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Mixed-lineage leukemia (MLL) fusions are transcriptional activators that induce leukemia, with a dismal prognosis that mandates further elucidation of their transformation mechanism. In this study, knockdown of the direct MLL-ENL target gene polypyrimidine tract binding protein-1 (PTBP1) was rate limiting for cell proliferation and caused a metabolic phenotype associated with reduced glucose consumption and lactate production. This effect was accompanied by a reduction of splice isoform-2 of pyruvate kinase M (PKM2). Because PKM2 restricts glycolytic outflow to provide anabolic intermediates, we tested the consequences of glucose, energy, and Ser/Gly starvation for cell physiology. Administration of deoxyglucose, energetic decoupling with rotenone, and inhibition of Ser biosynthesis by CBR5884 had a significantly stronger influence on self-renewal and survival of transformed cells than on normal controls. In particular, inhibition of Ser synthesis, which branches off glycolysis caused accumulation of reactive oxygen species, DNA damage, and apoptosis, predominantly in leukemic cells. Depletion of exogenous Ser/Gly affected proliferation and self-renewal of murine and human leukemia samples, even though they are classified as nonessential amino acids. Response to Ser/Gly starvation correlated with glucose transport, but did not involve activation of the AMPK energy homeostasis system. Finally, survival times in transplantation experiments were significantly extended by feeding recipients a Ser/Gly-free diet. These results suggest selective starvation as an option for supportive leukemia treatment.

IntroductionunCoVer—Unravelling data for rapid evidence-based response to COVID-19—is a Horizon 2020-funded network of 29 partners from 18 countries capable of collecting and using real-world data (RWD) derived from the response and provision of care to patients with COVID-19 by health systems across Europe and elsewhere. unCoVer aims to exploit the full potential of this information to rapidly address clinical and epidemiological research questions arising from the evolving pandemic.Methods and analysisFrom the onset of the COVID-19 pandemic, partners are gathering RWD from electronic health records currently including information from over 22 000 hospitalised patients with COVID-19, and national surveillance and screening data, and registries with over 1 900 000 COVID-19 cases across Europe, with continuous updates. These heterogeneous datasets will be described, harmonised and integrated into a multi-user data repository operated through Opal-DataSHIELD, an interoperable open-source server application. Federated data analyses, without sharing or disclosing any individual-level data, will be performed with the objective to reveal patients’ baseline characteristics, biomarkers, determinants of COVID-19 prognosis, safety and effectiveness of treatments, and potential strategies against COVID-19, as well as epidemiological patterns. These analyses will complement evidence from efficacy/safety clinical trials, where vulnerable, more complex/heterogeneous populations and those most at risk of severe COVID-19 are often excluded.Ethics and disseminationAfter strict ethical considerations, databases will be available through a federated data analysis platform that allows processing of available COVID-19 RWD without disclosing identification information to analysts and limiting output to data aggregates. Dissemination of unCoVer’s activities will be related to the access and use of dissimilar RWD, as well as the results generated by the pooled analyses. Dissemination will include training and educational activities, scientific publications and conference communications.

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues, which are impeding time-course studies. Here, we show that the metabolic profiling of the minute amounts of sweat sampled from fingertips addresses this challenge. Sweat sampling from fingertips is non-invasive, robust and can be accomplished repeatedly by untrained personnel. The sweat matrix represents a rich source for metabolic phenotyping. We confirm the feasibility of short interval sampling of sweat from the fingertips in time-course studies involving the consumption of coffee or the ingestion of a caffeine capsule after a fasting interval, in which we successfully monitor all known caffeine metabolites as well as endogenous metabolic responses. Fluctuations in the rate of sweat production are accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. To conclude, metabotyping using sweat from fingertips combined with mathematical network modelling shows promise for broad applications in precision medicine by enabling the assessment of dynamic metabolic patterns, which may overcome the limitations of purely compositional biomarkers., Biomonitoring of sweat from fingertips overcomes current limitations in time-resolved metabolomic profiling of humans and may prove to become a powerful, noninvasive tool for precision medicine. Here, in a feasibility study of short interval sampling of sweat from fingertips, the authors assay individual dynamic metabolic patterns of endogenous and exogenous molecules.

Multiple Myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). It develops from a premalignant stage, monoclonal gammopathy of undetermined significance (MGUS), often via an intermediate stage, smoldering MM (SMM). The mechanisms of MM progression have not yet been fully understood, all the more because patients with MGUS and SMM already carry similar initial mutations as found in MM cells. Over the last years, increased importance has been attributed to the tumor microenvironment and its role in the pathophysiology of the disease. Adaptations of MM cells to hypoxic conditions in the BM have been shown to contribute significantly to MM progression, independently from the genetic predispositions of the tumor cells. Searching for consequences of hypoxia-induced adaptations in primary human MM cells, CD138-positive plasma cells freshly isolated from BM of patients with different disease stages, comprising MGUS, SMM, and MM, were analyzed by proteome profiling, which resulted in the identification of 6218 proteins. Results have been made fully accessible via ProteomeXchange with identifier PXD010600. Data previously obtained from normal primary B cells were included for comparative purposes. A principle component analysis revealed three clusters, differentiating B cells as well as MM cells corresponding to less and more advanced disease stages. Comparing these three clusters pointed to the alteration of pathways indicating adaptations to hypoxic stress in MM cells on disease progression. Protein regulations indicating immune evasion strategies of MM cells were determined, supported by immunohistochemical staining, as well as transcription factors involved in MM development and progression. Protein regulatory networks related to metabolic adaptations of the cells became apparent. Results were strengthened by targeted analyses of a selected panel of metabolites in MM cells and MM-associated fibroblasts. Based on our data, new opportunities may arise for developing therapeutic strategies targeting myeloma disease progression.

