A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Distortion Effect on the UHPC Box Girder with Vertical Webs: Theoretical Analysis and Case Study.

Distortion deformation usually imposes a potential threat to bridge safety. In order to comprehensively understand the distortion effect on thin-walled ultra-high performance concrete (UHPC) box girders, an innovative approach encompassing the governing distortion differential equation is introduced in this study based on the general definition of distortion angle within the cross-section plane. The analytical results obtained from the proposed method are in accordance with those obtained from the energy method, and exhibit favorable agreement with experimental findings documented in the existing literature. Furthermore, a finite element model is developed on the ANSYS 2021 R1 software platform with the employment of a Shell 63 element. Numerical outcomes are also in good agreement with the experimental data, affirming the validity and reliability of the findings. In addition, parameter analysis results indicate that the distortion angle remains approximately constant at a location approximately 1/10 of the span from the mid-span cross-section of the box girder, regardless of changes in the span-to-depth ratio. Increasing the web thickness yields a notable reduction in the distortion effects, and decreasing the wall thickness can effectively mitigate the distortion-induced transverse bending moment. Compared with normal-strength concrete box girders, UHPC box girders can reduce the distortion angle within the span range, which is beneficial for maintaining the overall stability of the box girders. The outcomes obtained from this study yield engineers an enhanced understanding of distortion effect on the UHPC girder performance.

In-situ self-crosslinking strategy for super-tough polylactic acid/ bio-based polyurethane blends.

As a bio-based degradable plastic, polylactic acid (PLA) is highly commercialized, but its inherent brittleness limits its widespread use. In-situ polymerization techniques are effective in improving the toughness of PLA. However, the enhancement of the toughening effect in polyurethanes (PUs) through in-situ self-crosslinking still requires improvement and heavily relies on petroleum-derived feedstocks in certain approaches. In this paper, 1,3-polypropanediol (PO3G) of bio-based origin rather than conventional polyols like polyethylene glycol (PEG) and poly propylene glycol (PPG) was used. PLA/PO3G-PU blends were prepared via an in-situ self-crosslinking strategy. With a notch impact and tensile strength of 55.95 kJ/m2 and 47.77 MPa (a retention rate of 68.9 % compared with pure PLA), respectively, PLA/PO3G-PU blends achieved a better balance between stiffness and toughness. This work provides a new option for PLA to achieve a stiffness-toughness balance and get rid of dependence on petrochemical resources.

ATG12-ATG5-TECPR1: an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage.

ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12-ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12-ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

Inducin Triggers LC3-Lipidation and ESCRT-Mediated Lysosomal Membrane Repair.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes.

Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.

Eating while intoxicated: characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy.

Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.

V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy.

Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.

Polyurethanes Based on Polylactic Acid for 3D Printing and Shape-Memory Applications.

Polylactic acid (PLA) has received increased attention in the development of shape-memory polymers and biomedical materials owing to its excellent physical properties and good biocompatibility and biodegradability. However, the inherent brittleness and high shape-recovery temperature of this material limit its application in the human body. Herein, we fabricated a PLA-based thermoplastic polyurethane (PLA-TPU) prepared from modified PLA-diol, dicyclohexylmethane-4,4'-diisocyanate, and 1,4-butanediol to solve the limitations of pure PLA. The glass transition temperature (Tg) of the designed TPU can be tailored from 6 to 40.5 °C by adjusting the content of hard segments or molecular weight of soft segments. The shape of the designed TPU can be fixed at room temperature and recovered at temperatures above 37 °C. Moreover, the prepared PLA-TPUs exhibited recyclability, three-dimensional printing capability, non-cytotoxicity, blood compatibility, and biodegradability. The shape of PLA-TPU/nano-Fe3O4 composites can be recovered by exposure to near-infrared light. These results collectively indicate that PLA-TPUs and their composites may have potential applications as intelligent flexible medical scaffolds for surgical and medical implantation equipment.

Bio-Based, Recyclable and Self-Healing Polyurethane Composites with High Energy Dissipation and Shape Memory.

