4 research outputs found

    A mechanistic target of rapamycin complex 1/2 (mTORC1)/V-Akt murine thymoma viral oncogene homolog 1 (AKT1)/cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in patients with atopic dermatitis

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    Background Filaggrin, which is encoded by the filaggrin gene (FLG), is an important component of the skin's barrier to the external environment, and genetic defects in FLG strongly associate with atopic dermatitis (AD). However, not all patients with AD have FLG mutations. Objective We hypothesized that these patients might possess other defects in filaggrin expression and processing contributing to barrier disruption and AD, and therefore we present novel therapeutic targets for this disease. Results We describe the relationship between the mechanistic target of rapamycin complex 1/2 protein subunit regulatory associated protein of the MTOR complex 1 (RAPTOR), the serine/threonine kinase V-Akt murine thymoma viral oncogene homolog 1 (AKT1), and the protease cathepsin H (CTSH), for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in patients with AD. In keratinocyte cell cultures RAPTOR upregulation or AKT1 short hairpin RNA knockdown reduced expression of the protease CTSH. Skin of CTSH-deficient mice and CTSH short hairpin RNA knockdown keratinocytes showed reduced filaggrin processing, and the mouse had both impaired skin barrier function and a mild proinflammatory phenotype. Conclusion Our findings highlight a novel and potentially treatable signaling axis controlling filaggrin expression and processing that is defective in patients with AD

    Electroless Au Plating of CMOS Microelectrodes: Fabrication, Characterisation and Electrochemical Measurement

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    An essential step in developing amperometric sensors directly on CMOS integrated circuits (ICs) is to cover the exposed uppermost metal layer (aluminium pads) with a thin layer of noble metal to form the basis of the sensing electrode. A simple and scalable method to achieve the gold layer is through electroless plating. Despite the popularity of electroless plating in e.g. PCB manufacturing, there is a lack of information on how it can be applied to Al microelectrodes, and what the electrochemical performances of Au-coated microelectrodes are. This paper presents a detailed process for electroless gold plating of CMOS microelectrodes, with a step-by-step characterisation of the surface roughness, thickness, and elemental composition to optimise the deposition parameters (e.g. deposition time and temperature) for achieving a smooth and uniform gold coverage of the microelectrodes. A gold layer with a rms surface roughness of 53.6 ± 7.9 nm is achieved on the microelectrodes and successfully characterised by cyclic voltammetry in a ferri/ferrocyanide solution. Sonication, oxygen plasma, and continuous cyclic voltammetry are applied to the Au-coated microelectrodes to determine their mechanical and electrochemical stability

    Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance

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    Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis
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