Toxoplasma gondii is an important ubiquitous protozoan parasite, which can infect almost all warm-blooded vertebrates, including humans. The diagnosis of T. gondii infection is crucial for the prevention, surveillance, and control of its transmission. Here, a triplex real-time PCR assay targeting the B1 gene and 529rep element was used to determine the presence of T. gondii in feathered game (Anas platyrhynchos and Phasianus colchicus) hunted in the Czech Republic. The prevalence of T. gondii was 5.4% in wild ducks (n = 280) and 3.4% in common pheasants (n = 350). Additionally, genotyping of 28 T. gondii-positive samples revealed the presence of archetypal genotypes II and III as well as non-archetypal genotypes combining both type II and III alleles. Our results suggest that consumption of feathered game could pose a risk of T. gondii transmission to humans in the Czech Republic.

This publication shows that C/EBP (CCAAT enhancer–binding protein) transcription factors are mandatory for maintaining the myeloid leukemic phenotype, suggesting a therapeutic option by interfering with the non-oncogenic C/EBP dependence., Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL–transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL–transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPβ deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPβ expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPβ, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention. The authors would like to thank Kratos/Shimadzu (Manchester, UK) for providing the MALDI-MS instrumentation used in this study and Dr. Steven Pelech (Kinexus Bioinformatics Corp, Vancouver, BC, Canada) for initial instruction in antibody microarray kinomic analysis. This work was financially supported by the Medical Scientific Fund of the Mayor of the City of Vienna, by the ‘Initiative Krebsforschung’ of the Medical University of Vienna, and by the Herzfelder Familienstiftung, Vienna, Austria. Sí

Elevated blood pressure remains a major cause of cardiovascular disease, disability, and premature death in Austria, with suboptimal rates of detection, treatment and control also in recent years. Management of hypertension is a common challenge for physicians with different spezializations. In an attempt to standardize diagnostic and therapeutic strategies and, ultimately, to increase the rate of patients with controlled blood pressure and to decrease the burden of cardiovascular disease, 13 Austrian medical societies reviewed the evidence regarding prevention, detection, workup, treatment and consequences of high blood pressure in general and in various clinical scenarios. The result is presented as the first national consensus on blood pressure. The authors and societies involved are convinced that a joint national effort is needed to decrease hypertension-related morbidity and mortality in our country.

Eleven-nineteen leukemia (ENL) is a chromatin reader present in complexes stimulating transcriptional elongation. It is fused to mixed-lineage leukemia (MLL) in leukemia, and missense mutations have been identified in Wilms tumor and acute myeloid leukemia. Here we demonstrate that ENL overcomes polycomb silencing through recruitment of PAF1 via the conserved YEATS domain, which recognizes acetylated histone H3. PAF1 was responsible for antirepressive activities of ENL in vitro, and it determined the transforming potential of MLL-ENL. MLL-ENL target loci showed supraphysiological PAF1 binding, hyperubiquitination of histone H2B and hypomodification with H2AUb, resulting in accelerated transcription rates. YEATS mutations induced a gain of function, transforming primary hematopoietic cells in vitro and in transplantation assays through aberrant transcription and H2B ubiquitination of Hoxa9 and Meis1 Mechanistically, H3 and PAF1 competed for ENL interaction, with activating mutations favoring PAF1 binding, whereas the MLL moiety provided a constitutive PAF1 tether allowing MLL fusions to circumvent H3 competition.

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

The consumption of fruits and vegetables is increasing worldwide because of the positive impact of these foods on human health. Ready-to-eat, raw whole, and frozen fruits and vegetables were purchased from markets and examined for the presence of nontuberculous mycobacteria (NTM) using culture, real-time PCR (qPCR), and sequencing. Using qPCR, Mycobacterium sp. at 10(0) to 10(4) ge/g (genome equivalents per gram) was found in almost all of the 178 samples; members of the M. avium complex were found only sporadically. Culture and sequencing revealed the presence of 22 viable NTM isolates in 17 samples. In addition to NTM commonly found in the environment, several rarely described isolates of viable NTM were recovered. The presence of Mycobacterium shigaense, which has been previously isolated only from human patients, was found in lettuce, the first time that this species has been found in an environmental sample. Mycobacterium parmense, Mycobacterium palustre, and Mycobacterium llatzerense, which have been previously isolated from human patients and occasionally from soil and water, were recovered from leafy green vegetables. Strawberries and cut salad mixes contained Mycobacterium algericum, Mycobacterium fallax, and Mycobacterium minnesotense. NTM are primarily nonpathogenic. However, consumption of fruits or vegetables contaminated with NTM could represent a health risk for immunocompromised people, children, and the elderly.

Mixed lineage leukemia caused by MLL fusion proteins is still a mostly incurable disease. Research on novel treatment strategies has gained momentum in the last years with the elucidation of the molecular mechanisms underlying the transforming potential of these powerful oncoproteins. This review summarizes the recent developments in this area including new attempts to treat MLL in a rational way by exploiting the biochemical vulnerabilities of the leukemogenic process.