Rubber composites make an important contribution to eliminating vibration and noise owing to their unique viscoelasticity. However, it is important to find alternative bio-based products with high damping properties owing to the shortage of petrochemical resources and poor performance. The ability to self-heal is an additional characteristic that is highly desirable because it can further increase the service life and safety of such products. In this study, a bio-based polylactic acid thermoplastic polyurethane (PLA-TPU) and its composites (PLA-TPU/AO-80) are synthesized. The reversible sacrificial hydrogen bonds in the composites increase the peak value of the loss factor (tan δmax ) from 0.87 to 2.12 with a high energy dissipation efficiency of 99% at 50% strain. After being heated for 15 min, the healed sample recovers 81.98% of its comprehensive mechanical properties due to the reorganization of the hydrogen bonds. Its tensile strength remains at 93.4% after recycling five times. Moreover, its shape memory properties show a response temperature close to the human body temperature making it an ideal candidate for medical applications.

Nonenzymatic Browning of Amorphous Maltose/Whey Protein Isolates Matrix: Effects of Water Sorption and Molecular Mobility.

Nonenzymatic browning (NEB) reactions often affect the nutritional quality and safety properties of amorphous food solids. Developing a proper approach to control the NEB reaction has been of particular interest in the food industry. An NEB reaction in an amorphous maltose/Whey protein isolates (WPI) matrix containing L-lysine and D-xylose as reactants were studied at ambient temperatures aw ≤ 0.44 and 45~65 °C. The results indicated that the presence of NEB reactants barely disturbed the water sorption behavior of the matrix. The Guggenheim-Anderson-de Boer (GAB) constants and Qst values of the studied samples were affected by storage conditions as the migration of sorbed water among monolayers occurred. The rate of color changes and 5-hydoxymethylfurfural (5-HMF) accumulation on the matrix were accelerated at high ambient temperatures aw, reflecting the extent of NEB reaction increases. Since the strength concept (S) could give a measure of molecular mobility, the extent of the NEB reaction was governed by the molecular mobility of the matrix as the activation energy (Ea) of 5-HMF production minimized at solids with high S values. We found that the S concept had a considerable potential usage in controlling the NEB reaction on amorphous sugar-protein solids. This data set has practical significance in the comprehensive understanding of manipulating the diffusion-limited chemical reactions on low-moisture food solids.

Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry.

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Bio-Based Polyurethane and Its Composites towards High Damping Properties.

The operation of mechanical equipment inevitably generates vibrations and noise, which are harmful to not only the human body but also to the equipment in use. Damping materials, which can convert mechanical energy into thermal energy, possess excellent damping properties in the glass transition region and can alleviate the problems caused by vibration and noise. However, these materials mainly rely on petroleum-based resources, and their glass transition temperatures (Tg) are lower than room temperature. Therefore, bio-based materials with high damping properties at room temperature must be designed for sustainable development. Herein, we demonstrate the fabrication of bio-based millable polyurethane (BMPU)/hindered phenol composites that could overcome the challenges of sustainable development and exhibit high damping properties at room temperature. BMPUs with a high Tg were prepared from modified poly (lactic acid)-based polyols, the unsaturated chain extender trimethylolpropane diallylether, and 4,4'-diphenylmethane diisocyanate, and 3,9-Bis-{1,1-dimethyl-2[ß-(3-tert-butyl-4-hydroxy-5-methylphenyl-)propionyloxy]ethyl}-2,4,8,10-tetraoxaspiro [5,5]-undecane (AO-80) was added to prepare BMPU/AO-80 composites. Finally, the properties of the BMPUs and BMPU/AO-80 composites were systematically evaluated. After adding 30 phr of AO-80, the Tg and maximum loss factor (tan δmax) of BMPU/AO-80 composites increased from 7.8 °C to 13.5 °C and from 1.4 to 2.0, respectively. The tan δmax showed an improvement of 43%. Compared with other polyurethanes, the prepared BMPU/AO-80 composites exhibited higher damping properties at room temperature. This study proposes a new strategy to reduce society's current dependence on fossil resources and design materials featuring high damping properties from sustainable raw materials.

Physicochemical properties and Strength analysis of vitreous encapsulated solids for the safe delivery of ß-Carotene.

