Mitochondrial matrix RTN4IP1/OPA10 is an oxidoreductase for coenzyme Q synthesis.

Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.

A tunnel coaxial 3D hyperspectral scanning system for underground mine investigation.

A hyperspectral scanning system was developed for three-dimensional (3D) surface mapping in underground spaces, such as mine shafts and tunnels. A hyperspectral line-scanning camera was mounted on the rotating driver unit coaxial with the tunnel to image both the mine wall and the ceiling. Uniform light was illuminated on the target surface to be imaged using a halogen lamp rotating together with the hyperspectral imaging sensor. Inertial Measuring Unit (IMU) was also attached to the sensor unit together with the hyperspectral camera so that sensor's geometric information could be acquired simultaneously during imaging. All sensor and controller units were mounted on a cart-type platform for easy movement in the tunnel, and a battery mounted on the platform supplied power for system operation and the halogen light source. The developed scanning system was tested in an actual mine, and 3D hyperspectral images of the internal surface of the mine shaft were successfully obtained.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice.

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Identification of New Non-BBB Permeable Tryptophan Hydroxylase Inhibitors for Treating Obesity and Fatty Liver Disease.

Serotonin (5-hydroxytryptophan) is a hormone that regulates emotions in the central nervous system. However, serotonin in the peripheral system is associated with obesity and fatty liver disease. Because serotonin cannot cross the blood-brain barrier (BBB), we focused on identifying new tryptophan hydroxylase type I (TPH1) inhibitors that act only in peripheral tissues for treating obesity and fatty liver disease without affecting the central nervous system. Structural optimization inspired by para-chlorophenylalanine (pCPA) resulted in the identification of a series of oxyphenylalanine and heterocyclic phenylalanine derivatives as TPH1 inhibitors. Among these compounds, compound 18i with an IC50 value of 37 nM was the most active in vitro. Additionally, compound 18i showed good liver microsomal stability and did not significantly inhibit CYP and Herg. Furthermore, this TPH1 inhibitor was able to actively interact with the peripheral system without penetrating the BBB. Compound 18i and its prodrug reduced body weight gain in mammals and decreased in vivo fat accumulation.

Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice.

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Configurable topological textures in strain graded ferroelectric nanoplates.

Topological defects in matter behave collectively to form highly non-trivial structures called topological textures that are characterised by conserved quantities such as the winding number. Here we show that an epitaxial ferroelectric square nanoplate of bismuth ferrite subjected to a large strain gradient (as much as 105 m-1) associated with misfit strain relaxation enables five discrete levels for the ferroelectric topological invariant of the entire system because of its peculiar radial quadrant domain texture and its inherent domain wall chirality. The total winding number of the topological texture can be configured from - 1 to 3 by selective non-local electric switching of the quadrant domains. By using angle-resolved piezoresponse force microscopy in conjunction with local winding number analysis, we directly identify the existence of vortices and anti-vortices, observe pair creation and annihilation and manipulate the net number of vortices. Our findings offer a useful concept for multi-level topological defect memory.

Ferroelectric domain states of a tetragonal BiFeO3 thin film investigated by second harmonic generation microscopy.

We investigate the ferroelectric state of a tetragonal BiFeO3 thin film grown on a LaAlO3 (001) substrate using an optical second harmonic generation (SHG) microscope. Whereas the ferroelectric state of this material hosts nanometer-sized domains which again form micrometer-sized domains of four different configurations, we could figure out the characteristic features of each domain from the SHG mapping with various sizes of the probe beam, i.e., from 0.7 to 3.9 µm in its diameter. In particular, we demonstrate that a single micrometer-sized domain contributes to the SHG as a coherent summation of the constituent nanometer-sized domains, and multi-micrometer-sized domains contribute to the SHG as an incoherent summation of each micro-domain.

Impact of Isovalent and Aliovalent Doping on Mechanical Properties of Mixed Phase BiFeO3.

In this study, we report the effect of doping in morphotropic BiFeO3 (BFO) thin films on mechanical properties, revealing variations in the elasticity across the competing phases and their boundaries. Spectroscopic force-distance (F-D) curves and force mapping images by AFM are used to characterize the structure and elastic properties of three BFO thin-film candidates (pure-BFO, Ca-doped BFO, La-doped BFO). We show that softening behavior is observed in isovalent La-doped BFO, whereas hardening is seen in aliovalent Ca-doped BFO. Furthermore, quantitative F-D measurements are extended to show threshold strengths for phase transitions, revealing their dependence on doping in the system. First-principles simulation methods are also employed to understand the observed mechanical properties in pure and doped BFO thin films and to provide microscopic insight on them. These results provide key insight into doping as an effective control parameter to tune nanomechanical properties and suggest an alternative framework to control coupled ferroic functionalities at the nanoscale.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.