Field-grown strawberries, the environment of strawberry farms and fresh strawberries from marketplaces were examined for bacterial, viral, and protozoan pathogens. The presence of bacteria was determined using culture and real-time PCR (qPCR), presence of protozoa and viruses using qPCR and reverse transcription qPCR, respectively. The highest proportion of positivity was observed for Escherichia coli both in field and purchased strawberries (up to 48.6%). Finding of Cronobacter ranged from 0.6% to 9% both for field and market strawberries. The prevalence of other pathogens (Listeria monocytogenes, Giardia intestinalis, Cryptosporidium sp., and Norovirus) in strawberries was below 4.5%; HAV was not detected at all. Positivity of the environment was determined to be lower than 2.1% for all microorganisms, except for E. coli. The concentration of pathogens in most samples did not exceed 100 CFU/g using culture and 1.8 × 102 GE/g of strawberries or swabbing area 6.1 × 102 GE/mL or swabbing area of environmental samples using qPCR. All studied farms applied preventive measures such as drip irrigation, avoidance of organic fertilizers, and use of mulch foils or gloves for workers to decrease contamination of strawberries. Despite this, certain pathogens were found in fresh strawberries. Even at low concentrations, these pathogens can be a source of infection for consumers. Thus, their presence in strawberries is of particular significance as these are mostly consumed fresh and without any thermal processing. PRACTICAL APPLICATION: Nonlegislatively monitored pathogens of bacterial, viral and parasitic origin were found in strawberries. Monitoring the presence of these pathogens in ready-to-eat food is therefore meaningful and important in terms of food safety, especially in relation to pathogens with low infectious dose (for example, viruses, parasites).; © 2018 Institute of Food Technologists®.

Fresh vegetables and herbs are usually prepared and eaten raw without cooking or heating, which leads to a high risk of foodborne infection. The aim of the present study was to assess the contamination of raw vegetables, herbs, and the environment of food chains. Vegetable and herb samples originating both from the Czech Republic as well as from other countries were examined. The work was focused on the detection of commonly found, but also less frequently monitored foodborne pathogens, including viruses of the genus Norovirus (NoVs), hepatitis A virus (HAV), Listeria monocytogenes and Cronobacter spp. bacteria, and the parasites Cryptosporidium spp. and Giardia intestinalis. All samples were analyzed using individual RT-qPCR/qPCR assays; bacterial pathogens were also simultaneously detected using culture methods. The prevalence of the studied microorganisms in 623 samples ranged from 0.6% to 44.3% for individual pathogens. None of the samples were positive for the presence of HAV. Analysis of 157 environmental samples from 12 farms revealed the presence of NoVs in the environment of four farms. NoVs were detected in water samples as well as on the hands and gloves of workers. Escherichia coli was detected in all farms in the environmental samples and in eight farms in water samples. However, no sample of water exceeded the level of 100 CFU/mL for E. coli. None of the samples of water were positive for the presence of the studied parasites. Vegetables and herbs available from Czech markets and farms may pose a certain risk of foodborne disease, especially in the case of NoVs and parasites. PRACTICAL APPLICATION: This study provides valuable information on the microbiological quality of raw vegetables and herbs available from Czech markets and farms. Good hygienic practices aimed at reducing the incidence of pathogenic agents on fresh produce should not be neglected. Emphasis should be placed on the control of irrigation water, especially with respect to norovirus contamination. It is appropriate to combine culture methods and qPCR methods for the detection of bacterial agents.; © 2018 Institute of Food Technologists®.

Proteome profiling profoundly contributes to the understanding of cell response mechanisms to drug actions. Such knowledge may become a key to improve personalized medicine. In the present study, the effects of the natural remedy curcumin on breast cancer model systems were investigated. MCF-7, ZR-75-1 and TGF-β1 pretreated fibroblasts, mimicking cancer-associated fibroblasts (CAFs), were treated independently as well as in tumor cell/CAF co-cultures. Remarkably, co-culturing with CAF-like cells (CLCs) induced different proteome alterations in MCF-7 and ZR-75-1 cells, respectively. Curcumin significantly induced HMOX1 in single cell type models and co-cultures. However, other curcumin effects differed. In the MCF-7/CLC co-culture, curcumin significantly down-regulated RC3H1, a repressor of inflammatory signaling. In the ZR-75-1/CLC co-culture, curcumin significantly down-regulated PEG10, an anti-apoptotic protein, and induced RRAGA, a pro-apoptotic protein involved in TNF-alpha signaling. Furthermore, curcumin induced AKR1C2, an important enzyme for progesterone metabolism. None of these specific curcumin effects were observed in single cell type cultures. All high-resolution mass spectrometry data are available via ProteomeXchange with the identifier PXD008719. The present data demonstrate that curcumin induces proteome alterations, potentially accounting for its known antitumor effects, in a strongly context-dependent fashion. Biological significance Better means to understand and potentially predict individual variations of drug effects are urgently required. The present proteome profiling study of curcumin effects demonstrates the massive impact of the cell microenvironment on cell responses to drug action. Co-culture models apparently provide more biologically relevant information regarding curcumin effects than single cell type cultures.

