sulfate-glycoside. In addition, we showed that sulfated flavonoids represent a substantial sulfur pool in Flaveria, larger than the thiols glutathione and cysteine. The individual species possess different sulfated flavonoids, but there is no correlation between the qualitative pattern and type of photosynthesis. Similar to other sulfur-containing secondary compounds, the concentration of sulfated flavonoids in leaves is reduced by sulfur starvation. The new LC-MS method will enable qualitative and quantitative detection of these secondary metabolites in plants as a pre-requisite to addressing their functions.

Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus.

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34mgL-1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sga15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.

Optimized two-dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA-cisplatin-protein adducts in ovarian cancer cells.

Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin-binding proteins. The method used a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), two-dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA-cisplatin-containing protein adducts in the acidic pH range (pH 4-7). Based on this analytical method we extended the identification of intracellular cisplatin-protein adducts to the alkaline pH range (pH 6-10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA-cisplatin-binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D-3-phosphoglycerate 1-dehydrogenase.

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis.

Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.

Quantification of Polyphosphate in Microalgae by Raman Microscopy and by a Reference Enzymatic Assay.

Polyphosphates have occurred in living cells early in evolution and microalgae contain these important polymers in their cells. Progress in research of polyphosphate metabolism of these ecologically as well as biotechnologically important microorganisms is hampered by the lack of rapid quantification methods. Experiments with the green alga Chlorella vulgaris presented here compared polyphosphate extraction in water, methanol-chloroform, and phenol-chloroform followed by polyphosphate purification by binding to silica columns or ethanol precipitation. The phenol-chloroform extraction of C. vulgaris followed by ethanol precipitation of polyphosphate was shown to be superior to the other tested method variants. Recovery test of added polyphosphate standard to algal biomass showed that the method is accurate. Using this biochemical assay as a validated reference, we show that 2-dimensional, confocal Raman microscopy can serve as a linear proxy for polyphosphate in C. vulgaris with R2 up to 0.956. With this, polyphosphate quantification can be shortened by use of Raman microscopy from days to hours and, additionally, information about intracellular distribution of polyphosphate and heterogeneity among individual cells in algal culture can be obtained. This offers new insights into the dynamics and role of these polymers crucial for phosphorus uptake and storage. This analytical capability is of particular practical importance because algae aid phosphorus sequestration from wastewater and the thus enriched biomass may serve as organic fertilizer. Both these applications have a strong potential in a future sustainable, circular bioeconomy.

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity.

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (1O2). Pure 1O2 was generated by thermolysis at 37°C of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4+2] cycloaddition of 1O2, a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2+2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris(hydroxymethyl)aminomethane (TRIS) or glutathione (GSH). This suggests that 1O2 markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5mM GSH, but not in 25mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again.

Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris.

CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation.

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Assessing the contribution of the two protein disulfide isomerases PDIA1 and PDIA3 to cisplatin resistance.

Intracellular binding of cisplatin to non-DNA partners, such as proteins, has received increasing attention as an additional mode of action and as mechanism of resistance. We investigated two cisplatin-interacting isoforms of protein disulfide isomerase regarding their contribution to acquired cisplatin resistance using sensitive and resistant A2780/A2780cis ovarian cancer cells. Cisplatin cytotoxicity was assessed after knockdown of either protein disulfide isomerase family A member 1 (PDIA1) or protein disulfide isomerase family A member 3 (PDIA3). Whereas PDIA1 knockdown led to increased cytotoxicity in resistant A2780cis cells, PDIA3 knockdown showed no influence on cytotoxicity. Coincubation with propynoic acid carbamoyl methyl amide 31 (PACMA31), a PDIA1 inhibitor, resensitized A2780cis cells to cisplatin treatment. Determination of the combination index revealed that the combination of cisplatin and PACMA31 acts synergistically. Our results warrant further evaluation of PDIA1 as promising target for chemotherapy, and its inhibition by PACMA31 as a new therapeutic approach.

Combination of two-dimensional gel electrophoresis and a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue allows the identification of intracellular cisplatin-protein adducts.

Cisplatin is one of the most widely used anticancer agents, but a major problem for successful chemotherapy is the development of drug resistance of tumor cells against cisplatin. Resistance to cisplatin is a multifactorial problem. A method to detect and identify intracellular cisplatin-protein adducts was developed using a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), 2DE, and ESI-MS/MS. We identified several CFDA-cisplatin-protein adducts including members of the protein disulfide isomerase family (PDI). These are the first results of the detection of intracellular CFDA-cisplatin-protein adducts, which may help to understand the resistance mechanism of cisplatin.

IFN-γ licenses CD11b(+) cells to induce progression of systemic lupus erythematosus.

Autoantibodies are a hallmark of autoimmune diseases, such as rheumatoid arthritis, autoimmune hepatitis, and systemic lupus erythematosus (SLE). High titers of anti-nuclear antibodies are used as surrogate marker for SLE, however their contribution to pathogenesis remains unclear. Using murine model of SLE and human samples, we studied the effect of immune stimulation on relapsing of SLE. Although autoantibodies bound to target cells in vivo, only additional activation of CD8(+) T cells converted this silent autoimmunity into overt disease. In mice as well as in humans CD8(+) T cells derived IFN-γ enhanced expression of Fc-receptors on CD11b(+) cells. High expression of Fc-receptors allowed CD11b(+) cells to bind to antibody covered target cells and to destroy them in vivo. We found that autoantibodies induce clinically relevant disease when adaptive immunity, specific for disease non-related antigen, is activated.

Circulating microRNA-122, -21 and -223 as potential markers of liver injury following warm ischaemia and reperfusion in rats.

The liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) are commonly used but not specific markers to quantify hepatic injury. In this in vivo study it was determined whether hepatic expression and serum levels of the microRNAs (miRNA) miR-122, -21 and -223 are altered and correlated with the release of liver enzymes after warm hepatic ischaemia and reperfusion (IR). Male Wistar rats were subjected to either 45 min of partial (70%) hepatic ischaemia and 240 min of reperfusion (n=7) or sham operation (n=5). Expression levels of miR-122, -21 and -223 were analysed in serum and liver tissue by quantitative polymerase chain reaction and tested for correlation with serum activities of AST, ALT and LDH. The relative expression levels of circulating miR-122 increased after IR and correlated with the serum activity of AST, ALT and LDH. Neither increased serum level of miR-21 nor elevated relative hepatic expression of miR-223 correlated with the serum activity of liver enzymes. The hepatic expression of miR-122 was unaffected by IR. The correlation between circulating miR-122 expression levels and liver enzyme activity qualifies miR-122 as a potential biomarker of warm hepatic IR injury.