Publications by authors named "Bart Janssen"

64 Publications

The Use of Differential Scanning Fluorimetry to Assess Strigolactone Receptor Function.

Methods Mol Biol 2021 ;2309:233-243

The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.

Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (T) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the T tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.
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http://dx.doi.org/10.1007/978-1-0716-1429-7_18DOI Listing
August 2021

Gene Regulation Network Analysis on Human Prostate Orthografts Highlights a Potential Role for the Regulon in Clinical Prostate Cancer.

Cancers (Basel) 2021 Apr 26;13(9). Epub 2021 Apr 26.

GenomeScan B.V. Plesmanlaan 1D, 2333 BZ Leiden, The Netherlands.

Background: Prostate cancer (PCa) is the second most common tumour diagnosed in men. Tumoral heterogeneity in PCa creates a significant challenge to develop robust prognostic markers and novel targets for therapy. An analysis of gene regulatory networks (GRNs) in PCa may provide insight into progressive PCa. Herein, we exploited a graph-based enrichment score to integrate data from GRNs identified in preclinical prostate orthografts and differentially expressed genes in clinical resected PCa. We identified active regulons (transcriptional regulators and their targeted genes) associated with PCa recurrence following radical prostatectomy.

Methods: The expression of known transcription factors and co-factors was analysed in a panel of prostate orthografts ( = 18). We searched for genes (as part of individual GRNs) predicted to be regulated by the highest number of transcriptional factors. Using differentially expressed gene analysis (on a per sample basis) coupled with gene graph enrichment analysis, we identified candidate genes and associated GRNs in PCa within the UTA cohort, with the most enriched regulon being which was further validated in two additional cohorts, namely EMC and ICGC cohorts. Cox regression analysis was performed to evaluate the association of the regulon activity with disease-free survival time in the three clinical cohorts as well as compared to three published prognostic gene signatures (TMCC11, BROMO-10 and HYPOXIA-28).

Results: 1308 regulons were correlated to transcriptomic data from the three clinical prostatectomy cohorts. The regulon was identified as the top enriched regulon in the UTA cohort and again validated in the EMC cohort as the top-ranking regulon. In both UTA and EMC cohorts, the regulon was significantly associated with cancer recurrence. Active regulon also correlated with disease recurrence in the ICGC cohort. Furthermore, Kaplan-Meier analysis confirmed shorter time to recurrence in patients with active regulon for all three clinical cohorts (UTA, EMC and ICGC), which was not the case for three published prognostic gene signatures (TMCC11, BROMO-10 and HYPOXIA-28). In multivariate analysis, the regulon status significantly predicted disease recurrence in the UTA and EMC, but not ICGC datasets, while none of the three published signatures significantly prognosticate for cancer recurrence.

Conclusions: We have characterised gene regulatory networks from preclinical prostate orthografts and applied transcriptomic data from three clinical cohorts to evaluate the prognostic potential of the regulon.
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http://dx.doi.org/10.3390/cancers13092094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123677PMC
April 2021

The molecular and genetic regulation of shoot branching.

Plant Physiol 2021 Feb 22. Epub 2021 Feb 22.

The New Zealand Institute for Plant and Food Research Limited, Auckland, New Zealand.

The key regulatory genes and the role of multiple plant hormones coordinate the process of axillary meristem initiation and subsequent growth into a branch.
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http://dx.doi.org/10.1093/plphys/kiab071DOI Listing
February 2021

Electron-event representation data enable efficient cryoEM file storage with full preservation of spatial and temporal resolution.

IUCrJ 2020 Sep 7;7(Pt 5):860-869. Epub 2020 Aug 7.

Molecular Medicine Program, The Hospital for Sick Children, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.

Direct detector device (DDD) cameras have revolutionized electron cryomicroscopy (cryoEM) with their high detective quantum efficiency (DQE) and output of movie data. A high ratio of camera frame rate (frames per second) to camera exposure rate (electrons per pixel per second) allows electron counting, which further improves the DQE and enables the recording of super-resolution information. Movie output also allows the correction of specimen movement and compensation for radiation damage. However, these movies come at the cost of producing large volumes of data. It is common practice to sum groups of successive camera frames to reduce the final frame rate, and therefore the file size, to one suitable for storage and image processing. This reduction in the temporal resolution of the camera requires decisions to be made during data acquisition that may result in the loss of information that could have been advantageous during image analysis. Here, experimental analysis of a new electron-event representation (EER) data format for electron-counting DDD movies is presented, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables the recording of DDD movies at the raw camera frame rate without sacrificing either spatial or temporal resolution. Experimental data demonstrate that the method retains super-resolution information and allows the correction of specimen movement at the physical frame rate of the camera while maintaining manageable file sizes. The EER format will enable the development of new methods that can utilize the full spatial and temporal resolution of DDD cameras.
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http://dx.doi.org/10.1107/S205225252000929XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467176PMC
September 2020

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing.

Leukemia 2021 01 3;35(1):47-61. Epub 2020 Mar 3.

Department of Hematology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands.

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.
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http://dx.doi.org/10.1038/s41375-020-0762-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787979PMC
January 2021

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

J Biol Chem 2020 03 17;295(13):4181-4193. Epub 2020 Feb 17.

The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand. Electronic address:

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2 and DAD2, having substitutions in the core α/β hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2 variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2 and DAD2 revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2 structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.
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http://dx.doi.org/10.1074/jbc.RA119.011509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105320PMC
March 2020

Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus.

Genes Chromosomes Cancer 2020 06 13;59(6):366-374. Epub 2020 Feb 13.

Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands.

Melanoma demonstrates altered patterns of DNA methylation that are associated with genetic instability and transcriptional repression of numerous genes. Active DNA demethylation is mediated by TET enzymes that catalyze conversion of 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Loss of hmC occurs in melanoma and correlates with disease progression. Here we analyzed the genomic distribution of hmC along with mC in nevus and melanoma using oxidative bisulfite chemistry combined with high-density arrays. HmC was enriched relative to mC at enhancers, 5'UTR regions and CpG shores in nevus and melanoma samples, pointing to specific TET enzyme activity. The proportion of interrogated CpG sites with high hmC levels was lower in melanoma (0.54%) than in nevus (2.0%). Depletion of hmC in melanoma was evident across all chromosomes and intragenic regions, being more pronounced in metastatic than in non-metastatic tumors. The patterns of hmC distribution in melanoma samples differed significantly from those in nevus samples, exceeding differences in mC patterns. We identified specific CpG sites and regions with significantly lower hmC levels in melanoma than in nevus that might serve as diagnostic markers. Differentially hydroxymethylated regions localized to cancer-related genes, including the PTEN gene promoter, suggesting that deregulated DNA hydroxymethylation may contribute to melanoma pathogenesis.
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http://dx.doi.org/10.1002/gcc.22837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318264PMC
June 2020

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BMC Genomics 2018 Apr 16;19(1):257. Epub 2018 Apr 16.

The New Zealand Institute for Plant & Food Research Ltd (PFR), Private Bag 92169, Auckland, 1142, New Zealand.

Background: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.

Results: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models.

Conclusions: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.
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http://dx.doi.org/10.1186/s12864-018-4656-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902842PMC
April 2018

Inhibition of strigolactone receptors by -phenylanthranilic acid derivatives: Structural and functional insights.

J Biol Chem 2018 04 9;293(17):6530-6543. Epub 2018 Mar 9.

From the New Zealand Institute for Plant and Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand,

The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified -phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets.
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http://dx.doi.org/10.1074/jbc.RA117.001154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925799PMC
April 2018

Expression of MdCCD7 in the scion determines the extent of sylleptic branching and the primary shoot growth rate of apple trees.

J Exp Bot 2018 04;69(9):2379-2390

The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.

Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.
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http://dx.doi.org/10.1093/jxb/erx404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913623PMC
April 2018

Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment.

Sci Rep 2017 08 14;7(1):8089. Epub 2017 Aug 14.

Department of (Neuro) Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Tuberous Sclerosis Complex (TSC) is a rare genetic disorder that results from a mutation in the TSC1 or TSC2 genes leading to constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1). TSC is associated with autism, intellectual disability and severe epilepsy. Cortical tubers are believed to represent the neuropathological substrates of these disabling manifestations in TSC. In the presented study we used high-throughput RNA sequencing in combination with systems-based computational approaches to investigate the complexity of the TSC molecular network. Overall we detected 438 differentially expressed genes and 991 differentially expressed small non-coding RNAs in cortical tubers compared to autopsy control brain tissue. We observed increased expression of genes associated with inflammatory, innate and adaptive immune responses. In contrast, we observed a down-regulation of genes associated with neurogenesis and glutamate receptor signaling. MicroRNAs represented the largest class of over-expressed small non-coding RNA species in tubers. In particular, our analysis revealed that the miR-34 family (including miR-34a, miR-34b and miR-34c) was significantly over-expressed. Functional studies demonstrated the ability of miR-34b to modulate neurite outgrowth in mouse primary hippocampal neuronal cultures. This study provides new insights into the TSC transcriptomic network along with the identification of potential new treatment targets.
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http://dx.doi.org/10.1038/s41598-017-06145-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556011PMC
August 2017

Differentiation-Defective Human Induced Pluripotent Stem Cells Reveal Strengths and Limitations of the Teratoma Assay and In Vitro Pluripotency Assays.

Stem Cell Reports 2017 05;8(5):1340-1353

Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address:

The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assays are best selected on the basis of intended downstream applications.
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http://dx.doi.org/10.1016/j.stemcr.2017.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425621PMC
May 2017

Critical points for an accurate human genome analysis.

Hum Mutat 2017 08 16;38(8):912-921. Epub 2017 Jun 16.

Department of Human Genetics, Leiden University Medical Center, The Netherlands.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.
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http://dx.doi.org/10.1002/humu.23238DOI Listing
August 2017

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

Hum Mutat 2017 07 29;38(7):870-879. Epub 2017 May 29.

Leiden Genome Technology Center (LGTC), Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.
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http://dx.doi.org/10.1002/humu.23223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488171PMC
July 2017

Silencing of Profilin-1 suppresses cell adhesion and tumor growth via predicted alterations in integrin and Ca2+ signaling in T24M-based bladder cancer models.

Oncotarget 2016 Oct;7(43):70750-70768

Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

Bladder cancer (BC) is the second most common malignancy of the genitourinary system, characterized by the highest recurrence rate of all cancers. Treatment options are limited; thus a thorough understanding of the underlying molecular mechanisms is needed to guide the discovery of novel therapeutic targets. Profilins are actin binding proteins with attributed pleiotropic functions to cytoskeletal remodeling, cell adhesion, motility, even transcriptional regulation, not fully characterized yet. Earlier studies from our laboratory revealed that decreased tissue levels of Profilin-1 (PFN1) are correlated with BC progression to muscle invasive disease. Herein, we describe a comprehensive analysis of PFN1 silencing via shRNA, in vitro (by employing T24M cells) and in vivo [(with T24M xenografts in non-obese diabetic severe combined immunodeficient mice (NOD/SCID) mice]. A combination of phenotypic and molecular assays, including migration, proliferation, adhesion assays, flow cytometry and total mRNA sequencing, as well as immunohistochemistry for investigation of selected findings in human specimens were applied. A decrease in BC cell adhesion and tumor growth in vivo following PFN downregulation are observed, likely associated with the concomitant downregulation of Fibronectin receptor, Endothelin-1, and Actin polymerization. A decrease in the levels of multiple key members of the non-canonical Wnt/Ca2+ signaling pathway is also detected following PFN1 suppression, providing the groundwork for future studies, addressing the specific role of PFN1 in Ca2+ signaling, particularly in the muscle invasive disease.
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http://dx.doi.org/10.18632/oncotarget.12218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342587PMC
October 2016

Structural biology: Signal locked in.

Nature 2016 08 3;536(7617):402-4. Epub 2016 Aug 3.

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http://dx.doi.org/10.1038/nature19418DOI Listing
August 2016

CNDP1 genotype and renal survival in pediatric nephropathies.

J Pediatr Endocrinol Metab 2016 Jul;29(7):827-33

Background: The risk of developing type II diabetic nephropathy (DN) is lower in patients carrying the CNDP1 Mannheim polymorphism (homozygosity for the five leucine repeat), resulting in decreased activity of the histidine-dipeptide metabolizing enzyme carnosinase. The role of CNDP1 in other nephropathies is still unknown.

Methods: To evaluate the impact of the CNDP1 Mannheim allele on pediatric chronic kidney disease (CKD), we prospectively followed the long-term clinical outcome of 272 children with non-diabetic kidney disease (glomerulopathies n=32, non-glomerular kidney disease n=240).

Results: Renal failure progression was independent of CNDP1 genotype in the total cohort of CKD children. However, in patients with glomerulopathies, only 39% of patients homozygous for the CNDP1 Mannheim polymorphism attained the primary renal endpoint as compared to 77% of patients with any other CNDP1 genotype (p=0.06).

Conclusions: Our findings in pediatric CKD patients suggest that the nephroprotective effect of the CNDP1 Mannheim variant is not restricted to patients with diabetic nephropathy.
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http://dx.doi.org/10.1515/jpem-2015-0262DOI Listing
July 2016

Integrative analysis of extracellular and intracellular bladder cancer cell line proteome with transcriptome: improving coverage and validity of -omics findings.

Sci Rep 2016 05 11;6:25619. Epub 2016 May 11.

Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

Characterization of disease-associated proteins improves our understanding of disease pathophysiology. Obtaining a comprehensive coverage of the proteome is challenging, mainly due to limited statistical power and an inability to verify hundreds of putative biomarkers. In an effort to address these issues, we investigated the value of parallel analysis of compartment-specific proteomes with an assessment of findings by cross-strategy and cross-omics (proteomics-transcriptomics) agreement. The validity of the individual datasets and of a "verified" dataset based on cross-strategy/omics agreement was defined following their comparison with published literature. The proteomic analysis of the cell extract, Endoplasmic Reticulum/Golgi apparatus and conditioned medium of T24 vs. its metastatic subclone T24M bladder cancer cells allowed the identification of 253, 217 and 256 significant changes, respectively. Integration of these findings with transcriptomics resulted in 253 "verified" proteins based on the agreement of at least 2 strategies. This approach revealed findings of higher validity, as supported by a higher level of agreement in the literature data than those of individual datasets. As an example, the coverage and shortlisting of targets in the IL-8 signalling pathway are discussed. Collectively, an integrative analysis appears a safer way to evaluate -omics datasets and ultimately generate models from valid observations.
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http://dx.doi.org/10.1038/srep25619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863247PMC
May 2016

FEI's direct electron detector developments: Embarking on a revolution in cryo-TEM.

J Struct Biol 2015 Nov 9;192(2):179-87. Epub 2015 Oct 9.

FEI Company, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.

In early 2011 FEI Company launched the "Falcon", its first commercial direct electron detector product intended for application in 3-D electron microscopy in the life sciences. In this paper we discuss the principle of direct electron detection and its implementation in Falcon cameras. We describe the signal formation in the sensor and its impact on the detection quantum efficiency (DQE) of the sensor. Insights into the signal formation led us to improved camera designs. Three significant improvements are discussed. (1) Back thinning of the sensor. This is implemented in the second-generation Falcon (Falcon 2), where the sensor thickness is reduced to 50 μm, and in the latest generation Falcon 3 detector with further back-thinning down to 30 μm. (2) The introduction of electron counting, a signal processing technology implemented in Falcon 3. (3) Dose fractionation mode, which allows the user to access intermediate results during the illumination of the sample.
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http://dx.doi.org/10.1016/j.jsb.2015.09.014DOI Listing
November 2015

Copy number alterations and allelic ratio in relation to recurrence of rectal cancer.