Breast cancer is still the most common type of cancer in women; an important role in carcinogenesis is actually attributed to cancer-associated fibroblasts. In this study, we investigated whether it is possible to assess the functional state of cancer-associated fibroblasts through tumor tissue proteome profiling. Tissue proteomics was performed on tumor-central, tumor-near, and tumor-distant biopsy sections from breast adenocarcinoma patients, which allowed us to identify 2074 proteins. Data were interpreted referring to reference proteome profiles generated from primary human mammary fibroblasts comprising 4095 proteins. These cells were analyzed in quiescent cell state as well as after in vitro treatment with TGFβ or IL-1β, stimulating wound healing or inflammatory processes, respectively. Representative for cancer cells, we investigated the mammary carcinoma cell line ZR-75-1, identifying 5212 proteins. All mass analysis data have been made fully accessible via ProteomeXchange, DOI PXD001311 and PXD001323-8. Comparison of tissue proteomics data with all of those reference profiles revealed predominance of cancer cell-derived proteins within the tumor and fibroblast-derived proteins in the tumor-distant tissue sections. Remarkably, proteins characteristic for acute inflammation were hardly identified in the tissue samples. In contrast, several proteins found by us to be induced by TGFβ in mammary fibroblasts, including fibulin-5, SLC2A1, and MUC18, were positively identified in all tissue samples, with relatively higher abundance in tumor neighboring tissue sections. These findings indicate a predominance of cancer-associated fibroblasts with wound healing activities localized around tumors.

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblast-like cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively up-regulated in MGUS and MM. These proteins include laminin α4, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α5β5, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor β and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies.

A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB-NOTCH signaling.

CD83 is one of the best-known surface markers for fully mature dendritic cells (mature DCs), and its cell-type- and maturation-specific regulation makes the CD83 promoter an interesting tool for the genetic modulation of DCs. To determine the mechanisms regulating this DC- and maturation-specific CD83 expression, chromatin immunoprecipitation (ChIP)-on-chip microarray, biocomputational, reporter, electrophoretic mobility shift assay (EMSA), and ChIP analyses were performed. These studies led to the identification of a ternary transcriptional activation complex composed of an upstream regulatory element, a minimal promoter, and an enhancer, which have not been reported in this arrangement for any other gene so far. Notably, these DNA regions contain a complex framework of interferon regulatory factor (IRF)- and NF-κB transcription factor-binding sites mediating their arrangement. Mutation of any of the IRF-binding sites resulted in a significant loss of promoter activity, whereas overexpression of NF-κB transcription factors clearly enhanced transcription. We identified IRF-1, IRF-2, IRF-5, p50, p65, and cRel to be involved in regulating maturation-specific CD83 expression in DCs. Therefore, the characterization of this promoter complex not only contributes to the knowledge of DC-specific gene regulation but also suggests the involvement of a transcriptional module with binding sites separated into distinct regions in transcriptional activation as well as cell-type- and maturation-specific transcriptional targeting of DCs.

Proteome profiling is the method of choice to identify marker proteins whose expression may be characteristic for certain diseases. The formation of such marker proteins results from disease-related pathophysiologic processes. In healthy individuals, peripheral blood mononuclear cells (PBMCs) circulate in a quiescent cell state monitoring potential immune-relevant events, but have the competence to respond quickly and efficiently in an inflammatory manner to any invasion of potential pathogens. Activation of these cells is most plausibly accompanied by characteristic proteome alterations. Therefore we investigated untreated and inflammatory activated primary human PBMCs by proteome profiling using a ‘top down’ 2D-PAGE approach in addition to a ‘bottom up’ LC–MS/MS-based shotgun approach. Furthermore, we purified primary human T-cells and monocytes and activated them separately. Comparative analysis allowed us to characterize a robust proteome signature including NAMPT and PAI2 which indicates the activation of PBMCs. The T-cell specific inflammation signature included IRF-4, GBP1and the previously uncharacterized translation product of GBP5; the corresponding monocyte signature included PDCD5, IL1RN and IL1B. The involvement of inflammatory activated PBMCs in certain diseases as well as the responsiveness of these cells to anti-inflammatory drugs may be evaluated by quantification of these marker proteins. This article is part of a Special Issue entitled: Integrated omics., Graphical abstract Highlights ► Reproducible identification of marker proteins using 2D-gels and shotgun proteomics ► Application of sub-cellular fractionation including cytoplasmic, nuclear and secreted proteins ► Application of metabolic labeling of primary human cells ► Comparison of proteome profiles across different cell types ► Support of functional data interpretation of clinical proteomics studies

Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data. Significance Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.

Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. Both wild-type MLL and MLL fusion proteins interact with the tumor suppressor menin and with the Hoxa9 locus in vivo. Here, we show that MLL sequences between amino acids 5 and 44 are required for interaction with menin and for the transformation of hematopoietic progenitors. Blocking the MLL-menin interaction by the expression of a dominant negative inhibitor composed of amino terminal MLL sequences down-regulates Meis1 expression and inhibits cell proliferation, suggesting that targeting this interaction may be an effective therapeutic strategy for leukemias with MLL rearrangements. [Cancer Res 2007;67(15):7275–83]

The nontuberculous mycobacteria are typically environmental organisms residing in soil and water. These microorganisms can cause a wide range of clinical diseases; pulmonary disease is most frequent, followed by lymphadenitis in children, skin and soft tissue disease, and rare extra pulmonary or disseminated infections.Mycobacterium aviumcomplex is the second most common cause of pulmonary mycobacterioses afterM. tuberculosis. This review covers the clinical and laboratory diagnosis of infection caused by the members of this complex and particularities for the treatment of different disease types and patient populations.