ß-Carotene (ß-Car) is insoluble compounds in water and liable to degradation, which has health benefits for human beings. Although layer-by-layer (LBL) emulsions provide a better protection for ß-Car towards environmental stresses, the handling and transportation of LBL emulsions still faces restrictions. In this paper, therefore, the LBL emulsions including ß-Car were carefully prepared and encapsulated to obtain vitreous encapsulated solids (VES) using trehalose and maltodextrins (MD) as wall materials. Morphological results indicated that the LBL emulsions were formed a spheric shape, in where the polyelectrolyte shell was 30 nm. The MD exhibited the characteristics of not easy to absorb moisture, suitable carrier, and good stabilizer, which could improve the stability of VES systems at studied environmental stresses. Despite compositional effects from MD and environmental stresses, LBL emulsions changed the water sorption behavior of VES as oil dispersion in storage. Strength analysis indicated that LBL emulsions lubricated systems and increased the molecular mobility of wall materials. Structural collapse, rapid color changes, and ß-Car loss were confirmed in VES systems at 0.56 aw from 25 to 45 °C after 30 days of storage. Besides, a relationship between S and ß-Car loss kinetics was established, where the ß-Car degraded more rapidly in a sample with quicker molecular mobility of wall materials Therefore, the controlling of molecular mobility in wall materials can slow down the ß -Car degradation and improve the quality and stability of lipophilic nutrients delivery systems with high total solids.

Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation.

Design and synthesis of pseudo-natural products (PNPs) through recombination of natural product (NP) fragments in unprecedented arrangements enables the discovery of novel biologically relevant chemical matter. With a view to wider coverage of NP-inspired chemical and biological space, we describe the combination of this principle with macrocycle formation. PNP-macrocycles were synthesized efficiently in a stereoselective one-pot procedure including the 1,3-dipolar cycloadditions of different dipolarophiles with dimeric cinchona alkaloid-derived azomethine ylides formed in situ. The 20-membered bis-cycloadducts embody 18 stereocenters and an additional fragment-sized NP-structure. After further functionalization, a collection of 163 macrocyclic PNPs was obtained. Biological investigation revealed potent inducers of the lipidation of the microtubule associated protein 1 light chain 3 (LC3) protein, which plays a prominent role in various autophagy-related processes.

Monitoring the Response of Multiple Signal Network Components to Acute Chemo-Optogenetic Perturbations in Living Cells.

Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy-based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo-optogenetic approach that is based on switch-like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback-controlled regulation in the cell contraction signal network that includes GEF-H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants.

Biobased and Recyclable Polyurethane for Room-Temperature Damping and Three-Dimensional Printing.

Petroleum-based polymer materials heavily rely on nonrenewable petrochemical resources, and damping materials are an important category of them. As far as green chemistry, recycling, and damping materials are concerned, there is an urgent need for renewable and recyclable biobased materials with high damping performance. Thus, this study designs and synthesizes a series of polylactic acid-based thermoplastic polyurethanes (PLA-based TPUs) composed of modified polylactic acid polyols, 4,4'-diphenylmethane diisocyanate, and 1,4-butanediol. PLA-based TPUs, as prepared, display excellent mechanical properties, damping performance, and biocompatibility. Otherwise, they can be used for three-dimensional printing (3D printing). Under multiple recycling, the overall performance of PLA-based TPUs is still maintained well. Overall, PLA-based TPUs, as designed in this article, show a potential application in damping materials under room temperature and personalized shoes via 3D printing and could realize resource recycling and material reuse.

Imaging of Spatial Cycling of Rab GTPase in the Cell.

Rab GTPases (>60 members in human) function as master regulators of intracellular membrane trafficking. To fulfill their functions, Rab proteins need to localize on specific membranes in cells. It remains elusive how the distinct spatial distribution of Rab GTPases in the cell is regulated. To make a global assessment on the subcellular localization of Rab1, we determined kinetic parameters of the spatial cycling of Rab1 in live cells using photoactivatable fluorescent proteins and live cell imaging. We found that the switching between GTP- and GDP-binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), GDP dissociation inhibitor (GDI) and GDI displacement factor (GDF), is a major determinant for Rab1's ability to effectively cycle between cellular compartments and eventually for its subcellular distribution. Herein, we describe the method for monitoring Rab1 dynamics in live cells. This approach can be used to study spatial cycling of other Rab GTPases.