Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

Structural Insights into Water-Based Spider Silk Protein-Nanoclay Composites with Excellent Gas and Water Vapor Barrier Properties.

Nature reveals a great variety of inorganic-organic composite materials exhibiting good mechanical properties, high thermal and chemical stability, and good barrier properties. One class of natural bio-nanocomposites, e.g. found in mussel shells, comprises protein matrices with layered inorganic fillers. Inspired by such natural bio-nanocomposites, the cationic recombinant spider silk protein eADF4(κ16) was processed together with the synthetic layered silicate sodium hectorite in an all-aqueous setup. Drop-casting of this bio-nanocomposite resulted in a thermally and chemically stable film reflecting a one-dimensional crystal. Surprisingly, this bio-nanocomposite coating was, though produced in an all-aqueous process, completely water insoluble. Analyzing the structural details showed a low inner free volume due to the well-oriented self-assembly/alignment of the spider silk proteins on the nanoclay surface, yielding high oxygen and water vapor barrier properties. The here demonstrated properties in combination with good biocompatibility qualify this new bio-nanocomposite to be used in packaging applications.

Reproducibility of aortic annulus measurements by computed tomography.

OBJECTIVES: To evaluate a systematic approach for measurement of aortic annulus dimensions by cardiac computed tomography. METHODS: CT data sets of 64 patients were evaluated. An oblique cross-section aligned with the aortic root was created by systematically identifying the caudal insertion points of the three aortic cusps and sequentially aligning them in a double oblique plane. Aortic annulus dimensions, distances of coronary ostia and a suitable fluoroscopic projection angle were independently determined by two observers. RESULTS: Interobserver intraclass correlation coefficients (ICC) for aortic annulus diameters were excellent (ICC 0.89-0.93). Agreement for prosthesis size selection was excellent (ĸ = 0.86 for mean, ĸ = 0.84 for area-derived and ĸ = 0.91 for circumference-derived diameter). Mean distances of the left/right coronary ostium were 13.4 ± 2.4/14.4 ± 2.8 mm for observer 1 and 13.2 ± 2.7/13.5 ± 3.2 mm for observer 2 (p = 0.30 and p = 0.0001, respectively; ICC 0.76/0.77 for left/right coronary artery). A difference of less than 10° for fluoroscopic projection angle was achieved in 84.3% of patients. CONCLUSIONS: A systematic approach to generate a double oblique imaging plane exactly aligned with the aortic annulus demonstrates high interobserver and intraobserver agreements for derived measurements which are not influenced by aortic root calcification. KEY POINTS: • Systematic approach to generate a double oblique imaging plane for TAVI evaluation. • This method is straightforward and software independent. • An approach with high reproducibility, not influenced by aortic root calcification.

The frequencies and clinical implications of mutations in 33 kinase-related genes in locally advanced rectal cancer: a pilot study.

BACKGROUND: Locally advanced rectal cancer (LARC: T3/4 and/or node-positive) is treated with preoperative/neoadjuvant chemoradiotherapy (CRT), but responses are not uniform. The phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and related pathways are implicated in rectal cancer tumorigenesis. Here, we investigated the association between genetic mutations in these pathways and LARC clinical outcomes. METHODS: We genotyped 234 potentially clinically relevant nonsynonymous mutations in 33 PI3K and MAPK pathway-related genes, including PIK3CA, PIK3R1, AKT, STK11, KRAS, BRAF, MEK, CTNNB1, EGFR, MET, and NRAS, using the Sequenom platform. DNA samples were extracted from pretreatment LARC biopsy samples taken from 201 patients who were then treated with long-course neoadjuvant CRT followed by surgical resection. RESULTS: Sixty-two mutations were detected in 15 genes, with the highest frequencies occurring in KRAS (47 %), PIK3CA (14 %), STK11 (6.5 %), and CTNNB1 (6 %). Mutations were detected in BRAF, NRAS, AKT1, PIK3R1, EGFR, GNAS, MEK1, PDGFRA, ALK, and TNK2, but at frequencies of <5 %. As expected, a pathologic complete response (pCR) was associated with improved 5-year recurrence-free survival (RFS; hazard ratio, 0.074; 95 % CI 0.01-0.54; p = 0.001). Mutations in PI3K pathway-related genes (odds ratio, 5.146; 95 % CI 1.17-22.58; p = 0.030), but not MAPK pathway-related genes (p = 0.911), were associated with absence of pCR after neoadjuvant CRT. In contrast, in patients who did not achieve pCR, mutations in PI3K pathway-related genes were not associated with recurrence-free survival (p = 0.987). However, in these patients, codon 12 (G12D/G12 V/G12S) and 13 mutations in KRAS were associated with poor recurrence-free survival (hazard ratio, 1.579; 95 % confidence ratio, 1.00-2.48; p = 0.048). CONCLUSIONS: Mutations in kinase signaling pathways modulate treatment responsiveness and clinical outcomes in LARC and may constitute rational targets for novel therapies.

Automated attenuation-based selection of tube voltage and tube current for coronary CT angiography: reduction of radiation exposure versus a BMI-based strategy with an expert investigator.

BACKGROUND: Recently developed automated algorithms use the topogram and the corresponding attenuation information before coronary CT angiography (CTA) to allow for an individualized anatomic-based selection of tube current (mAs) and voltage (kV). OBJECTIVES: The value of these algorithms in reducing the associated radiation exposure was evaluated. METHODS: One hundred patients underwent coronary CTA with dual-source CT with prospectively electrocardiogram-triggered axial data acquisition. In all patients, tube parameters (current and voltage) were suggested by both an experienced investigator according to the patient's body mass index (BMI; calculated as weight divided by height squared; kg/m(2)) and by an automated software according to attenuation values of the initial topogram. The first 50 consecutive patients (group 1) underwent coronary CTA with dual-source CT with tube parameters suggested by the experienced investigator (BMI-based tube parameters), whereas in another 50 consecutive patients (group 2) CT data acquisition was performed with tube settings of the automated software. Subsequently, subjective image quality (4-point rating score from 0 = nondiagnostic to 3 = excellent image quality), image noise (SD of CT number within the aortic root), as well as signal- and contrast-to-noise ratios and mean effective radiation doses, were compared between both groups. RESULTS: Both groups showed comparable image quality parameters (group 1 vs 2: noise, 28.1 ± 6.0 HU vs 29.9 ± 5.4 HU, P = .12; signal-to-noise ratio, 16.4 ± 3.9 vs 16.8 ± 4.1, P = .54; contrast-to-noise ratio, 18.6 ± 4.1 vs 19.2 ± 4.3, P = .49; 4-point rating score, 2.8 ± 0.3 vs 2.9 ± 0.3, P = .81). Tube voltage, current, and mean effective radiation dose for groups 1 and 2 were 111 ± 12 kV and 108 ± 12 kV (P = .18), 361 ± 32 mAs and 320 ± 48 mAs (P < .001), and 2.3 mSv (25th; 75th percentile, 1.5; 2.8 mSv) and 1.4 mSv (25th; 75th percentile, 1.1; 1.9 mSv) (P < .001), respectively. CONCLUSIONS: Automated attenuation-based selections of individualized tube parameters are superior to BMI-based selections with expert oversight and show a potential for reduction of radiation exposure in coronary CTA, and image quality is maintained.

Effects of hepatocyte nuclear factor-1A and -4A on pancreatic stone protein/regenerating protein and C-reactive protein gene expression: implications for maturity-onset diabetes of the young.

BACKGROUND: There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A. METHODS: Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use. RESULTS: Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects. CONCLUSION: Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects.

Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization.

Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP.

RNASET2--an autoantigen in anaplastic large cell lymphoma identified by protein array analysis.

Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted.

Clinical application of a systems model of apoptosis execution for the prediction of colorectal cancer therapy responses and personalisation of therapy.

OBJECTIVE: Key to the clinical management of colorectal cancer is identifying tools which aid in assessing patient prognosis and determining more effective and personalised treatment strategies. We evaluated whether an experimental systems biology strategy which analyses the susceptibility of cancer cells to undergo caspase activation can be exploited to predict patient responses to 5-fluorouracil-based chemotherapy and to case-specifically identify potential alternative targeted treatments to reactivate apoptosis. DESIGN: We quantified five essential apoptosis-regulating proteins (Pro-Caspases 3 and 9, APAF-1, SMAC and XIAP) in samples of Stage II (n = 13) and III (n=17) tumour and normal colonic (n = 8) tissue using absolute quantitative immunoblotting and employed systems simulations of apoptosis signalling to predict the susceptibility of tumour cells to execute apoptosis. Additional systems analyses assessed the efficacy of novel apoptosis-inducing therapeutics such as XIAP antagonists, proteasome inhibitors and Pro-Caspase-3-activating compounds in restoring apoptosis execution in apoptosis-incompetent tumours. RESULTS: Comparisons of caspase activity profiles demonstrated that the likelihood of colorectal tumours to undergo apoptosis decreases with advancing disease stage. Systems-level analysis correctly predicted positive or negative outcome in 85% (p=0.004) of colorectal cancer patients receiving 5-fluorouracil based chemotherapy and significantly outperformed common uni- and multi-variate statistical approaches. Modelling of individual patient responses to novel apoptosis-inducing therapeutics revealed markedly different inter-individual responses. CONCLUSIONS: Our study represents the first proof-of-concept example demonstrating the significant clinical potential of systems biology-based approaches for predicting patient outcome and responsiveness to novel targeted treatment paradigms.