Skin and Artificial Skin Models in Electrical Sensing Applications.

Skin electrical properties play a significant role in recording biopotentials by using electrophysiological sensors. To test and evaluate sensor systems, it is commonly accepted to employ artificial skin models due to complications associated with testing on living tissues. The first goal of this Review is to provide a systematic understanding of the relation between skin structure and skin electrochemical behavior at an appropriate depth for electrophysiological sensing applications through a focus on skin structure, electrochemical properties of skin, and theoretical models (equivalent circuits) representing skin electrochemical behavior. The second goal is to review artificial skin models mimicking the electrochemical properties of skin and to give suggestions for future studies on relevant skin models based on a comparison between the behavior of skin and that of artificial skin models. The Review aims to help the reader to analyze the relation between the structure, elements of the equivalent circuits, and the resulting impedance data for both skin and artificial skin models.

Loss of the Ash2l subunit of histone H3K4 methyltransferase complexes reduces chromatin accessibility at promoters.

Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.

Induction of senescence upon loss of the Ash2l core subunit of H3K4 methyltransferase complexes.

Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence.

Hematopoietic stem and progenitor cell proliferation and differentiation requires the trithorax protein Ash2l.

Post-translational modifications of core histones participate in controlling the expression of genes. Methylation of lysine 4 of histone H3 (H3K4), together with acetylation of H3K27, is closely associated with open chromatin and gene transcription. H3K4 methylation is catalyzed by KMT2 lysine methyltransferases that include the mixed-lineage leukemia 1-4 (MLL1-4) and SET1A and B enzymes. For efficient catalysis, all six require a core complex of four proteins, WDR5, RBBP5, ASH2L, and DPY30. We report that targeted disruption of Ash2l in the murine hematopoietic system results in the death of the mice due to a rapid loss of mature hematopoietic cells. However, lin-Sca1+Kit+ (LSK) cells, which are highly enriched in hematopoietic stem and multi-potent progenitor cells, accumulated in the bone marrow. The loss of Ash2l resulted in global reduction of H3K4 methylation and deregulated gene expression, including down-regulation of many mitosis-associated genes. As a consequence, LSK cells accumulated in the G2-phase of the cell cycle and were unable to proliferate and differentiate. In conclusion, Ash2l is essential for balanced gene expression and for hematopoietic stem and multi-potent progenitor cell physiology.

Modes of Interaction of KMT2 Histone H3 Lysine 4 Methyltransferase/COMPASS Complexes with Chromatin.

Regulation of gene expression is achieved by sequence-specific transcriptional regulators, which convey the information that is contained in the sequence of DNA into RNA polymerase activity. This is achieved by the recruitment of transcriptional co-factors. One of the consequences of co-factor recruitment is the control of specific properties of nucleosomes, the basic units of chromatin, and their protein components, the core histones. The main principles are to regulate the position and the characteristics of nucleosomes. The latter includes modulating the composition of core histones and their variants that are integrated into nucleosomes, and the post-translational modification of these histones referred to as histone marks. One of these marks is the methylation of lysine 4 of the core histone H3 (H3K4). While mono-methylation of H3K4 (H3K4me1) is located preferentially at active enhancers, tri-methylation (H3K4me3) is a mark found at open and potentially active promoters. Thus, H3K4 methylation is typically associated with gene transcription. The class 2 lysine methyltransferases (KMTs) are the main enzymes that methylate H3K4. KMT2 enzymes function in complexes that contain a necessary core complex composed of WDR5, RBBP5, ASH2L, and DPY30, the so-called WRAD complex. Here we discuss recent findings that try to elucidate the important question of how KMT2 complexes are recruited to specific sites on chromatin. This is embedded into short overviews of the biological functions of KMT2 complexes and the consequences of H3K4 methylation.

In Vitro and In Vivo Characterization of Biodegradable Reactive Isocyanate-Terminated Three-Armed- and Hyperbranched Block Copolymeric Tissue Adhesives.

Tissue adhesives are an attractive class of biomaterials, which can serve as a treatment for meniscus tears. In this study, physicochemical and adhesive properties of novel biodegradable three-armed- and hyperbranched block copolymeric adhesives are evaluated. Additionally, their degradation in vitro and in vivo, and the tissue reaction after subcutaneous injection in rats are assessed. The developed adhesives have sufficient adhesive strength to meniscus tissue after curing (66-88 kPa). Networks based on the three-armed adhesive have tensile properties that are in the same range as human meniscus. After 26 weeks, networks based on the hyperbranched adhesive show a faster mass loss (25.4%) compared to networks prepared from the three-armed ones (5.5%). Both adhesives induce an inflammatory reaction, however, no necrosis and only initial toxic effects on peripheral tissues are observed. The proposed materials are suitable candidates for the use as resorbable tissue adhesives for meniscus repair.

Development of a fast curing tissue adhesive for meniscus tear repair.

Isocyanate-terminated adhesive amphiphilic block copolymers are attractive materials to treat meniscus tears due to their tuneable mechanical properties and good adhesive characteristics. However, a drawback of this class of materials is their relatively long curing time. In this study, we evaluate the use of an amine cross-linker and addition of catalysts as two strategies to accelerate the curing rates of a recently developed biodegradable reactive isocyanate-terminated hyper-branched adhesive block copolymer prepared from polyethylene glycol (PEG), trimethylene carbonate, citric acid and hexamethylene diisocyanate. The curing kinetics of the hyper-branched adhesive alone and in combination with different concentrations of spermidine solutions, and after addition of 2,2-dimorpholinodiethylether (DMDEE) or 1,4-diazabicyclo [2.2.2] octane (DABCO) were determined using FTIR. Additionally, lap-shear adhesion tests using all compositions at various time points were performed. The two most promising compositions of the fast curing adhesives were evaluated in a meniscus bucket handle lesion model and their performance was compared with that of fibrin glue. The results showed that addition of both spermidine and catalysts to the adhesive copolymer can accelerate the curing rate and that firm adhesion can already be achieved after 2 h. The adhesive strength to meniscus tissue of 3.2-3.7 N was considerably higher for the newly developed compositions than for fibrin glue (0.3 N). The proposed combination of an adhesive component and a cross-linking component or catalyst is a promising way to accelerate curing rates of isocyanate-terminated tissue adhesives.

The effect of tissue surface modification with collagenase and addition of TGF-ß3 on the healing potential of meniscal tears repaired with tissue glues in vitro.

The aim of the current in vitro study was to investigate if tissue surface modification with collagenase and addition of the TGF-ß3 can increase the number of cells present in meniscus tears repaired with the use of newly developed tissue adhesives based on isocyanate-terminated block copolymers. Cylindrical explants were harvested from the inner part of bovine menisci. To simulate a full-thickness tear, the central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing. The repair constructs were then cultured with or without addition of TGF-ß3, and assessed for their histological appearance. The histological staining of the constructs confirmed that both developed adhesives were not cytotoxic. After 28 days, meniscus cells were present in direct contact with the glues. The addition of TGF-ß3 to the culture medium resulted in the presence of cells that formed a sheath inside the simulated tear and in increased cell numbers at the edges of annulus of the explants. In the group in which the tissue was incubated in collagenase and cultured in medium containing TGF-ß3, thicker layers of cells were observed. These results suggest that repairing the torn meniscus with tissue adhesives after pre-treatment of the tissue with collagenase and stimulation with TGF-ß3 is a very promising treatment method, especially when treating the inner avascular part of the meniscus. Nevertheless, longer-term in vitro and in vivo studies are needed to confirm the beneficial effects of this combination therapy.

Regulation of acetate kinase isozymes and its importance for mixed-acid fermentation in Lactococcus lactis.

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.