Background The KMT2A/MLL1 lysine methyltransferase complex is an epigenetic regulator of selected developmental genes, in part through the SET domain-catalysed methylation of H3K4. It is essential for normal embryonic development and haematopoiesis and frequently mutated in cancer. The catalytic properties and targeting of KMT2A/MLL1 depend on the proteins with which it complexes and the post-translational protein modifications which some of these proteins put in place, though detailed mechanisms remain unclear. Results KMT2A/MLL1 (both native and FLAG-tagged) and Msk1 (RPS6KA5) co-immunoprecipitated in various cell types. KMT2A/MLL1 and Msk1 knockdown demonstrated that the great majority of genes whose activity changed on KTM2A/MLL1 knockdown, responded comparably to Msk1 knockdown, as did levels of H3K4 methylation and H3S10 phosphorylation at KTM2A target genes HoxA4, HoxA5. Knockdown experiments also showed that KMT2A/MLL1 is required for the genomic targeting of Msk1, but not vice versa. Conclusion The KMT2A/MLL1 complex is associated with, and functionally dependent upon, the kinase Msk1, part of the MAP kinase signalling pathway. We propose that Msk1-catalysed phosphorylation at H3 serines 10 and 28, supports H3K4 methylation by the KMT2A/MLL1 complex both by making H3 a more attractive substrate for its SET domain, and improving target gene accessibility by prevention of HP1- and Polycomb-mediated chromatin condensation. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0103-3) contains supplementary material, which is available to authorized users.

The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy. However, literature on human SCs is scarce and it is unknown to which extent human SC cultures reflect the SC repair phenotype developing after PNI in patients. We performed high-resolution proteome profiling and RNA-sequencing on highly enriched human SC and fibroblast cultures, control and ex vivo degenerated nerve explants to identify novel molecules and functional processes active in repair SCs. In fact, we found cultured SCs and degenerated nerves to share a similar repair SC-associated expression signature, including the upregulation of JUN, as well as two prominent functions, i.e., myelin debris clearance and antigen presentation via MHCII. In addition to myelin degradation, cultured SCs were capable of actively taking up cell-extrinsic components in functional phagocytosis and co-cultivation assays. Moreover, in cultured SCs and degenerated nerve tissue MHCII was upregulated at the cellular level along with high expression of chemoattractants and co-inhibitory rather than -stimulatory molecules. These results demonstrate human SC cultures to execute an inherent program of nerve repair and support two novel repair SC functions, debris clearance via phagocytosis-related mechanisms and type II immune-regulation. GLIA 2016;64:2133-2153.

High blood pressure is a major modifiable risk factor for all clinical manifestations of coronary artery disease (CAD). In people without known cardiovascular disease, the lowest systolic (down to 90-114 mmHg) and the lowest diastolic (down to 60-74 mmHg) pressures are associated with the lowest risk for developing CAD. Although diastolic blood pressure is the strongest predictor of CAD in younger and middle-aged people, this relationship becomes inverted and pulse pressure shows the strongest direct relationship with CAD in people above 60 years of age.Pathophysiological mechanisms of blood pressure as a risk factor for CAD are complex and include the influence of blood pressure as a physical force on the development of the atherosclerotic plaque, and the relationship between pulsatile hemodynamics/arterial stiffness and coronary perfusion. Treatment of arterial hypertension has been proven to prevent coronary events in patients without clinical CAD. In patients with established CAD, the effect of blood pressure lowering per se is beneficial, probably more than specific drugs or drug classes. The important exceptions are beta blockers (BBs), which are superior to all other drug classes for use after a recent myocardial infarction. Blood pressure targets in patients with established CAD have created controversy in the light of the so-called J-curve phenomenon, which describes an increase in coronary events at lower diastolic blood pressures. One explanation for this observation is that perfusion of the left ventricle occurs predominantly during diastole, and that coronary autoregulation may be exhausted with low diastolic blood pressure in the setting of left ventricular hypertrophy and atherosclerotic narrowing of the epicardial coronaries. The worst situation is a high systolic blood pressure in the presence of a low diastolic blood pressure, both a hallmark of increased aortic stiffness. However, the lowering of systolic blood pressure is clearly beneficial in this setting, even at the price of further lowering diastolic pressure. Primary blood pressure goal in patients with established CAD is below 140/90 mmHg. Recent studies suggest that a lower systolic blood pressure may be appropriate, whereas caution is advised with diastolic blood pressure below 60 mmHg.

In order to systematically analyze proteins fulfilling effector functionalities during inflammation, here we present a comprehensive proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-β and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying a false discovery rate of less than 0.01 at both, peptide and protein level, a total of 8370 protein groups assembled from 117,599 peptides was identified; mass spectrometry data have been made fully accessible via ProteomeXchange with identifier PXD003406 to PXD003417.Comparative proteome analysis allowed us to determine common and cell type-specific inflammation signatures comprising novel candidate marker molecules and related expression patterns of transcription factors. Cardinal features of inflammation such as interleukin 1-β processing and the interferon response differed substantially between the investigated cells. Furthermore, cells also exerted similar inflammation-related tasks; however, by making use of different sets of proteins. Hallmarks of inflammation thus emerged, including angiogenesis, extracellular matrix reorganization, adaptive and innate immune responses, oxidative stress response, cell proliferation and differentiation, cell adhesion and migration in addition to monosaccharide metabolic processes, representing both, common and cell type-specific responsibilities of cells during inflammation.

