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1.  Development of Carbocyanine Dyes for PRMT Inhibition and Imaging 
Summary
Protein arginine methylation regulates multiple biological processes. Deregulation of protein arginine methyltransferase (PRMT) activities has been observed in many disease phenotypes. Small molecule probes that target PRMTs with strong affinity and selectivity can be used as valuable tools to dissect biological mechanisms of arginine methylation and establish the role of PRMT proteins in a disease process. In this work, we report synthesis and evaluation of a class of carbocyanine compounds containing indolium, benz[e]indolium or benz[c,d]indolium heterocyclic moieties that bind to the predominant arginine methyltransferase PRMT1 and inhibit its methyltransferase activity at low micromolar potencies. In particular, the developed molecules have long wavelength colorimetric and fluorometric photoactivities, which can be used for optical and near-infrared fluorescence imaging in cells or biological tissues. Together, these new chemical probes have potential application in PRMT studies both as enzyme inhibitors and as fluorescent dyes for microscope imaging.
doi:10.1016/j.ejmech.2012.06.017
PMCID: PMC4151304  PMID: 22749641
2.  Labeling Lysine Acetyltransferase Substrates with Engineered Enzymes and Functionalized Cofactor Surrogates 
Elucidating biological and pathological functions of protein lysine acetyltransferases (KATs) greatly depends on the knowledge of the dynamic and spatial localization of their enzymatic targets in the cellular proteome. Here we report the design and application of chemical probes for facile labeling and detection of substrates of the three major human KAT enzymes. In this approach, we attempted to create engineered KATs in junction with synthetic Ac-CoA surrogates to effectively label KAT substrates even in the presence of competitive nascent cofactor acetyl-CoA. The functionalized and transferable acyl moiety of the Ac-CoA analogs further allowed the labeled substrates to be probed with alkynyl or azido-tagged fluorescent reporters by the copper-catalyzed azide–alkyne cycloaddition. The synthetic co-factors, in combination with either native or rationally engineered KAT enzymes, provide a versatile chemical biology strategy to label and profile cellular targets of KATs at the proteomic level.
doi:10.1021/ja311636b
PMCID: PMC3703473  PMID: 23659802
3.  Systematic literature review and validity evaluation of the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Functional Composite (MSFC) in patients with multiple sclerosis 
BMC Neurology  2014;14:58.
Background
There are a number of instruments that describe severity and progression of multiple sclerosis and they are increasingly used as endpoints to assess the effectiveness of therapeutic interventions. We examined to what extent the psychometric properties of two accepted instruments – EDSS and MSFC – meet methodological standards and the value they have in clinical trials.
Methods
We conducted a systematic literature search in relevant databases [MEDLINE (PubMed), ISI Web of Science, EMBASE, PsycINFO & PSYNDEX, CINAHL] yielding 3,860 results. Relevant full-text publications were identified using abstract and then full-text reviews, and the literature was reviewed.
Results
For evaluation of psychometric properties (validity, reliability, sensitivity of change) of EDSS and MSFC, 120 relevant full-text publications were identified, 54 of them assessed the EDSS, 26 the MSFC and 40 included both instruments. The EDSS has some documented weaknesses in reliability and sensitivity to change. The main limitations of the MSFC are learning effects and the z-scores method used to calculate the total score. However, the methodological criterion of validity applies sufficiently for both instruments.
For use in clinical studies, we found the EDSS to be preferred as a primary and secondary outcome measure in recent studies (50 EDSS, 9 MSFC).
Conclusions
Recognizing their strengths and weaknesses, both EDSS and MSFC are suitable to detect the effectiveness of clinical interventions and to monitor disease progression. Almost all publications identify the EDSS as the most widely used tool to measure disease outcomes in clinical trials. Despite some limitations, both instruments are accepted as endpoints and neither are discussed as surrogate parameters in identified publications. A great advantage of the EDSS is its international acceptance (e.g. by EMA) as a primary endpoint in clinical trials and its broad use in trials, enabling cross-study comparisons.
doi:10.1186/1471-2377-14-58
PMCID: PMC3986942  PMID: 24666846
Multiple sclerosis; Expanded Disability Status Scale (EDSS); Multiple Sclerosis Functional Composite (MSFC); Psychometric properties; Validity; Reliability; Sensitivity of change
4.  Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH 
Bioorganic & medicinal chemistry  2012;20(9):2957-2961.