BMC Genomics 2015 Jun 6;16:438. Epub 2015 Jun 6.

Department of Pathology, L1-Q, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.

Background: In rectal cancer, total mesorectal excision surgery combined with preoperative (chemo)radiotherapy reduces local recurrence rates but does not improve overall patient survival, a result that may be due to the harmful side effects and/or co-morbidity of preoperative treatment. New biomarkers are needed to facilitate identification of rectal cancer patients at high risk for local recurrent disease. This would allow for preoperative (chemo)radiotherapy to be restricted to high-risk patients, thereby reducing overtreatment and allowing personalized treatment protocols. We analyzed genome-wide DNA copy number (CN) and allelic alterations in 112 tumors from preoperatively untreated rectal cancer patients. Sixty-six patients with local and/or distant recurrent disease were compared to matched controls without recurrence. Results were validated in a second cohort of tumors from 95 matched rectal cancer patients. Additionally, we performed a meta-analysis that included 42 studies reporting on CN alterations in colorectal cancer and compared results to our own data.

Results: The genomic profiles in our study were comparable to other rectal cancer studies. Results of the meta-analysis supported the hypothesis that colon cancer and rectal cancer may be distinct disease entities. In our discovery patient study cohort, allelic retention of chromosome 7 was significantly associated with local recurrent disease. Data from the validation cohort were supportive, albeit not statistically significant, of this finding.

Conclusions: We showed that retention of heterozygosity on chromosome 7 may be associated with local recurrence in rectal cancer. Further research is warranted to elucidate the mechanisms and effect of retention of chromosome 7 on the development of local recurrent disease in rectal cancer.
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http://dx.doi.org/10.1186/s12864-015-1550-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458034PMC
June 2015

Environmental control of branching in petunia.

Plant Physiol 2015 Jun 24;168(2):735-51. Epub 2015 Apr 24.

New Zealand Institute for Plant and Food Research, Limited, Sandringham, Auckland 1025, New Zealand

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal.
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http://dx.doi.org/10.1104/pp.15.00486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453797PMC
June 2015

Regulation of axillary shoot development.

Curr Opin Plant Biol 2014 Feb 27;17:28-35. Epub 2013 Nov 27.

The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand. Electronic address:

Axillary meristems are formed in leaf axils and their growth into branches is a highly controlled process that is an important contributor to plant architecture. Here we discuss work that improves our understanding of the initiation and growth of axillary meristems. Recent results have implicated brassinosteroid signalling in the formation of axillary meristems. Our knowledge of axillary meristem outgrowth has also advanced, particularly in the areas of strigolactone signal production and perception, which have been shown to respond to environmental inputs. Auxins and cytokinins have also been linked to the control of axillary shoot development, revealing a complex network of signals that combine to regulate the outgrowth of an axillary meristem into a branch.
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http://dx.doi.org/10.1016/j.pbi.2013.11.004DOI Listing
February 2014

Strigolactone and karrikin signal perception: receptors, enzymes, or both?

Front Plant Sci 2012 28;3:296. Epub 2012 Dec 28.

Plant Development Team, Breeding and Genomics, Plant & Food Research Institute of New Zealand Auckland, New Zealand.

The signaling molecules strigolactone (SL) and karrikin are involved in seed germination, development of axillary meristems, senescence of leaves, and interactions with arbuscular mycorrhizal fungi. The signal transduction pathways for both SLs and karrikins require the same F-box protein (MAX2) and closely related α/β hydrolase fold proteins (DAD2 and KAI2). The crystal structure of DAD2 has been solved revealing an α/β hydrolase fold protein with an internal cavity capable of accommodating SLs. DAD2 responds to the SL analog GR24 by changing conformation and binding to MAX2 in a GR24 concentration-dependent manner. DAD2 can also catalyze hydrolysis of GR24. Structure activity relationships of analogs indicate that the butenolide ring common to both SLs and karrikins is essential for biological activity, but the remainder of the molecules can be significantly modified without loss of activity. The combination of data from the study of DAD2, KAI2, and chemical analogs of SLs and karrikins suggests a model for binding that requires nucleophilic attack by the active site serine of the hydrolase at the carbonyl atom of the butenolide ring. A conformational change occurs in the hydrolase that results in interaction with the F-box protein MAX2. Downstream signal transduction is then likely to occur via SCF (Skp-Cullin-F-box) complex-mediated ubiquitination of target proteins and their subsequent degradation. The role of the catalytic activity of the hydrolase is unclear but it may be integral in binding as well as possibly allowing the signal to be cleared from the receptor. The α/β hydrolase fold family consists mostly of active enzymes, with a few notable exceptions. We suggest that DAD2 and KAI2 represent an intermediate stage where some catalytic activity is retained at the same time as a receptor role has evolved.
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http://dx.doi.org/10.3389/fpls.2012.00296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531792PMC
January 2013

DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.

Curr Biol 2012 Nov 6;22(21):2032-6. Epub 2012 Sep 6.

Plant & Food Research, Private Bag 92169, Auckland 1142, New Zealand.

Strigolactones are a recently discovered class of plant hormone involved in branching, leaf senescence, root development, and plant-microbe interactions. They are carotenoid-derived lactones, synthesized in the roots and transported acropetally to modulate axillary bud outgrowth (i.e., branching). However, a receptor for strigolactones has not been identified. We have identified the DAD2 gene from petunia, an ortholog of the rice and Arabidopsis D14 genes, and present evidence for its roles in strigolactone perception and signaling. DAD2 acts in the strigolactone pathway, and the dad2 mutant is insensitive to the strigolactone analog GR24. The crystal structure of DAD2 reveals an α/β hydrolase fold containing a canonical catalytic triad with a large internal cavity capable of accommodating strigolactones. In the presence of GR24 DAD2 interacts with PhMAX2A, a central component of strigolactone signaling, in a GR24 concentration-dependent manner. DAD2 can hydrolyze GR24, with mutants of the catalytic triad abolishing both this activity and the ability of DAD2 to interact with PhMAX2A. The hydrolysis products can neither stimulate the protein-protein interaction nor modulate branching. These observations suggest that DAD2 acts to bind the mobile strigolactone signal and then interacts with PhMAX2A during catalysis to initiate an SCF-mediated signal transduction pathway.
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http://dx.doi.org/10.1016/j.cub.2012.08.007DOI Listing
November 2012

Genome-wide association study identifies three new melanoma susceptibility loci.

Nat Genet 2011 Oct 9;43(11):1108-13. Epub 2011 Oct 9.

Section of Epidemiology and Biostatistics, Leeds Institute of Molecular Medicine, Leeds Cancer Research UK Centre, St James’s University Hospital, Leeds, UK.

We report a genome-wide association study for melanoma that was conducted by the GenoMEL Consortium. Our discovery phase included 2,981 individuals with melanoma and 1,982 study-specific control individuals of European ancestry, as well as an additional 6,426 control subjects from French or British populations, all of whom were genotyped for 317,000 or 610,000 single-nucleotide polymorphisms (SNPs). Our analysis replicated previously known melanoma susceptibility loci. Seven new regions with at least one SNP with P < 10(-5) and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Texas, USA). Additional replication came from case-control series from the UK and The Netherlands. Variants at three of the seven loci replicated at P < 10(-3): an SNP in ATM (rs1801516, overall P = 3.4 × 10(-9)), an SNP in MX2 (rs45430, P = 2.9 × 10(-9)) and an SNP adjacent to CASP8 (rs13016963, P = 8.6 × 10(-10)). A fourth locus near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication (rs1485993, overall P = 4.6 × 10(-7) under a fixed-effects model and P = 1.2 × 10(-3) under a random-effects model). These newly associated variants showed no association with nevus or pigmentation phenotypes in a large British case-control series.
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http://dx.doi.org/10.1038/ng.959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251256PMC
October 2011

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy.

PLoS One 2010 Oct 20;5(10):e13421. Epub 2010 Oct 20.

Department of Internal Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Background: Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82).

