A compact system for simultaneous stimulation and recording for closed-loop myoelectric control

Background.Despite important advancements in control and mechatronics of myoelectric prostheses, the communication between the user and his/her bionic limb is still unidirectional, as these systems do not provide somatosensory feedback. Electrotactile stimulation is an attractive technology to close the control loop since it allows flexible modulation of multiple parameters and compact interface design via multi-pad electrodes. However, the stimulation interferes with the recording of myoelectric signals and this can be detrimental to control.The work in this study was supported by the project ROBIN (8022-00243A and 8022-00226B) funded by the Independent Research Fund Denmark

Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair

CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN

SummaryTumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk

Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival