Allergenicity evaluation of quinoa proteins - A study in Brown Norway rats.

The popularity of quinoa seeds has increased in the last decade due to their high nutritional value and natural gluten-free composition. Consumption of new proteins may pose a risk of introducing new allergies. In the present study the immunogenicity and sensitising capacity of quinoa proteins were assessed in a dose-response experiment in Brown Norway rats in comparison to proteins from spinach and peanut. Cross-reactivity between quinoa proteins and known allergens was evaluated by in silico analyses followed by analyses with 11 selected protein extracts and their anti-sera by means of ELISAs and immunoblotting. Further, an in vitro simulated gastro-duodenal digestion was performed. Quinoa proteins were found to have an inherent medium to high immunogenicity and sensitising capacity, being able to induce specific IgG1 and IgE levels higher than spinach but lower than peanut and elicit reactions of clinical relevance similar to peanut. Quinoa proteins were generally shown to resist digestion and retain capacity to bind quinoa-specific antibodies. Quinoa proteins were shown to be cross-reactive with peanut and tree nut allergens as high sequence homology and antibody cross-binding were demonstrated. Present study suggests that quinoa pose a medium to high level of allergenicity that should be further investigated in human studies.

Dose and route of administration determine the efficacy of prophylactic immunotherapy for peanut allergy in a Brown Norway rat model.

Introduction: Allergen-specific immunotherapy (IT) is emerging as a viable option for treatment of peanut allergy. Yet, prophylactic IT remains unexplored despite early introduction of peanut in infancy was shown to prevent allergy. There is a need to understand how allergens interact with the immune system depending on the route of administration, and how different dosages of allergen may protect from sensitisation and a clinical active allergy. Here we compared peanut allergen delivery via the oral, sublingual (SL), intragastric (IG) and subcutaneous (SC) routes for the prevention of peanut allergy in Brown Norway (BN) rats. Methods: BN rats were administered PBS or three different doses of peanut protein extract (PPE) via either oral IT (OIT), SLIT, IGIT or SCIT followed by intraperitoneal (IP) injections of PPE to assess the protection from peanut sensitisation. The development of IgE and IgG1 responses to PPE and the major peanut allergens were evaluated by ELISAs. The clinical response to PPE was assessed by an ear swelling test (EST) and proliferation was assessed by stimulating splenocytes with PPE. Results: Low and medium dose OIT (1 and 10 mg) and all doses of SCIT (1, 10, 100 µg) induced sensitisation to PPE, whereas high dose OIT (100 mg), SLIT (10, 100 or 1000 µg) or IGIT (1, 10 and 100 mg) did not. High dose OIT and SLIT as well as high and medium dose IGIT prevented sensitisation from the following IP injections of PPE and suppressed PPE-specific IgE levels in a dose-dependent manner. Hence, administration of peanut protein via different routes confers different risks for sensitisation and protection from peanut allergy development. Overall, the IgE levels toward the individual major peanut allergens followed the PPE-specific IgE levels. Discussion: Collectively, this study showed that the preventive effect of allergen-specific IT is determined by the interplay between the specific site of PPE delivery for presentation to the immune system, and the allergen quantity, and that targeting and modulating tolerance mechanisms at specific mucosal sites may be a prophylactic strategy for prevention of peanut allergy.

Camel Milk Cannot Prevent the Development of Cow's Milk Allergy-A Study in Brown Norway Rats.

SCOPE: Currently there are no specific recommendations for the use of any particular infant formula in the prevention of cow's milk allergy (CMA). Recently, there has been an increasing interest in alternative infant formulas based on milk proteins from other sources than the cow, including milk from other mammalians such as goat, sheep, donkey, horse, and camel. Whereas these have been studied for their usability in CMA management, there are no studies of their CMA preventive capacity. Thus, the aim of this study is to evaluate whether camel milk can prevent CMA and vice versa. METHODS AND RESULTS: The capacity of camel milk in preventing CMA and vice versa is evaluated in a well-established prophylactic Brown Norway rat model. IgG1, IgE, and IgA responses, allergy elicitation, intestinal and mLN gene expression, and protein uptake are analyzed. The study demonstrates that camel and cow's milk in general has an insignificant cross-preventive capacity. Yet, whereas cow's milk is shown to have a low transient capacity to prevent sensitization and clinically active camel milk allergy, camel milk does not show this effect for CMA. CONCLUSIONS: This study suggests that due to lack of cross-tolerance camel milk cannot be used for CMA prevention.

Novel foods: allergenicity assessment of insect proteins.

Insects represent a promising source of proteins and have been reported as a great potential for being used as novel food and feed proteins. This makes them a valuable source of nutrients to face the increasing demand of food necessitated by the growing global population. The current European food legislation on novel food (EU Reg. 2015/2283), which entered into force in 2018, provides the provisions that should be considered in the applications for the authorisation of novel foods in the European market. Insects, intended as an alternative source of food proteins for human consumption, are considered novel foods. Since food allergens are mostly proteins, the analysis and identification of the potential allergenicity of novel proteins should be a fundamental activity that enables the applicants to fulfil the requirements for the application and authorisation to bring a novel food into the European market and ensures a high level of food safety for the European consumers. The main aims of the work of the EU-FORA fellow were to: (i) Review, assess and identify gaps in the current strategies for predicting allergenicity of novel foods and new alternative protein sources; and (ii) Familiarise, understand and perform an allergenicity assessment of a novel food protein source by: (a) Working on an allergenicity assessment case study of insect proteins from black soldier fly larva (Hermetia Illucens); and (b) Taking into consideration other risk assessment aspects of insects as novel food, including toxicological, nutritional and microbial risks. The project contributed to the continuous learning of the fellow on practical assays and methodologies for the in silico, in vitro and in vivo analysis principles and complemented personal skills related to the food risk assessment requirement for the preparation and submission of an application for authorisation of a novel food.

Alternatives to Cow's Milk-Based Infant Formulas in the Prevention and Management of Cow's Milk Allergy.

Cow's milk-based infant formulas are the most common substitute to mother's milk in infancy when breastfeeding is impossible or insufficient, as cow's milk is a globally available source of mammalian proteins with high nutritional value. However, cow's milk allergy (CMA) is the most prevalent type of food allergy among infants, affecting up to 3.8% of small children. Hypoallergenic infant formulas based on hydrolysed cow's milk proteins are commercially available for the management of CMA. Yet, there is a growing demand for more options for infant feeding, both in general but especially for the prevention and management of CMA. Milk from other mammalian sources than the cow, such as goat, sheep, camel, donkey, and horse, has received some attention in the last decade due to the different protein composition profile and protein amino acid sequences, resulting in a potentially low cross-reactivity with cow's milk proteins. Recently, proteins from plant sources, such as potato, lentil, chickpeas, quinoa, in addition to soy and rice, have gained increased interest due to their climate friendly and vegan status as well as potential lower allergenicity. In this review, we provide an overview of current and potential future infant formulas and their relevance in CMA prevention and management.

Cell Adhesion Assessment Reveals a Higher Force per Contact Area on Fibrous Structures Compared to Flat Substrates.

The distribution and density of ligands have a determinant role in cell adhesion on planar substrates. At the same time, planar surfaces are nonphysiological for most cells, and cell behavior on planar and topographical surfaces is significantly different, with fibrous structures being the most natural environment for cells. Despite phenomenological examinations, the role of adhesion ligand density in the fibrous scaffold for cell adhesion strength has so far not been assessed. Here, we established a method to measure the amount of cell ligands on biofunctionalized electrospun meshes and planar substrate coatings with the same chemical composition. With this as a basis for systematic comparison and pure polyester as benchmark substrates, we have cultured L929 mouse fibroblasts and measured the adhesion force to surfaces of different chemistry and topography. In every case, having fibrous structures have led to an increased adhesion force per area also at a lower ligand density, which remarks the importance of such structures in a natural extracellular environment. Conversely, cells migrate more on planar surfaces than on the tested fibrous substrates. We thus established a platform to study cell-matrix interactions on different surfaces in a precise and reproducible manner as a new tool to assess and quantify cell-matrix interactions toward 3D scaffolds.

Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia.

The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.

Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

The mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules - the main integrin ligand fibronectin and galectin-8, a lectin that binds ß-galactoside residues  - as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed. This article has an associated First Person interview with the first author of the paper.

Production of allergen-specific immunotherapeutic agents for the treatment of food allergy.

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

A comparative analysis of detachment forces and energies in initial and mature cell-material interaction.