Methodology/principal Findings: Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient's subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients.

Conclusions: These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013421PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958112PMC
October 2010

Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration.

Clin Chim Acta 2011 Jan 21;412(3-4):263-7. Epub 2010 Oct 21.

Centre for Paediatric and Adolescent Medicine, University of Heidelberg, Division of Metabolic Diseases, Heidelberg, Germany.

Background: We reported an association of a particular allele of the carnosinase (CNDP1 Mannheim) gene with reduced serum carnosinase (CN1) activity and absence of nephropathy in diabetic patients. Carnosine protects against the adverse effects of high glucose levels but serum carnosine concentration was generally low.

Methods: We measured the concentration of two further histidine dipeptides, anserine and homocarnosine, via HPLC. CN1 activity was measured fluorometically and for concentration we developed a capture ELISA.

Results: We found an association between the CNDP1 Mannheim allele and reduced serum CN1 activity for all three dipeptides but no correlation to serum concentrations although anserine and homocarnosine inhibited carnosinase activity. Patients with liver cirrhosis have low CN1 activity (0.24 ± 0.17 μmol/ml/h, n=7 males; normal range: 3.2 ± 1.1, n=104; p<0.05) and CN1 concentrations (2.3 ± 1.5 μg/ml; normal range: 24.9 ± 8.9, p<0.05) but surprisingly, histidine dipeptide concentrations in serum are not increased compared to controls.

Conclusions: Serum histidine dipeptide concentrations are not correlated to CN1 activity. The protective effect of low CN1 activity might be related either to turnover of CN1 substrates or a protective function of dipeptides might be localized in other tissues.
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http://dx.doi.org/10.1016/j.cca.2010.10.016DOI Listing
January 2011

Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis).

New Phytol 2010 Nov;188(3):803-13

New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.

• CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). • Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. • Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. • The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial.
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http://dx.doi.org/10.1111/j.1469-8137.2010.03394.xDOI Listing
November 2010

Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon.

BMC Evol Biol 2010 May 20;10:151. Epub 2010 May 20.

Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand.

Background: Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.

Results: Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.

Conclusions: We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.
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http://dx.doi.org/10.1186/1471-2148-10-151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2886070PMC
May 2010

N-glycosylation of carnosinase influences protein secretion and enzyme activity: implications for hyperglycemia.

Diabetes 2010 Aug 11;59(8):1984-90. Epub 2010 May 11.

15th Medical Clinic, University Medical Centre Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

Objective: The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion. Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1 secretion is changed under hyperglycemic conditions.

Results: N-glycosylation of CN-1 was either inhibited by tunicamycin in pCSII-CN-1-transfected Cos-7 cells or by stepwise deletion of its three putative N-glycosylation sites. CN-1 protein expression, N-glycosylation, and enzyme activity were assessed in cell extracts and supernatants. The influence of hyperglycemia on CN-1 enzyme activity in human serum was tested in homozygous (CTG)(5) diabetic patients and healthy control subjects. Tunicamycin completely inhibited CN-1 secretion. Deletion of all N-glycosylation sites was required to reduce CN-1 secretion efficiency. Enzyme activity was already diminished when two sites were deleted. In pCSII-CN-1-transfected Cos-7 cells cultured in medium containing 25 mmol/l d-glucose, the immature 61 kilodaltons (kDa) CN-1 immune reactive band was not detected. This was paralleled by an increased GlcNAc expression in cell lysates and CN-1 expression in the supernatants. Homozygous (CTG)(5) diabetic patients had significantly higher serum CN-1 activity compared with genotype-matched, healthy control subjects.

Conclusions: We conclude that apart from the (CTG)(n) polymorphism in the signal peptide of CN-1, N-glycosylation is essential for appropriate secretion and enzyme activity. Since hyperglycemia enhances CN-1 secretion and enzyme activity, our data suggest that poor blood glucose control in diabetic patients might result in an increased CN-1 secretion even in the presence of the (CTG)(5) allele.
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http://dx.doi.org/10.2337/db09-0868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911063PMC
August 2010
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