Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Alpine and lowland grazing differentially alter the reproductive tract redox milieu and amino acid composition in cattle.

An alpine environment is unique due to pasture biodiversity, with an abundant content of natural antioxidant polyphenols. The present study investigated the effects of lowland and alpine grazing on the oviduct and uterine tissue redox status and amino acid concentrations in plasma and reproductive fluids. In the first experiment, heifers grazed on lowland (H-LOW: n = 13) and on alpine (H-ALP: n = 15) pastures. In the second experiment, heifers grazed on the same lowland (HS-LOW: n = 6) and on a different alpine (HS-ALP: n = 6) pasture. The abundance of mRNA transcripts for antioxidant enzymes in the oviduct (glutathione S-transferase alpha 2, glutathione synthetase (GSS)) and the endometrium (catalase, glutathione-disulfide reductase, GSS) was less (P <  0.05), and for glutathione peroxidase 4 in the endometrium greater (P =  0.006) in the H-LOW than in the H-ALP group. The abundance of mRNA transcript for catalase was less in the endometrium in the H-LOW than in the H-ALP (P =  0.001) group. Catalase and NAD(P)H quinone dehydrogenase 1 concentrations in the oviduct were greater in the HS-LOW than in the HS-ALP group (P <  0.05). Of 32 amino acids analysed, there were differences in concentrations in the H-LOW and H-ALP group of 13, seven and 15 in plasma, oviduct and uterine fluids, respectively (P <  0.05). Comparing the HS-LOW to the HS-ALP groups, there were 13, one and three amino acids in the plasma, oviduct and uterine fluids, respectively, that were differentially abundant (P <  0.05). The grazing systems had some effect on the redox status and amino acid patterns in reproductive tissues.

Predicting enhancers in mammalian genomes using supervised hidden Markov models.

BACKGROUND: Eukaryotic gene regulation is a complex process comprising the dynamic interaction of enhancers and promoters in order to activate gene expression. In recent years, research in regulatory genomics has contributed to a better understanding of the characteristics of promoter elements and for most sequenced model organism genomes there exist comprehensive and reliable promoter annotations. For enhancers, however, a reliable description of their characteristics and location has so far proven to be elusive. With the development of high-throughput methods such as ChIP-seq, large amounts of data about epigenetic conditions have become available, and many existing methods use the information on chromatin accessibility or histone modifications to train classifiers in order to segment the genome into functional groups such as enhancers and promoters. However, these methods often do not consider prior biological knowledge about enhancers such as their diverse lengths or molecular structure. RESULTS: We developed enhancer HMM (eHMM), a supervised hidden Markov model designed to learn the molecular structure of promoters and enhancers. Both consist of a central stretch of accessible DNA flanked by nucleosomes with distinct histone modification patterns. We evaluated the performance of eHMM within and across cell types and developmental stages and found that eHMM successfully predicts enhancers with high precision and recall comparable to state-of-the-art methods, and consistently outperforms those in terms of accuracy and resolution. CONCLUSIONS: eHMM predicts active enhancers based on data from chromatin accessibility assays and a minimal set of histone modification ChIP-seq experiments. In comparison to other 'black box' methods its parameters are easy to interpret. eHMM can be used as a stand-alone tool for enhancer prediction without the need for additional training or a tuning of parameters. The high spatial precision of enhancer predictions gives valuable targets for potential knockout experiments or downstream analyses such as motif search.

Biofabrication of a co-culture system in an osteoid-like hydrogel matrix.

Biofabrication aims to develop functional, biological constructs using automated processes (additive manufacturing, AM) involving different cell types and biomaterials (Groll  et al 2016  Biofabrication 13001 1-6). As bone tissue is based on the crosstalk between osteoblasts and osteoclasts at least, evaluating cell-cell and cell-material interactions is of interest to understand bone remodeling. There is increasing interest in the role of osteoclasts not only considering bone resorption, but also their influence on the proliferation, migration and differentiation of osteoblasts. Osteoid-like, non-mineralized matrix is used here for the 3D cultivation of osteoblast and osteoclast progenitor cells to evaluate interactions in an early stage of bone formation. The AM technology bioplotting was used to tailor a 3D environment with defined properties. These results could be helpful to transfer this approach to the fabrication of bone tissue in regenerative medicine approaches. Gelatin is derived from collagen, which is the main phase of osteoid. Oxidized alginate-gelatin crosslinked hydrogel was used to immobilize osteoblastic (ST2) and osteoclastic (RAW) progenitor cells. Cell viability and number, the expression of different proteins like alkaline phosphatase (ALP), osteopontin (OPN) and tartrate resistant acid phosphatase (TRAP) were investigated. Release of vascular endothelial growth factor (VEGF) by the immobilized cells was analyzed. Microscopy techniques were used to evaluate cell morphology during an incubation period of 21 days. The biofabrication process was compatible with the cells. Cells migrated, proliferated and expressed their specific proteins indicating cell differentiation. The co-culture showed increased OPN concentration, which is a major protein of the osteoid involved in the mineralization process. TRAP activity was increased compared to single culture. ST2 single culture showed higher ALP activity compared to the co-culture. VEGF concentration of the co-culture was strongly increased. The results indicate the importance of using co-cultures to fabricate bone tissue by biofabrication. Especially the influence of the osteoblast/osteoclast crosstalk, in an early stage of bone formation, is shown here, using a 3D hydrogel based cell culture model created by biofabrication.

Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D.

Alginate-based hydrogels are extensively used matrices for cell encapsulation, but they need to be modified to recapitulate chemical, microstructural, and mechanical properties of the native extracellular matrix. Like other cell types, mesenchymal stem cells exhibit rounded and clustered morphologies when they are embedded in alginate hydrogels. In this study, we use covalently cross-linked oxidized alginate-gelatin hydrogels to encapsulate human adipose-derived stem cells in order to investigate cell growth, viability, and morphology during osteogenic differentiation taking advantage of the different physicochemical properties of this modified alginate-based hydrogel in comparison to those of the pristine alginate hydrogel. We investigate the effect of hydrogel compositions on stem cell behavior in 3D. Higher viability and the spreading morphology of encapsulated cells with interconnected networks were observed in high gelatin containing compositions. More filopodial protrusions from multicellular nodules were noticed during osteogenic differentiation in the hydrogels having a high amount of gelatin, confirming their suitability for cell encapsulation and bone tissue engineering applications.

Bioplotting of a bioactive alginate dialdehyde-gelatin composite hydrogel containing bioactive glass nanoparticles.

Alginate dialdehyde-gelatin (ADA-GEL) constructs incorporating bioactive glass nanoparticles (BGNPs) were produced by biofabrication to obtain a grid-like highly-hydrated composite. The material could induce the deposition of an apatite layer upon immersion in a biological-like environment to sustain cell attachment and proliferation. Composites were formulated with different concentrations of BGNPs synthetized from a sol-gel route, namely 0.1% and 0.5% (w/v). Strontium doped BGNPs were also used. EDS analysis suggested that the BGNPs loading promoted the growth of bone-like apatite layer on the surface when the constructs were immersed in a simulated body fluid. Moreover, the composite constructs could incorporate with high efficiency ibuprofen as a drug model. Furthermore, the biofabrication process allowed the successful incorporation of MG-63 cells into the composite material. Cells were distributed homogeneously within the hydrogel composite, and no differences were found in cell viability between ADA-GEL and the composite constructs, proving that the addition of BGNPs did not influence cell fate. Overall, the composite material showed potential for future applications in bone tissue engineering.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

Fabrication of Cell-Loaded Two-Phase 3D Constructs for Tissue Engineering.

Hydrogel optimisation for biofabrication considering shape stability/mechanical properties and cell response is challenging. One approach to tackle this issue is to combine different additive manufacturing techniques, e.g., hot-melt extruded thermoplastics together with bioplotted cell loaded hydrogels in a sequential plotting process. This method enables the fabrication of 3D constructs mechanically supported by the thermoplastic structure and biologically functionalised by the hydrogel phase. In this study, polycaprolactone (PCL) and polyethylene glycol (PEG) blend (PCL-PEG) together with alginate dialdehyde gelatine hydrogel (ADA-GEL) loaded with stromal cell line (ST2) were investigated. PCL-PEG blends were evaluated concerning plotting properties to fabricate 3D scaffolds, namely miscibility, wetting behaviour and in terms of cell response. Scaffolds were characterised considering pore size, porosity, strut width, degradation behaviour and mechanical stability. Blends showed improved hydrophilicity and cell response with PEG blending increasing the degradation and decreasing the mechanical properties of the scaffolds. Hybrid constructs with PCL-PEG blend and ADA-GEL were fabricated. Cell viability, distribution, morphology and interaction of cells with the support structure were analysed. Increased degradation of the thermoplastic support structure and proliferation of the cells not only in the hydrogel, but also on the thermoplastic phase, indicates the potential of this novel material combination for biofabricating 3D tissue engineering scaffolds.

Evaluation of an alginate-gelatine crosslinked hydrogel for bioplotting.

Using additive manufacturing to create hydrogel scaffolds which incorporate homogeneously distributed, immobilized cells in the context of biofabrication approaches represents an emerging and expanding field in tissue engineering. Applying hydrogels for additive manufacturing must consider the material processing properties as well as their influence on the immobilized cells. In this work alginate-dialdehyde (ADA), a partially oxidized alginate, was used as a basic material to improve the physico-chemical properties of the hydrogel for cell immobilization. At first, the processing ability of the gel using a bioplotter and the compatibility of the process with MG-63 osteoblast like cells were investigated. The metabolic and mitochondrial activities increased at the beginning of the incubation period and they balanced at a relatively high level after 14-28 days of incubation. During this incubation period the release of vascular endothelial growth factor-A also increased. After 28 days of incubation the cell morphology showed a spreading morphology and cells were seen to move out of the scaffold struts covering the whole scaffold structure. The reproducible processing capability of alginate-gelatine (ADA-GEL) and the compatibility with MG-63 cells were proven, thus the ADA-GEL material is highlighted as a promising matrix for applications in biofabrication.

Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties.

Microencapsulation of cells by using biodegradable hydrogels offers numerous attractive features for a variety of biomedical applications including tissue engineering. This study highlights the fabrication of microcapsules from an alginate-gelatin crosslinked hydrogel (ADA-GEL) and presents the evaluation of the physico-chemical properties of the new microcapsules which are relevant for designing suitable microcapsules for tissue engineering. Alginate di-aldehyde (ADA) was synthesized by periodate oxidation of alginate which facilitates crosslinking with gelatin through Schiff's base formation between the free amino groups of gelatin and the available aldehyde groups of ADA. Formation of Schiff's base in ADA-GEL and aldehyde groups in ADA was confirmed by FTIR and NMR spectroscopy, respectively. Thermal degradation behavior of films and microcapsules fabricated from alginate, ADA and ADA-GEL was dependent on the hydrogel composition. The gelation time of ADA-GEL was found to decrease with increasing gelatin content. The swelling ratio of ADA-GEL microcapsules of all compositions was significantly decreased, whereas the degradability was found to increase with the increase of gelatin ratio. The surface morphology of the ADA-GEL microcapsules was totally different from that of alginate and ADA microcapsules, observed by SEM. Two different buffer solutions (with and without calcium salt) have an influence on the stability of microcapsules which had a significant effect on the gelatin release profile of ADA-GEL microcapsules in these two buffer solutions.