Single cell force spectroscopy (SCFS) enables data on interaction forces to be acquired during the very early adhesion phase. However, SCFS detachment forces and energies have not been compared so far with the forces and energies after maturation of the cell-material contact on a single cell level and with comparable time resolution. We used FluidFM® to physically attach single cells to the cantilever by aspiration through a microfluidic channel, in order to achieve the higher forces required for detaching maturely adhering cells. Combining these two approaches allowed us to compare cell adhesion in the initial and maturation phases of adhesion for two exemplary cell-substrate combinations - L929 fibroblasts on fibronectin and MC3T3 osteoblasts on collagen type I. Uncoated glass substrates were used as a reference. For both cell lines, SCFS measurements after contact times of 5, 15 and 30 s revealed significantly higher maximum detachment forces (MDFs) and energies on glass compared to the protein-coated surfaces in the 0.5-4 nN (1-40 fJ) range. FluidFM® measurements after 1, 2 and 3 days of culture revealed a significant absolute increase in the MDFs and detachment energies for both cell lines on protein-coated substrates to values of about 600 nN and 10 pJ. On glass, the MDFs were similar for MC3T3 cells, while they were significantly lower for L929 cells. For both cell types, the differences in detachment energy were significant. These differences underline the importance of investigating early and mature adhesion states to obtain a holistic assessment of the cell-material interactions.

Role of Executive Function in Response to a Problem Solving Based Psychoeducational Intervention in Adolescents with Psychosis: The PIENSA Trial Revisited.

An improvement in negative symptoms and a reduction in the number of visits to the emergency department have been reported in a problem solving based psychoeducational group intervention (PE) for adolescents with psychosis relative to a nonstructured group (NS). One of the factors that may play a role on the response to PE treatment is executive function (EF), a crucial cognitive domain for problem-solving performance. We aimed to examine the role of EF in response to PE treatment versus an NS group. We examined the associations between changes in cognition and in clinical/functional variables within each treatment group using Spearman-ranked and partial correlation analyses. A total of 22 individuals (mean age: 16.3) were randomized to PE (N = 10) and NS (N = 12). We found an association between improvements in EF performance and a reduction in positive symptoms (rs = -0.756, p = 0.030 for semantic fluency), reduction in negative symptoms (r = 0.758, p = 0.029 for semantic; rs = -0,733, p = 0.025 for verbal fluency), and reduction in the number of visits to the emergency department (r = -0,743, p = 0.035 for semantic fluency) in the PE group. No associations were found in the NS group. Our results suggest that EF may play a role in the specific improvements observed in the PE group. This may have implications in the development of new areas of clinical intervention focusing on the role of cognitive functioning in response to psychosocial treatments in psychosis.

Easy-to-Prepare Coating of Standard Cell Culture Dishes for Cell-Sheet Engineering Using Aqueous Solutions of Poly(2-n-propyl-oxazoline).

Cell-sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become nonadhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. Polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for block copolymers, physisorbed onto substrates in a monomolecular thin film to achieve this. Consequently, such substrates, and the polymers required for film formation, can only be prepared in a chemical lab with profound macromolecular expertise. In this study, we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt % aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells, and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.

Comparison of the Allergenicity and Immunogenicity of Camel and Cow's Milk-A Study in Brown Norway Rats.

BACKGROUND: When breastfeeding is impossible or insufficient, the use of cow's milk-based hypoallergenic infant formulas is an option for infants suffering from or at risk of developing cow's milk allergy. As the Camelidae family has a large evolutionary distance to the Bovidae family and as camel milk differs from cow's milk protein composition, there is a growing interest in investigating the suitability of camel milk as an alternative to cow's milk-based hypoallergenic infant formulas. METHODS: The aim of the study was to compare the allergenicity and immunogenicity of camel and cow's milk as well as investigating their cross-reactivity using a Brown Norway rat model. Rats were immunised intraperitoneally with one of four products: camel milk, cow's milk, cow's milk casein or cow's milk whey fraction. Immunogenicity, sensitising capacity, antibody avidity and cross-reactivity were evaluated by means of different ELISAs. The eliciting capacity was evaluated by an ear swelling test. RESULTS: Camel and cow's milk showed similarity in their inherent immunogenicity, sensitising and eliciting capacity. Results show that there was a lower cross-reactivity between caseins than between whey proteins from camel and cow's milk. CONCLUSIONS: The study showed that camel and cow's milk have a low cross-reactivity, indicating a low protein similarity. Results demonstrate that camel milk could be a promising alternative to cow's milk-based hypoallergenic infant formulas.

A new strategy to measure intercellular adhesion forces in mature cell-cell contacts.

Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. METHODS: Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. RESULTS: The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. CONCLUSION: Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

RBM5-AS1 Is Critical for Self-Renewal of Colon Cancer Stem-like Cells.

Cancer-initiating cells (CIC) undergo asymmetric growth patterns that increase phenotypic diversity and drive selection for chemotherapeutic resistance and tumor relapse. WNT signaling is a hallmark of colon CIC, often caused by APC mutations, which enable activation of ß-catenin and MYC Accumulating evidence indicates that long noncoding RNAs (lncRNA) contribute to the stem-like character of colon cancer cells. In this study, we report enrichment of the lncRNA RBM5-AS1/LUST during sphere formation of colon CIC. Its silencing impaired WNT signaling, whereas its overexpression enforced WNT signaling, cell growth, and survival in serum-free media. RBM5-AS1 has been little characterized previously, and we determined it to be a nuclear-retained transcript that selectively interacted with ß-catenin. Mechanistic investigations showed that silencing or overexpression of RBM5-AS1 caused a respective loss or retention of ß-catenin from TCF4 complexes bound to the WNT target genes SGK1, YAP1, and MYC Our work suggests that RBM5-AS1 activity is critical for the functional enablement of colon cancer stem-like cells. Furthermore, it defines the mechanism of action of RBM5-AS1 in the WNT pathway via physical interactions with ß-catenin, helping organize transcriptional complexes that sustain colon CIC function. Cancer Res; 76(19); 5615-27. ©2016 AACR.

Deposition of 5-Methylcytosine on Enhancer RNAs Enables the Coactivator Function of PGC-1α.

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

Psychoeducational Group Intervention for Adolescents With Psychosis and Their Families: A Two-Year Follow-Up.

OBJECTIVE: To investigate whether the beneficial effects of a structured, psychoeducational, parallel-group program for adolescents with early-onset psychosis and their families observed immediately after the intervention were maintained 2 years later. METHOD: The present study examines the longitudinal efficacy of a randomized controlled trial based on a psychoeducational, problem-solving, structured group intervention for adolescents with early-onset psychosis and their families (PE) and compares it with that of a nonstructured group intervention (NS) after a 2-year follow-up. We analyzed whether the differences between PE and NS found after the intervention persisted 2 years later. Intergroup differences in number and duration of hospitalizations, symptoms, and functioning were also assessed. RESULTS: After 2 years of follow-up, we were able to reassess 89% of patients. In the PE group, 13% of patients had visited the emergency department, compared with 50% in the NS group (p = .019). However, no statistically significant differences were found between the groups for negative symptoms or number and duration of hospitalizations. A significant improvement in Positive and Negative Syndrome Scale (PANSS) general symptoms was observed in the PE group. CONCLUSION: Our psychoeducational group intervention showed sustained effects by diminishing the number of visits to emergency departments 2 years after the intervention. Our findings indicate that this psychoeducational intervention could provide patients with long-lasting resources to manage crises more effectively. Clinical trial registration information-Intervention Module AGES (AGES-CM); http://clinicaltrials.gov/; NCT02101372.

Coordination of m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Pluripotency and Reprogramming.

Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity.

Substrate stiffness and composition specifically direct differentiation of induced pluripotent stem cells.

Substrate stiffness, biochemical composition, and matrix topography deeply influence cell behavior, guiding motility, proliferation, and differentiation responses. The aim of this work was to determine the effect that the stiffness and protein composition of the underlying substrate has on the differentiation of induced pluripotent stem (iPS) cells and the potential synergy with specific soluble cues. With that purpose, murine iPS-derived embryoid bodies (iPS-EBs) were seeded on fibronectin- or collagen I-coated polyacrylamide (pAA) gels of tunable stiffness (0.6, 14, and 50 kPa) in the presence of basal medium; tissue culture polystyrene plates were employed as control. Specification of iPS cells toward the three germ layers was analyzed, detecting an increase of tissue-specific gene markers in the pAA matrices. Interestingly, soft matrix (0.6 kPa) coated with fibronectin favored differentiation toward cardiac and neural lineages and, in the case of neural differentiation, the effect was potentiated by the addition of specific soluble factors. The generation of mature astrocytes, neural cells, and cardiomyocytes was further proven by immunofluorescence and transmission electron microscopy. In summary, this work emphasizes the importance of using biomimetic matrices to accomplish a more specific and mature differentiation of stem cells for future therapeutic applications.