SummaryActivation of the MLL-ENL-ERtm oncogene initiates aberrant proliferation of myeloid progenitors. Here, we show induction of a fail-safe mechanism mediated by the DNA damage response (DDR) machinery that results in activation of the ATR/ATM-Chk1/Chk2-p53/p21CIP1 checkpoint and cellular senescence at early stages of cellular transformation caused by a regulatable MLL-ENL-ERtm in mice. Furthermore, we identified the transcription program underlying this intrinsic anticancer barrier, and DDR-induced inflammatory regulators that fine-tune the signaling toward senescence, thereby modulating the fate of MLL-ENL-immortalized cells in a tissue-environment-dependent manner. Our results indicate that DDR is a rate-limiting event for acquisition of stem cell-like properties in MLL-ENL-ERtm-mediated transformation, as experimental inhibition of the barrier accelerated the transition to immature cell states and acute leukemia development.

Mixed lineage leukemia is a very aggressive blood cancer that predominantly occurs in pediatric patients. In contrast to other types of childhood acute leukemias, mixed lineage leukemia presents with a dismal prognosis and despite the availability of advanced treatment methods cure rates have stagnated over the last years. Mixed lineage leukemia is characterized by the presence of MLL fusion proteins that are the result of chromosomal translocations affecting the MLL gene at 11q23. These events juxtapose the amino-terminus of the histone methyltransferase MLL with a variety of different fusion partners that destroy normal histone methyltransferase function of MLL and replace it by heterologous functions contributed by the fusion partner. The resulting chimeras are transcriptional regulators that take control of targets normally controlled by MLL with the clustered HOX homeobox genes as prominent examples. Recent studies suggested that MLL fusion partners activate transcription by two different mechanisms. Some of these proteins are themselves chromatin modifiers that introduce histone acetylation whereas other fusion partners can recruit histone methyltransferases. In particular, histone H3 specific methylation at lysine 79 catalyzed by DOT1L has been recognized as a hallmark of chromatin activated by MLL fusion proteins. Interestingly, several frequent MLL fusion partners seem to coordinate DOT1L activity with a protein complex that stimulates the elongation phase of transcription by phosphorylating the carboxy-terminal repeat domain of RNA polymerase II. The discovery of these novel enzymatic activities that are essentially involved in MLL fusion protein function presents potential new targets for a rational drug development.

Interpretation of proteome data with a focus on biomarker discovery largely relies on comparative proteome analyses. Here, we introduce a database-assisted interpretation strategy based on proteome profiles of primary cells. Both 2-D-PAGE and shotgun proteomics are applied. We obtain high data concordance with these two different techniques. When applying mass analysis of tryptic spot digests from 2-D gels of cytoplasmic fractions, we typically identify several hundred proteins. Using the same protein fractions, we usually identify more than thousand proteins by shotgun proteomics. The data consistency obtained when comparing these independent data sets exceeds 99% of the proteins identified in the 2-D gels. Many characteristic differences in protein expression of different cells can thus be independently confirmed. Our self-designed SQL database (CPL/MUW - database of the Clinical Proteomics Laboratories at the Medical University of Vienna accessible via www.meduniwien.ac.at/proteomics/database) facilitates (i) quality management of protein identification data, which are based on MS, (ii) the detection of cell type-specific proteins and (iii) of molecular signatures of specific functional cell states. Here, we demonstrate, how the interpretation of proteome profiles obtained from human liver tissue and hepatocellular carcinoma tissue is assisted by the Clinical Proteomics Laboratories at the Medical University of Vienna-database. Therefore, we suggest that the use of reference experiments supported by a tailored database may substantially facilitate data interpretation of proteome profiling experiments.

Dendritic cells (DCs), the most potent and specialized antigen-presenting cells, play a key role in the regulation of the adaptive immunity. Immature DCs were generated by in vitro culturing of peripheral blood monocytes and functionally activated with the classical pathogen-associated molecular pattern lipopolysaccharide (LPS). Alternative activation resulting in Th-2 polarization was induced with lipid oxidation products derived from 1-palmitoyl-2-arachidoyl-sn-glycerol-3-phosphorylcholin (OxPAPC). Tolerogenic cells were obtained by treating DCs with human rhinovirus (HRV). The aim of this study was the identification of proteome profiles related to the functionally different dendritic cell phenotypes. Cytoplasmic proteins were analyzed by shotgun proteomics resulting in the identification of 1690 proteins. While mature and alternatively activated DCs displayed highly distinct protein expression profiles, HRV-treated DCs showed minor proteome alterations. As DCs exert many specific functions via secretion, we investigated the secretomes by a combination of 2D-PAGE and shotgun proteomics. We successfully identified a broad variety of cytokines (e.g., GM-CSF, TNF-alpha, interleukin-1beta, 6, 12 beta, 28B and 29), chemokines (e.g., CCL3, 5, 8, 17, 18, 19, 24, CXCL1, 2, 9 and 10) and growth factors (growth/differentiation factor 8, C-type lectin domain family 11 member A). The relative composition of secretome profiles, although comprising much less proteins, was found to be much more affected by functional alteration of cells than the cytoplasmic protein composition. In conclusion, we demonstrate that functional distinct subsets of DCs display distinct proteome profiles which comprise biomarker candidates. These proteins may prove useful for the interpretation of complex clinical proteomics data.

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

Purpose To report a complication of stent placement at a bifurcation in the infrapopliteal arteries. Case report A 60-year-old man presented with a complex subtotal stenosis in the anterior tibial artery and the tibioperoneal trunk. Two drug-eluting stents were implanted by means of the crush technique, successfully recanalizing the trifurcation. One week later, the patient was readmitted with a thrombotic occlusion of the popliteal artery. Fluoroscopy showed a fracture of the larger stent in the tibioperoneal trunk. After successful local thrombolysis, a stent fragment was located in the popliteal artery and removed with an Amplatz snare. Long-term follow-up was uneventful. Conclusion Fracture may be a more common complication of bifurcation stenting when undertaken in a territory at high risk for this complication.

The use of cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines.The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay.AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both cardenolides.The new cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.

In recent years, mass spectrometry-based proteomics has undergone significant development steps which may be divided into an exploratory phase, a consolidation phase and an application phase. We are in a stage now where we are able to apply mass spectrometric technologies to answer complex and clinically relevant questions. This is demonstrated here with respect to a current hot topic, namely the consideration of the cancer-supporting microenvironment as a target of new and more efficient anti-cancer therapy. Actually, the relevance of micro environmental stromal cells to tumor initiation and promotion has been clearly recognized. However, the individual kind and degree of stroma-derived tumor promotion can so far hardly be determined in patients, and hardly any therapeutic option exists to dismantle the cancer cells of the stroma-derived support. Quite remarkably, the response of stromal cells to standard chemotherapeutics is also rather unknown. In this Perspective, experimental strategies how to address such issues are outlined in detail. Different cell systems are presented as powerful models which allow identifying relevant marker molecules. Targeted proteomics is presented as method of choice for both, drug screening in vitro as well as monitoring drug responses in patients. By this means, a way of classifying different functional tumor promoting mechanisms, evaluating how current treatment strategies may affect cancer-associated fibroblasts, identifying effective drugs targeting these cancer-associated cells and, may be most importantly, demonstrating how combined therapeutic strategies may improve the efficiency of anti-cancer treatments are indicated.

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses, we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005 %, the accuracy was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.

Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia.

The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

Chromosome translocations involving the mixed lineage leukemia gene MLL are associated with aggressive acute leukemias in both children and adults. Leukemogenic MLL fusion proteins delete the MLL SET domain Lys4 methyltransferase activity and fuse MLL to 1 of >40 different translocation partners. Some MLL fusion proteins involve nuclear proteins that are transcriptional activators, whereas others have transcriptional activating activity but instead dimerize the truncated MLL molecule. Both types of MLL fusion proteins enforce persistent expression of Hox a9 and Meis1, which is pivotal for leukemogenesis through mechanisms that remain obscure. Here, we show that nuclear and dimerizable forms of MLL bind with a similar pattern to the Hox a9 locus that overlaps the distribution of wild-type MLL and deregulate transcription of three isoforms of Hox a9. Induction of MLL fusion protein activity is associated with increased levels of histone acetylation and Lys4 methylation at Hox target genes. In addition, the MLL-ENL-ER protein, but not dimerized MLL, also induces dimethylation of histone H3 at Lys79, suggesting alternative mechanisms for transcriptional activation. (Cancer Res 2005; 65(24): 11367-374)

The balance of the 2 cytokines, osteoprotegerin (OPG) and the receptor activator of nuclear factor κ B ligand (soluble (s)RANKL), is known to have considerable influence on bone formation and degradation. Plasma concentrations of OPG and (s)RANKL were determined in a total of 31 long-distance runners before and immediately after running distances of either 15 or 42.195 km, respectively. In both groups of endurance runners, a significant decrease of sRANKL was observed during the run, the extent of which correlated to the running distance. Furthermore, OPG increased only in runners covering the marathon distance of 42.195 km. We hypothesize that the known positive effect of long-distance running on the skeletal mass may be mediated by the OPG/sRANKL system.

Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.

Whether diabetes mellitus affects the prognosis of stroke patients, and whether admission hyperglycemia influences prognosis similarly in diabetic as in non-diabetic patients is assessed controversially. The aims of the study were: 1) to compare the course of diabetic and non-diabetic acute stroke patients, and 2) to assess the influence of admission serum glucose levels on case fatality.In 57 Austrian medical departments the hospital course of consecutive stroke patients was documented prospectively between June 1999 and October 2000.Two hundred and ninety-six (30%) of 992 patients had a history of diabetes mellitus. Intracerebral hemorrhage was more frequent in non-diabetic patients than diabetic (13% versus 5%, P=0.0001). Coronary heart disease was more frequent in diabetic than in non-diabetic patients (35% versus 24%, P=0.0003). The case fatality was 18% among non-diabetic and 16% among diabetic patients (P=0.3559). Among patients who were discharged alive, the Barthel Index increased from 50 to 90 in non-diabetic and from 45 to 75 in diabetic patients (P=0.0403). In non-diabetic patients, admission serum glucose9.2 mmol/L was associated with a more than 4-fold increase in case fatality, compared with patients with serum glucose5.7 mmol/L (P0.0001).Diabetic stroke patients need special care since they tend to have a poorer recovery than non-diabetic patients. Admission hyperglycemia in non-diabetic acute stroke patients predicts a poor prognosis.

