Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation

În epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1−/− murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1−/− epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling

CDK4 regulation by TNFR1 and JNK is required for NF-κB–mediated epidermal growth control

Nuclear factor κB (NF-κB) mediates homeostatic growth inhibition in the epidermis, and a loss of NF-κB function promotes proliferation and oncogenesis. To identify mechanisms responsible for these effects, we impaired NF-κB action in the epidermis by three different genetic approaches, including conditional NF-κB blockade. In each case, epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the G1 cell cycle kinase, CDK4. CDK4 up-regulation required intact TNFR1 and c-Jun NH2-terminal kinase (JNK) function. Cdk4 gene deletion concomitant with conditional NF-κB blockade demonstrated that CDK4 is required for growth deregulation. Therefore, epidermal homeostasis depends on antagonist regulation of CDK4 expression by NF-κB and TNFR1/JNK

Identification of proteins binding coding and non-coding human RNAs using protein microarrays

Background: The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. Results: We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. Conclusions: Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.Stem Cell and Regenerative Biolog

Dual frequency ultrasonic cavitation in various liquids: High-speed imaging and acoustic pressure measurements

Ultrasonic cavitation is used in various processes and applications, utilizing powerful shock waves and high-speed liquid jets generated by the collapsing bubbles. Typically, a single frequency source is used to produce the desired effects. However, optimization of the efficiency of ultrasound reactors is necessary to improve cavitation activity in specific applications such as for the exfoliation of two dimensional materials. This research takes the next step to investigate the effect of a dual frequency transducer system on the bubble dynamics, cavitation zone, pressure fields, acoustic spectra, and induced shock waves for four liquids with a range of physical properties. Using ultra-high-speed imaging and synchronized acoustic pressure measurements, the effect of ultrasonic dual frequencies on bubble dynamics was investigated. The addition of a high frequency transducer (1174 kHz) showed that the bubble fragments and satellite bubbles induced from a low frequency transducer (24 kHz) were able to extend their lifecycle and increase spatial distribution, thus, extending the boundaries of the cavitation zone. Furthermore, this combination of ultrasonic frequencies generated higher acoustic pressures (up to 180%) and enhanced the characteristic shock wave peak, indicating more bubble collapses and the generation of additional shock waves. The dual frequency system also enlarged the cavitation cloud size under the sonotrode. These observations specifically delineated the enhancement of cavitation activity using a dual frequency system pivotal for optimization of existing cavitation-based processing technologies

An eco-friendly solution for liquid phase exfoliation of graphite under optimised ultrasonication conditions

Ultrasonic assisted liquid phase exfoliation (ULPE) is a promising method for the large scale production of 2D materials. Currently, toxic solvents such as N-Methyl-2-pyrrolidone (NMP) are commonly used for the production of graphene. In this paper four solvents; three green solvents (water, ethanol and water/ethanol) plus NMP for comparison, were sonicated and examined in terms of their bubble dynamics and acoustic emissions. Advanced fundamental analysis was conducted using high-speed imaging synchronised with acoustic pressure measurements complemented by shadowgraphic photography of the emitted shockwaves, in order to determine a suitable eco-friendly solvent medium from a cavitation bubbles dynamics perspective. Thereafter, ULPE of graphite in the optimum solvent took place for 2 h under controlled ultrasonication parameters. The produced graphene samples were characterised by employing a series of techniques consisting of Ultraviolet–visible (UV–Vis) and Raman spectroscopy as well as transmission electron microscopy (TEM). A mixture of deionised water and ethanol was shown to produce a yield twice that of pure water, comprising of high quality few layer graphene (3–5 Ls) with an average area of ∼1.15 (μm)2 and stability of ∼78% for the duration of six months. This combination is a promising eco-friendly substitute for future commercial manufacturing of graphene

Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy