An α-helical motif containing the sequence L<i>XX</i>LL is required for ligand-dependent binding of transcriptional co-activators to nuclear receptors. By using a peptide inhibition assay, we have defined minimal "core" as an 8-amino acid spanning positions −2 +6 relative primary conserved leucine residue. In yeast two-hybrid assays, core sequences derived from steroid receptor co-activator (SRC1), 140-kDa interacting protein (RIP140), and CREB-binding (CBP) displayed differences in...

Abstract Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable binary control activity. Here we report on a method precise levels mammalian cells using engineered microRNA response elements (MREs). First, measure the efficacy thousands synthetic MRE variants under an endogenous by high-throughput sequencing. Guided this data, establish...

The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor 1 (SRC1) and its homologues the general coactivators CREB binding protein (CBP) p300. SRC1 contains an activation domain (AD1) which functions via recruitment CBP In this study, we have used yeast two-hybrid in vitro interaction-peptide inhibition experiments to map AD1 a 35-residue sequence potentially containing two alpha-helices. We also define 72-amino-acid necessary for...

Abstract Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient‐specific equivalents. Precise editing using homology‐directed repair (HDR) is ultimate goal, however low efficiencies have hindered progress. Reframing strategies highly efficient non‐homologous end joining (NHEJ) aimed at...

Gene editing facilitated by homology-directed repair (HDR) holds great potential for treating monogenetic disorders such as recessive dystrophic epidermolysis bullosa (RDEB). However, low efficiency and variability between loci must be overcome its widespread adoption into personalized therapies. To address these challenges, we developed a highly efficient versatile gene strategy RDEB that incorporates the small molecule inhibitor M3814 to enhance HDR. We focused on three causative COL7A1...

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range inflammatory and degenerative conditions. These lipoaspirate-derived part abundant accessible source heterogeneous vascular fraction (SVF). They typically isolated expanded from SVF via adherent cell culture at least 2 weeks as such represent relatively undefined population cells. We ex vivo ASC directly lipoaspirate using cocktail antibodies combined with immunomagnetic bead...

A set of 10 compounds, each combining the seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (seco-CBI) and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores, was designed prepared. These compounds were anticipated to cross-link between N3 adenine N2 guanine in minor groove DNA. The which differ chain length separating two alkylation subunits, configuration CBI portion, showed great variation cellular toxicity (over 4 orders magnitude a cell line panel) with most potent example...

The aim of this study was to further our understanding the transformation process by identifying differentially expressed proteins in melanocytes compared with melanoma cell lines. Tandem mass spectrometry incorporating iTRAQ reagents used as a screen identify and comparatively quantify expression membrane-enriched samples isolated from primary human or three cells Real-time PCR validate significant hits. Immunohistochemistry interest skin melanoma-infiltrated lymph nodes. Publically...

Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress a mechanism which transactivation domain of is abrogated while DNA unaffected. Specifically, purified as complex with mouse cells was found bind transcriptionally inactive intact complex, human...

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, additives, we have now obtained mean precise &gt;90% on cord blood HSCPs minimal toxicity without observed off-target editing. The main protocol modifications needed to achieve such...

microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from surface receptors influence expression miRNAs cells, and may have consequent effects on phenotype function. We set out to investigate whether common gamma chain cytokines modulated human miR-146a, which previous studies associated with different stages differentiation. also investigated how changes miR-146a...

Abstract B‐cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed drive of B cells into follicles, followed by their relocation specific zones the follicle during activation, and ultimately egress. However, molecular drivers these processes generating chemotactic signals that affect in human LNs not well understood. We used immunofluorescence microscopy, flow cytometry functional assays study mechanisms LNs, found subtle but...

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin formed from plasminogen, which found in high concentrations the blood converted by plasminogen activators. In circulation, levels of α2-antiplasmin rapidly efficiently inhibit activity. Certain myeloid immune cells have been shown bind on their cell surface via proteins that plasmin(ogen) kringle domains. Our earlier work showed T can activate but they do not themselves express plasminogen. Here,...

Overexpression of mutant p53 has been reported to promote tumorigenicity in several cancers. However, despite its potential importance, the signals regulating protein expression are not known. Here we show that a form is incapable binding DNA overexpressed acute promyelocytic leukemia NB4 cell line. Our results demonstrate treatment cells with bryostatin-1, which induces differentiation this line, leads hyperphosphorylation binding-impaired via mitogen-activated kinase. After...

Gene editing therapies are designed to minimise off-target editing. However, it is not widespread practice for common polymorphisms be considered when identifying potential sites in silico. Nevertheless, genetic variants should included as they have the alter existing, or generate new, sites. To facilitate consideration of designing targeted gene we developed PopOff, a web-based tool that integrates minor allele frequencies from gnomAD variant database into an analysis. We used PopOff...

One of the key functions human skin is to provide a barrier, protecting body from surrounding environment and maintaining homeostasis internal environment. A mature, stratified epidermis critical achieve barrier function particularly important when producing grafts in vitro for wound treatment. For decades epidermal stratification has been achieved by culturing keratinocytes at an air-liquid interface, triggering proliferating basal differentiate form all layers. We show here that...

Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease have increasingly led to adoption dual-Cas9 nickases (dual-Cas9n) owing their improved safety profile. However, rates precise obtained such...

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, cheap, easy to and widely utilized by laboratories analyzing the adipogenic potential cells. However, they not specific changes gene transcription. We have developed a gene-specific assay analyze when cell has switched its fate an lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), lineage-specific marker differentiation, enabled...

Simian virus 40 large T antigen has been shown to inhibit p53-mediated transcription once tethered p53-responsive promoters through interaction with p53. In this study we report that p53 stimulates by enhancing the recruitment of basal factors, TFIIA and TFIID, on promoter (the DA complex) inducing a conformational change in complex. Significantly, have demonstrated inhibits blocking ability We investigated mechanism for inhibition found complex formation was resistant T-antigen repression...

The ability to study migratory behavior of immune cells is crucial understanding the dynamic control system. Migration induced by chemokines often assumed be directional (chemotaxis), yet commonly used end-point migration assays are confounded detecting increased cell that lacks directionality (chemokinesis). To distinguish between chemotaxis and chemokinesis we classic "under-agarose assay" in combination with video-microscopy monitor CCR7+ human monocyte-derived dendritic T response a...

There is clinical interest in using human adipose tissue-derived mesenchymal stromal cells (ASC) to treat a range of inflammatory and regenerative conditions. Aspects ASC biology, including their potential paracrine effect, are likely be modulated, part, by microRNAs, small RNA molecules that embedded as regulators gene-expression most biological pathways. However, the effect standard isolation expansion protocols on microRNA expression not well explored. Here, an untouched enriched...