SUMMARY We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multidimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Summary We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

SUMMARYThe majority of land plants live in symbiosis with arbuscular mycorrhizal fungi from the phylum Glomeromycota. This symbiosis improves acquisition of phosphorus (P) by the host plant in exchange for carbohydrates, especially under low-P availability. The symbiosome, constituted by root cortex cells accommodating arbuscular mycorrhizal fungal hyphae, is the site at which bi-directional exchange of nutrients and metabolites takes place. Uptake of orthophosphate (Pi) in the symbiosome is facilitated by mycorrhizaspecific plant Pi transporters. Modifications of the potato Pi transporter 3 (StPT3) promoter were analysed in transgenic mycorrhizal roots, and it was found that the CTTC cis-regulatory element is necessary and sufficient for a transcriptional response to fungal colonization under low-Pi conditions. Phylogenetic footprinting also revealed binary combination of the CTTC element with the Pi starvation response-associated PHR1-binding site (P1BS) in the promoters of several mycorrhiza-specific Pi transporter genes. Scanning of the Lotus japonicus genome for gene promoters containing both cis-regulatory elements revealed a strong over-representation of genes involved in transport processes. One of these, LjVTI12, encoding a member of the SNARE family of proteins involved in membrane transport, exhibited enhanced transcript levels in Lotus roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Down-regulation of LjVTI12 by RNA interference resulted in a mycorrhiza-specific phenotype characterized by distorted arbuscule morphology. The results highlight cooperative cis-regulation which integrates mycorrhiza and Pi starvation signaling with vesicle trafficking in symbiosome development.

Background/Aims: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca²⁺ content. It has been described in literature that RBCs show an increased intracellular Ca²⁺ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca²⁺ uptake and PS exposure. Methods: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca²⁺ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). Results: The percentage of RBCs showing increased Ca²⁺ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca²⁺ content and the PS exposure of RBCs increased after 48 h incubation. Conclusion: No significant differences of intracellular Ca²⁺ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca²⁺ uptake at short-time incubation.

BACKGROUND AND PURPOSEFew neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease EXPERIMENTAL APPROACHWe generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP + ) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used. KEY RESULTSWe initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP + toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP + within the layered cultures played an important role in neuroprotection. CONCLUSIONS AND IMPLICATIONSOur new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD(+) serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261-279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival.

Background/Aims: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca²⁺-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca²⁺ content, scramblase- and PKCα-activation. In addition, the role of the Ca²⁺-activated K⁺ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca²⁺ content and PS exposure, respectively. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. Results: The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K⁺ content. Conclusion: PS exposure is mediated by the Ca²⁺-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca²⁺-dependent and a Ca²⁺-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca²⁺ entry.