Stroke patients with atrial fibrillation (AF) have a poorer neurological outcome than stroke patients without AF. Whether stroke patients with AF also have a higher rate of medical complications is unknown. The aim of the study was to compare the in-hospital course of acute stroke patients with and without AF.The Austrian Stroke registry was a prospective multi-centre study involving 57 medical departments documenting the hospital course of consecutive stroke patients from June 1999 to October 2000. AF was diagnosed in 304 (31%) of 992 patients. Patients with AF were older (79 versus 75 years, p0.0004) than no-AF patients. There were more cases of pneumonia (23% versus 9%, p0.0004), pulmonary oedema (12% versus 6%, p0.0004) and symptomatic intracerebral haemorrhage (8% versus 2%, p0.0004) in AF compared to no-AF. In-hospital mortality was higher in AF (25% versus 14%, p0.0004), and neurological outcome was poorer (65 versus 90 Barthel index, p0.0004). On multivariable logistic regression analysis, however, AF was no predictor for mortality, but a Barthel index of zero (odds ratio 5.30, 95% CI 3.10-9.08, p0.0001), a National Institutes of Health Stroke Scale21 or comatose (odds ratio 3.13, 95% CI 2.26-4.32, p0.0001), age75 years (odds ratio 3.15, 95% CI 1.85-5.37, p0.0001), heart rate100 min(-1) (odds ratio 2.15, 95% CI 1.26-3.66, p = 0.0049), obstructive pulmonary disease (odds ratio 2.58, 95% CI 1.03-6.48, p = 0.0442) and creatinine125 micromol/l (odds ratio 1.84, 95% CI 1.00-3.37, p = 0.0479).Stroke in AF is associated with a poor prognosis, an increased rate of medical and neurological complications and a higher in-hospital mortality than in no-AF.

OBJECTIVE: Intracranial pressure (ICP) monitoring is frequently used in intensive care treatment of patients with intracranial hemorrhage. Data demonstrating an improved outcome from this intervention are lacking. We analyzed standardized mortality ratios in patients with and without ICP monitoring to determine its efficacy. DESIGN: A nonrandomized study of case records of consecutively admitted intensive care unit (ICU) patients with intracranial hemorrhage. SETTING: General and medical ICU of a 900-bed tertiary-care hospital. PATIENTS: A total of 225 patients with intracranial hemorrhage (mainly nontraumatic) admitted consecutively between April 1997 and March 2000. MEASUREMENTS: Simplified Acute Physiology Score (SAPS) II, diagnosis, age, sex, use of ICP monitoring, and in-hospital mortality rates were collected from the hospital's ICU database. Expected mortality was provided by means of SAPS II. Standardized mortality ratios were calculated and compared in 119 patients with ICP monitoring and 106 patients without ICP monitoring. MAIN RESULTS: The case mix-adjusted hospital mortality in the group with ICP monitoring was in the expected range (standardized mortality ratio, 1.09 [95% confidence interval (CI), 0.87-1.31]). Patients without ICP monitoring had a significantly higher standardized mortality ratio than expected (1.26 [95% CI, 1.06-1.46]). CONCLUSIONS: A beneficial effect of ICP monitoring in patients with intracranial hemorrhage may be reflected in an improved standardized mortality ratio.

Chromosomal translocations that fuse the mixed lineage leukemia gene (MLL) to a variety of unrelated partner genes are frequent in pediatric leukemias. The novel combination of genetic material leads to the production of active oncoproteins that depend on the contributions of both constituents. In a search for a common function amongst the diverse group of MLL fusion partners we constructed artificial fusions joining MLL with generic transactivator and repressor domains (acidic blob, GAL4 transactivator domain, Herpes simplex VP16 activation domain, KRAB repressor domain). Of all constructs tested, only MLL-VP16 was able to transform primary bone marrow cells and to induce a block of early myeloid differentiation like an authentic MLL fusion. Interestingly, the transformation capability of the artificial MLL fusions was correlated with the transcriptional potential of the resulting chimeric protein but it was not related to the strength of the isolated transactivation domain that was joined to MLL. These results prove for the first time that a general biological function - transactivation - might be the common denominator of many MLL fusion partners.

The MLL gene at chromosome 11q23 is frequently rearranged in acute leukemia. Here we report the identification of a new MLL fusion partner in the case of an infant with AML-M4 and a t(11;17)(q23;q21) translocation. Fluorescence in situ hybridization (FISH) and RT-PCR analyses indicated a rearrangement of the MLL gene, but no fusion with previously identified MLL fusion partners at 17q, such as AF17 or MSF. Rapid amplification of cDNA ends (RACE) revealed an in-frame fusion of MLL to LASP1, a gene that is amplified and overexpressed in breast cancer. Retroviral transduction of myeloid progenitors demonstrated that MLL/LASP1 is the fourth known fusion of MLL with a cytoplasmic protein that has no in vitro transformation capability, thus establishing a potential subgroup among the MLL fusion proteins.