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Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that is clinically asymptomatic in its early stages of development. Non-invasive testing for pancreatic cancer biomarkers would significantly improve early detection and patient care. Extracellular vesicles (EVs) are circulating tumor fragments present in the blood and may express cancer specific biomarkers that would enable early detection of pancreatic cancer. We tested the utility of a blood test enumerating EVs positive for the pancreas-specific marker Glycoprotein 2 (GP2) and the putative pancreatic cancer marker Glypican-1 (GPC1) in patients with PDAC. Various levels of GPC1-positive and GP2/GPC1-positive EVs were detected in PDAC patients but were not significantly higher than benign pancreatic disease (BPD) patients. The sensitivity and specificity of the GPC1 EV test was 26.67% and 87.50% respectively, whereas the sensitivity and specificity for the GPC1+GP2 EV test was 23.33% and 90.00% respectively. Immunohistochemistry of GPC1 expression in a tissue microarray of PDAC and various controls also did not demonstrate specificity of GPC1 to PDAC. Hence, enumeration of GPC1-positive EVs, solely or in conjunction with GP2, was unable to effectively distinguish between BPD and pancreatic cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Individuals at risk (IAR) of familial pancreatic cancer (FPC) are good candidates for screening. Unfortunately, neither reliable imaging modalities nor biomarkers are available to detect high-grade precursor lesions or early cancer. Circulating levels of candidate biomarkers LCN2, TIMP1, Glypican-1, RNU2-1f, and miRNA-196b were analyzed in 218 individuals with sporadic pancreatic ductal adenocarcinoma (PDAC, n = 50), FPC ( n = 20), chronic pancreatitis ( n = 10), IAR with relevant precursor lesions ( n = 11) or non-relevant lesions ( n = 5), 20 controls, and IAR with ( n = 51) or without ( n = 51) lesions on pancreatic imaging. In addition, corresponding duodenal juice samples were analyzed for Glypican-1 ( n = 144) enrichment and KRAS mutations ( n = 123). The panel miR-196b/LCN2/TIMP1 could distinguish high-grade lesions and stage I PDAC from controls with absolute specificity and sensitivity. In contrast, Glypican-1 enrichment in serum exosomes and duodenal juice was not diagnostic. KRAS mutations in duodenal juice were detected in 9 of 12 patients with PDAC and only 4 of 9 IAR with relevant precursor lesions. IAR with lesions on imaging had elevated miR-196b/LCN2/TIMP1 levels ( p = 0.0007) and KRAS mutations in duodenal juice ( p = 0.0004) significantly more often than IAR without imaging lesions. The combination miR-196b/LCN2/TIMP1 might be a promising biomarker set for the detection of high-grade PDAC precursor lesions in IAR of FPC families.

Prostate cancer is responsible for hundreds of thousands of annual deaths worldwide. The current gold standard in early detection of prostate cancer, the prostate specific antigen test, boasts a high sensitivity but low specificity, resulting in many unnecessary prostate biopsies. Thus, emphasis has been placed on identifying new biomarkers to improve prostate cancer detection. Glypican-1 has recently been proposed as one such biomarker, however further exploration into its predictive power has been hindered by a lack of available, dependable glypican-1 immunoassays. Previously, we identified human glypican-1 as the antigenic target of the MIL-38 monoclonal antibody. Additionally, we have now generated another monoclonal antibody, 3G5, that also recognizes human glypican-1. Here we report the development of a reliable, custom Luminex ® assay that enables precise quantitation of circulating human glypican-1 in plasma and serum. Using this assay, we show for the first time that circulating glypican-1 levels can differentiate non-cancer (normal and benign prostatic hyperplasia) patients from prostate cancer patients, as well as benign prostatic hyperplasia patients alone from prostate cancer patients. Our findings strongly promote future investigation into the use of glypican-1 for early detection of prostate cancer., Competing Interests: CONFLICTS OF INTEREST The MIL-38 and 3G5 monoclonal antibodies are proprietary to Minomic International Ltd. The authors declare no other conflicts of interest.

Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult. Here, we present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless "sample-to-answer" liquid biopsy screening and improve early stage cancer diagnostics.

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S 35 ]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein., (Copyright © 2017. Published by Elsevier Inc.)

Objective: Glioblastoma (GBM) relapses locally or in a disseminated pattern and is highly resistant to chemoradiotherapy. Although dissemination is associated with poor prognosis for patients with GBM, the clinicopathologic factors that promote dissemination have not been elucidated. Glypican-1 (GPC-1) is a heparin sulfate proteoglycan that is attached to the extracytoplasmic surface of the cell membrane and regulates cell motility. The aim of this study was to determine whether GPC-1 expression correlated with GBM dissemination and patient prognosis., Methods: GPC-1 expression was examined by immunohistochemistry in 53 patients with GBM who received radiotherapy and temozolomide treatment. We assessed the relationship between dissemination and clinicopathologic factors, including GPC-1 expression. We also evaluated the relationship between GPC-1 expression and overall survival (OS) by uni- and multivariate analyses of a range of clinicopathologic factors, including age, Karnofsky Performance Status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) status., Results: Logistic regression analysis revealed that GPC-1 expression correlated with dissemination (P = 0.0116). Log-rank tests revealed that age, Karnofsky Performance Status, extent of resection, MGMT status, dissemination (P = 0.0008) and GPC-1 expression (P = 0.0011) were significantly correlated with OS. Multivariate analysis indicated that age, MGMT status, and GPC-1 expression were significantly correlated with OS. GPC-1 expression had the highest hazard ratio (2.392) among all regressors., Conclusions: GPC-1 expression significantly correlated with OS in patients with GBM who received radiotherapy and temozolomide treatment. GPC-1 expression can help predict the occurrence of dissemination and shorter OS in patients with GBM., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and in vitro assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. KRAS mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest, financial or otherwise, related to this study.

Muscle fiber number is determined around the time hatch with continued posthatch muscle growth being mediated by the adult myoblast, satellite cell, population of cells. Satellite cells are dynamic in their expression of proteins including the cell membrane associated proteoglycans, syndecan-4 and glypican-1. These proteoglycans play roles in organizing the extracellular environment in the satellite cell niche, cytoskeletal structure, cell-to-cell adhesion, satellite cell migration, and signal transduction. This review article focuses on syndecan-4 and glypican-1 as both are capable of regulating satellite cell responsiveness to fibroblast growth factor 2. Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation. Proteoglycans are composed of a central core protein defined functional domains, and covalently attached glycosaminoglycans and N-glycosylation chains. The functional association of these components with satellite cell function is discussed as well as an emerging role for microRNA regulation of syndecan-4 and glypican-1.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in humans, with a 5-year survival rate of <5%. Recently, glypican-1 (GPC1)-expressing circulating exosomes were found to be a promising diagnostic tool for PDAC. However, the aberrant expression of GPC1 has not been systematically evaluated in large-scale clinical samples of PDAC. Here, we performed a comprehensive analysis of GPC1 mRNA and protein expression features. Included in this study were 178 PDAC patients from the cancer genome atlas (TCGA) and 186 subjects whose tissues were used in immunohistochemical staining assays. We demonstrated that GPC1 mRNA was silenced in normal pancreata; however, it was re-expressed in PDAC tissues probably because of the promoter hypomethylation. The GPC1 protein was barely expressed in the normal and adjacent noncancerous pancreata. In tumor tissues, 59.7% (111/186) of the detected samples showed positive expression. Notably, GPC1 was elevated in 63.6% (34/55) of early stage cases. High levels of GPC1 were associated with poorer differentiation and larger tumor diameters. Kaplan-Meier analysis showed a significant difference in overall survival between the groups categorized by GPC1 expression (P = 0.0028). Multivariate analyses indicated that GPC1 was a significant risk factor for poor overall survival with a 1.82-fold increase in the hazard ratio (P = 0.0022). In conclusion, during pancreatic tumorigenesis, GPC1 was ectopically expressed and served as an independent poor prognostic factor. Our findings highlighted the alluring prospect of GPC1 as an early diagnostic and prognostic marker as well as a therapeutic target for PDAC., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that often presents clinically at an advanced stage and that may be confused with chronic pancreatitis (CP). Conversely, CP may be misdiagnosed as PDAC leading to unwarranted pancreas resection. Therefore, early PDAC diagnosis and clear differentiation between PDAC and CP are crucial for improved care. Exosomes are circulating microvesicles whose components can serve as cancer biomarkers. We compared exosomal glypican-1 (GPC1) and microRNA levels in normal control subjects and in patients with PDAC and CP. We report that exosomal GPC1 is not diagnostic for PDAC, whereas high exosomal levels of microRNA-10b, (miR-10b), miR-21, miR-30c, and miR-181a and low miR-let7a readily differentiate PDAC from normal control and CP samples. By contrast with GPC1, elevated exosomal miR levels decreased to normal values within 24 h following PDAC resection. All 29 PDAC cases exhibited significantly elevated exosomal miR-10b and miR-30c levels, whereas 8 cases had normal or slightly increased CA 19-9 levels. Thus, our exosomal miR signature is superior to exosomal GPC1 or plasma CA 19-9 levels in establishing a diagnosis of PDAC and differentiating between PDAC and CP., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis despite the development of multimodal therapy. Expression of glypican-1 (GPC1) has been reported to be elevated in a subset of patients with ESCC and associated with chemoresistance. This study aimed to determine the association of GPC1 with ESCC growth and potential usefulness of the GPC1 targeted therapy by monoclonal antibody (mAb) in ESCC. Expression of GPC1 was higher in ESCC tumor tissues than in adjacent non-tumoral tissues and normal tissues. Knockdown of GPC1 decreased growth of ESCC cells and induced apoptosis via inhibition of EGFR, AKT and p44/42-MAPK signaling pathways in vitro. Anti-GPC1 mAb strongly inhibited tumor growth via antibody-dependent cellular cytotoxicity dependent and independent manner in GPC1-positive ESCC xenograft models. Anti-GPC1 mAb also inhibited tumor growth of GPC1 positive ESCC patients derived tumor xenograft models. Furthermore, anti-GPC1 mAb showed a significant tumor growth inhibition with decreased angiogenesis compared with IgG treated controls in ESCC xenografted mice. Treatment with anti-GPC1 mAb was not toxic in mice. Anti-GPC1 mAb may have a potent anti-tumor effect and represent a novel treatment option for patients with GPC1-positive ESCC.

Blood flow patterns in proatherogenic and antiatherogenic regions are rather different. We hypothesize that the laminar flow with steady shear stress increased nitric oxide (NO) bioavailability while disturbed flow with low shear stress reduced it, which is mediating by glypican-1. Thus, we detected the expression of glypican-1 under different shear stress magnitudes, and tested whether the magnitude of shear stress determines the level of endothelial NO synthase (eNOS) via glypican-1 by using phosphatidylinositol phospholipase C (PI-PLC). Results revealed that the expression of glypican-1 depends on the magnitude and duration of shear stress loading. Activation of eNOS in HUVECs is downregulated by 4dyn/cm 2 of shear stress, but is upregulated by 15dyn/cm 2 . Removal of glypican-1 significantly suppressed the 15dyn/cm 2 shear stress-induced eNOS activity, and further reduced the 4dyn/cm 2 -inhibited eNOS activity. Therefore, eNOS activation depends on shear stress magnitudes and is mediated by glypican-1. The role of glypican-1 in mediating the eNOS activation under shear stress might involve in protecting the endothelial function against disturbed flow and enhancing the sensitive of the endothelial cell to laminar flow, supporting a potential role of glypican-1 against atherosclerosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Therapeutic angiogenesis is a highly appealing concept for treating tissues that become ischemic due to vascular disease. A major barrier to the clinical translation of angiogenic therapies is that the patients that are in the greatest need of these treatments often have long term disease states and co-morbidities, such as diabetes and obesity, that make them resistant to angiogenic stimuli. In this study, we identified that human patients with type 2 diabetes have reduced levels of glypican-1 in the blood vessels of their skin. The lack of this key co-receptor in the tissue may make the application of exogenous angiogenic growth factors or cell therapies ineffective. We created a novel therapeutic enhancer for growth factor activity consisting of glypican-1 delivered in a nanoliposomal carrier (a "glypisome"). Here, we demonstrate that glypisomes enhance FGF-2 mediated endothelial cell proliferation, migration and tube formation. In addition, glypisomes enhance FGF-2 trafficking by increasing both uptake and endosomal processing. We encapsulated FGF-2 or FGF-2 with glypisomes in alginate beads and used these to deliver localized growth factor therapy in a murine hind limb ischemia model. Co-delivery of glypisomes with FGF-2 markedly increased the recovery of perfusion and vessel formation in ischemic hind limbs of wild type and diabetic mice in comparison to mice treated with FGF-2 alone. Together, our findings support that glypisomes are effective means for enhancing growth factor activity and may improve the response to local angiogenic growth factor therapies for ischemia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Heparan sulfate (HS)-containing, S-nitrosylated (SNO) glypican-1 (Gpc-1) releases anhydromannose-containing HS (anMan-HS) by SNO-catalyzed autodegradation in endosomes. Transport of anMan-HS to the nucleus requires processing of the amyloid precursor protein (APP) to amyloid beta peptides (Aβ). To further examine the relationship between APP and Gpc-1 processing in normal fibroblasts we have suppressed Gpc-1 autodegradation by aminoguanidine inhibition of NO synthesis and prevented lysosomal degradation of anMan-HS by using chloroquine. Deconvolution immunofluorescence microscopy and SDS-PAGE using anMan- and APP/Aβ-specific antibodies and markers for nuclei and autophagosomes were used to identify subcellular localization of Aβ and its oligomeric state. Wild-type mouse embryonic fibroblasts (WT MEF) grown during NO-deprivation accumulated 95-98% of Aβ as oligomers in the nucleus. WT MEF treated with chloroquine accumulated both anMan-HS and Aβ, first in the nucleus then in autophagosomes. Maximal nuclear anMan-HS and Aβ accumulation was obtained after 4 and 7 h of growth, respectively. Both yielded similar banding patterns on SDS-PAGE which were also similar to the Aβ oligomers obtained after NO-deprivation. Nuclear Aβ accumulation was marginally increased (from 54 to 58%) by suppression of both release and degradation of anMan-HS. Nuclear exit of Aβ, accumulated during growth in aminoguanidine, was enhanced by ascorbate-induced reactivation of anMan-HS production. Transgenic Alzheimer disease mouse (Tg2576) MEF, which produces excess amount of Aβ was used for comparison. Overall, nuclear Aβ exit and lysosomal degradation was compromised by inhibition of the autophagosome-lysosome pathway in both WT and Tg2576 MEF, while only WT MEF was sensitive to suppression of Gpc-1 autodegradation.

