HELP-based matrices for stimuli-responsive controlled release of bioactive compounds

Direct delivery of bioactive substances to the sites of injury represents a key issue for therapies based on regenerative medicine and tissue repair [1]. Protein derived hydrogels represent an interesting system for this purpose because they possess several features that make them suitable to this purpose. A method for preparation of hydrogel matrices based on Human Elastin-like Polypeptide (HELP) has been set up [2]. HELPs are a family of elastin-like recombinant biopolymers modeled after the most regularly repeated domain in human tropoelastin, retaining peculiar properties as self-assembling and thermoresponsive behavior [3]. In this study we assayed two elastolytic activities from different sources to test their potential to specifically degrade the HELP matrix

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression

DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'- deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.Proteomic

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease

Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19 + B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3 + T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.The authors would like to thank Stephanie Scott for her organizational and administrational contribution. The study has been funded by the EU FP7 grant European Commission IBD-BIOM (contract # 305479), EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997), European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010), and Croatian Science Foundation grant EpiGlycoIgG (contract # 3361). Financial support from Portugal (PI: SSP): FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project (POCI-01/0145-FEDER-016601; PTDC/DTP-PIC/0560/2014) was received. SSP also acknowledges the European Crohn’s and Colitis Organization (ECCO) and the “Broad Medical Research program at Crohn’s and Colitis Foundation of America-CCFA” for funding. SSP acknowledges the Portuguese Group of Study on IBD (GEDII) for funding. A.M.D. [PD/BD/105982/2014] also acknowledges FCT for funding. IBD-BIOM consortium: Daniel Kolarich (Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany), Manfred Wuhrer (Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, the Netherlands), Dermot P. B. McGovern (F. Widjaja Family Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles), Iain K. Pemberton (IP Research Consulting SAS, Paris, France), Daniel IR Spencer (Ludger Ltd., Culham Science Centre, Oxford, UK, Daryl L. Fernandes (Ludger Ltd., Culham Science Centre, Oxford, UK), Rahul Kalla, Kate O’Leary, Alex T Adams, Hazel Drummond, Elaine Nimmo, Ray Boyapati, David C Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK), Ray Doran (Ludger Ltd., Culham Science Centre, Oxford, UK), Igor Rudan (all, Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK), Paolo Lionetti (Paediatric Gastroenterology Unit, AOU Meyer, Viale Pieraccini, Florence, Italy), Natalia Manetti (Department of Medical and Surgical Sciences, Division of Gastroenterology, University Hospital Careggi, Florence, Italy), Fabrizio Bossa (Department of Medical Sciences, Division of Gastroenterology, IRCCS-CSS Hospital, Viale Cappuccini, Rotondo, Italy), Paola Cantoro, Anna Kohn (Division of Gastroenterology, S. Camillo Hospital, Rome, Italy), Giancarlo Sturniolo (Gastrointestinal Unit, University of Padua, Padua, Italy), Silvio Danese (IBD Unit, Humanitas Research Institute, Rozzano, Milan, Italy), Mariek Pierik (Maastricht University Medical Centre (MUMC), Maastricht, the Netherlands), and David C. Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK). This independent research was generously supported by the following grants: EU FP7 research grant IBD-BIOM (contract # 305479) to JS, VA, GL, and VZ; EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997) to GL and VZ; European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010) to GL and VZ; Croatian Science Foundation grant EpiGlycoIgG (contract # 3361) to VZ; FEDER COMPETE 2020 POCI, Portugal 2020, and Portuguese funds through FCT (contracts # POCI-01/0145-FEDER-016601 and PTDC/DTP-PIC/0560/2014) to SP; and FTC (contract # PD/BD/105982/2014) to AMD

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease

Background Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn’s disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.</p

Stimuli-Induced Release of Compounds from Elastin Biomimetic Matrix

Stimuli-responsive hydrogel matrices have attracted great attention in biomedical and biotechnological fields for controlled delivery of bioactive compounds, as well as a vehicle for therapeutic cell spreading. Elastin-derived biomimetic polypeptides are recombinant macromolecules suitable for the realization of smart biomaterials. In this study, we explored the potential of an elastin biomimetic matrix to realize proteolytic stimuli-responsive systems to control the release of substances. Our approach showed that this matrix was susceptible to elastolytic degradation, and it has been successfully employed to obtain an efficient delivery of a model protein. This setup will constitute a therapeutic agent delivery platform to realize devices capable of responding and interacting with biological systems at the molecular level

Bioproduction of Anticancer Podophyllotoxin and Related Aryltretralin-Lignans in Hairy Root Cultures of Linum Flavum L.

Podophyllotoxin (PPT) is the unique natural precursor of Etoposide, a topoisomerase II inhibitor drug, used in more than a dozen anticancer chemotherapy treatments. Etoposide is appearing on the list of essential medicines of the World Health Organization. PPT is still exclusively extracted from the rhizome of Podophyllum species, its main natural source. The supply of Podophyllum hexandrum plants is limited, since the occurrence of these plant species is scarce, collection is destructive, and the plants need a long regeneration period. As a consequence, this species is now endangered and listed on Appendix II of the Convention on International Trading of Endangered Species. Chemical synthesis of PPT is difficult due to the presence of four contiguous chiral centers and the presence of a base sensitive trans-lactone moiety. Alternatives are being actively searched, but so far, no wild plants have been described with similar PPT production capacity as compared to Podophyllum. However, several plants producing PPT or other related aryltetralin lignans (ATL) have been identified in recent decades, including the Linaceae. Given its high lignan accumulation capacity, Linum flavum is considered a promising alternative source of PPT and other related ATL. However, unlike the common flax L. usitatissimum, L. flavum has a low agricultural potential (e.g., slow growth and dehiscence of fruits). Therefore, in vitro cultures of plant cells and/or tissues provide an interesting alternative to whole L. flavum plants for the production of these valuable ATL. In particular, L. flavum hairy roots (HRs) accumulate high levels of ATL and it is also possible to further increase this ATL accumulation by the selection of the best genotype, optimization of cultures media and conditions and choice of carbon sources, use of plant growth regulators, elicitor treatments, or precursors’ addition. To date, the ATL accumulation levels can still be perceived insufficient for L. flavum HRs before being used as a commercially viable biotechnological production system. To reach this goal, a better knowledge of the mechanisms that regulate the metabolic flux of intermediates in the different branches of the ATL metabolic pathway will be an important prerequisite to direct the biosynthesis toward the production of a high amount of the desired PPT. In the future, metabolic engineering aiming at constructing the PPT pathway in a heterologous host is very appealing, but for that approach in-depth knowledge of the biosynthetic pathway toward PPT and other related ATL is necessary