Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Deep learning supported machine vision system to precisely automate the wild blueberry harvester header.

An operator of a wild blueberry harvester faces the fatigue of manually adjusting the height of the harvester's head, considering spatial variations in plant height, fruit zone, and field topography affecting fruit yield. For stress-free harvesting of wild blueberries, a deep learning-supported machine vision control system has been developed to detect the fruit height and precisely auto-adjust the header picking teeth rake position. The OpenCV AI Kit (OAK-D) was used with YOLOv4-tiny deep learning model with code developed in Python to solve the challenge of matching fruit heights with the harvester's head position. The system accuracy was statistically evaluated with R2 (coefficient of determination) and σ (standard deviation) measured on the difference in distances between the berries picking teeth and average fruit heights, which were 72, 43% and 2.1, 2.3 cm for the auto and manual head adjustment systems, respectively. This innovative system performed well in weed-free areas but requires further work to operate in weedy sections of the fields. Benefits of using this system include automated control of the harvester's head to match the header picking rake height to the level of the fruit height while reducing the operator's stress by creating safer working environments.

Water-soluble adhesive for stable long-term ambulatory EEG recordings.

OBJECTIVE: Conventional methods used to adhere EEG electrodes are often uncomfortable. Here, we present a polymer-based water-soluble EEG adhesive that can be maintained for up to 6 days. The primary outcome measure of this study is the median electrode impedance at day 6. METHODS: Impedance measurements for 841 EEG recordings using a 21 channel 10-20 configuration were remotely logged daily for 6 days after connection. A novel electrode adhesive was used to attach EEG electrodes. Patients were instructed to maintain their electrodes on day 4. RESULTS: Median electrode impedances were significantly below 10kOhms for each day of recording, with a median value on day 6 of 4.18kOhms. Impedance values were significantly lower on day 5 than on day 4, demonstrating that the maintenance process can reduce impedance. Except for day 4-5, the median impedance increased each day. No significant difference was found on the first or final day between clinics or residences from areas of different geographic remoteness. CONCLUSIONS: EEG is able to be recorded in patients homes for 6 days with acceptable impedance and no significant effect of regionality or patients age. SIGNIFICANCE: To the best of our knowledge, this is the first report in the literature of impedance data from long-term ambulatory EEG studies.

Crystal structure of the hinge domain of Smchd1 reveals its dimerization mode and nucleic acid-binding residues.

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.

Microparticle dispensers for the controlled release of insect pheromones.

The potential utility of micrometer-sized particles as controlled-release devices for the volatilization of insect pheromones for mating disruption applications is evaluated in this study for two pheromone/model compound systems (codlemone/1-dodecanol and disparlure/1,2-epoxyoctadecane). To expedite the measurement of release rates from these particle devices, two techniques based on thermogravimetric analysis (TGA) have been exploited: isothermal TGA (I-TGA) at elevated temperatures (40-80 degrees C) with N(2) convection and volatilization temperature (VT) by dynamic TGA. A correlation between these two methods has been established. Samples that exhibit a higher VT provide a lower release rate from a particle substrate. Using these techniques, it has been demonstrated that chemical interactions between adsorbed liquids and particle surfaces may play a small role in defining release characteristics under conditions of low surface area, whereas parameters associated with total surface area and micropore structure appear to be much more significant in retarding evaporation for uncoated particles containing an adsorbed liquid. Additional regulation of release rates was achieved by coating the particle systems with water-soluble or water-dispersible polymers. By careful selection of particle porosity and coating composition, it is envisioned that the evaporation rate of pheromones can be tailored to specific insect control applications.