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from S-nitrosylated glypican-1 is generated in endosomes by an endogenously or ascorbate induced S-nitrosothiol-catalyzed reaction. Expression and processing of amyloid precursor protein (APP) is required to initiate formation and endosome-to-nucleus translocation of anMan-containing HS in wild-type mouse embryonic fibroblasts (WT MEF). HS is then transported to autophagosomes and finally degraded in lysosomes. To investigate how APP-derived amyloid β (Aβ) peptide affects intracellular trafficking of HS, we have studied nuclear transit as well as autophagosome/lysosome targeting and degradation in transgenic Alzheimer disease mouse (Tg2576) MEF which produce increased amounts of Aβ. Deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody showed anMan staining in the nuclei of Tg2576 MEF after 5 min of ascorbate treatment and after 15 min in WT MEF. There was also greater nuclear accumulation of HS in Tg2576 MEF as determined by (35)S-sulfate-labeling experiments. Tg2576 MEF was less sensitive to inhibition of NO production and copper-chelation than WT MEF. By using APP- and Aβ-recognizing antibodies, we observed nuclear translocation of Aβ peptide in Tg2576 MEF but not in WT MEF. HS remained in the nucleus of WT MEF for at least 8 h and was then transported to autophagosomes. By 8 h, HS had disappeared from the nuclei of Tg2576 MEF but colocalized poorly with the autophagosome marker LC3. Aβ also disappeared rapidly from the nuclei of Tg2576 MEF. Initially, it appeared in acidic vesicles and later it accumulated extracellularly. Thus, in Tg2576 MEF there is nuclear accumulation as well as secretion of Aβ and impaired degradation of HS., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The amyloid beta (Aβ) peptides (mainly Aβ40 and Aβ42), which are derived from the amyloid precursor protein (APP), can oligomerize into antibody A11-positive, neurotoxic species, believed to be involved in Alzheimer's disease. Interestingly, APP binds strongly to the heparan sulfate (HS) proteoglycan (PG) glypican-1 (Gpc-1) in vitro and both proteins are colocalized inside cells. In endosomes, APP is proteolytically processed to yield Aβ peptides. The HS chains of S-nitrosylated (SNO) Gpc-1 PG are cleaved into anhydromannose (anMan)-containing di- and oligosaccharides by an NO-dependent reaction in the same compartments. Here, we have studied the toxicity of oligomers/aggregates of Aβ40 and Aβ42, as well as Aβ40/42 mixtures that were formed in the presence of immobilized Gpc-1 PG or immobilized HS oligosaccharides. Afterwards, Aβ was displaced from the matrices, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for A11 immunoreactivity, for effects on growth of mouse N2a neuroblastoma cells and for membrane leakage in rat cortical neurons. HS generally promoted and accelerated Aβ multimerization into oligomers as well as larger aggregates that were mostly A11 positive and showed toxic effects. However, non-toxic Aβ was formed in the presence of Gpc-1 PG or when anMan-containing HS degradation products were simultaneously generated. Both toxic and non-toxic Aβ peptides were taken up by the cells but toxic forms appeared to enter the nuclei to a larger extent. The protection afforded by the presence of HS degradation products may reflect a normal intracellular function for the Aβ peptides.

Satellite cells are multipotential stem cells responsible for muscle growth and regeneration. Satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from 16-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and 16 wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and 16 wk cells treated with FGF2. Expression of myogenic regulatory factors MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory factor expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity., (© 2013.)

The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (<3kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤1% q-value, ≤5% PEP) - a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell-cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome., (Copyright © 2013 Elsevier B.V. All rights reserved.)