The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant.
doi:10.1016/j.bmc.2012.03.014
PMCID: PMC3355949  PMID: 22464680
stopped-flow; isoquinolinylboronic acid; binding constant; fluorescence
5.  A Transient Kinetic Analysis of PRMT1 Catalysis 
Biochemistry  2011;50(32):7033-7044.
Posttranslational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.
doi:10.1021/bi200456u
PMCID: PMC3153576  PMID: 21736313
PRMT1; arginine methylation; transient-state kinetics; conformational transition; fluorescent probe; stopped flow
6.  L-Carnitine-supplementation in advanced pancreatic cancer (CARPAN) - a randomized multicentre trial 
Nutrition Journal  2012;11:52.
Background
Cachexia, a >10% loss of body-weight, is one factor determining the poor prognosis of pancreatic cancer. Deficiency of L-Carnitine has been proposed to cause cancer cachexia.
Findings
We screened 152 and enrolled 72 patients suffering from advanced pancreatic cancer in a prospective, multi-centre, placebo-controlled, randomized and double-blinded trial to receive oral L-Carnitine (4 g) or placebo for 12 weeks. At entry patients reported a mean weight loss of 12 ± 2,5 (SEM) kg. During treatment body-mass-index increased by 3,4 ± 1,4% under L-Carnitine and decreased (−1,5 ± 1,4%) in controls (p < 0,05). Moreover, nutritional status (body cell mass, body fat) and quality-of-life parameters improved under L-Carnitine. There was a trend towards an increased overall survival in the L-Carnitine group (median 519 ± 50 d versus 399 ± 43 d, not significant) and towards a reduced hospital-stay (36 ± 4d versus 41 ± 9d,n.s.).
Conclusion
While these data are preliminary and need confirmation they indicate that patients with pancreatic cancer may have a clinically relevant benefit from the inexpensive and well tolerated oral supplementation of L-Carnitine.
doi:10.1186/1475-2891-11-52
PMCID: PMC3439338  PMID: 22824168
Pancreatic adenocarcinoma; L-Carnitine; Quality of life; Survival; Cancer cachexia; Fatique syndrome
7.  Scintillation Proximity Assay of Arginine Methylation 
Journal of Biomolecular Screening  2011;17(2):237-244.
Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein post-translational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.
doi:10.1177/1087057111414903
PMCID: PMC3236808  PMID: 21821785
protein arginine methyltransferases; PRMT; scintillation proximity assay; SPA; high-throughput screening; HTS
8.  Self-perceived quality of life predicts mortality risk better than a multi-biomarker panel, but the combination of both does best 
Background
Associations between measures of subjective health and mortality risk have previously been shown. We assessed the impact and comparative predictive performance of a multi-biomarker panel on this association.
Methods
Data from 4,261 individuals aged 20-79 years recruited for the population-based Study of Health in Pomerania was used. During an average 9.7 year follow-up, 456 deaths (10.7%) occurred. Subjective health was assessed by SF-12 derived physical (PCS-12) and mental component summaries (MCS-12), and a single-item self-rated health (SRH) question. We implemented Cox proportional-hazards regression models to investigate the association of subjective health with mortality and to assess the impact of a combination of 10 biomarkers on this association. Variable selection procedures were used to identify a parsimonious set of subjective health measures and biomarkers, whose predictive ability was compared using receiver operating characteristic (ROC) curves, C-statistics, and reclassification methods.
Results
In age- and gender-adjusted Cox models, poor SRH (hazard ratio (HR), 2.07; 95% CI, 1.34-3.20) and low PCS-12 scores (lowest vs. highest quartile: HR, 1.75; 95% CI, 1.31-2.33) were significantly associated with increased risk of all-cause mortality; an association independent of various covariates and biomarkers. Furthermore, selected subjective health measures yielded a significantly higher C-statistic (0.883) compared to the selected biomarker panel (0.872), whereas a combined assessment showed the highest C-statistic (0.887) with a highly significant integrated discrimination improvement of 1.5% (p < 0.01).
Conclusion
Adding biomarker information did not affect the association of subjective health measures with mortality, but significantly improved risk stratification. Thus, a combined assessment of self-reported subjective health and measured biomarkers may be useful to identify high-risk individuals for intensified monitoring.
doi:10.1186/1471-2288-11-103
PMCID: PMC3152941  PMID: 21749697
Health-related quality of life; multiple biomarker panel; all-cause mortality; SF-12; population-based cohort
9.  Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro 
Background
IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells.
Methods
We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay.
Results
IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration.
Conclusion
Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.
doi:10.1186/1756-9966-27-77
PMCID: PMC2626583  PMID: 19036171

Results 1-9 (9)