Target Substrate Research Articles

Inhibition of USP1 enhances anticancer drugs-induced cancer cell death through downregulation of survivin and miR-216a-5p-mediated upregulation of DR5

Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.

Schiff Base Aggregation-Induced Emission Luminogens for Sensing Applications: A Review.

Fluorescence sensing can not only identify a target substrate qualitatively but also achieve the purpose of quantitative detection through the change of the fluorescence signal. It has the advantages of immense sensitivity, rapid response, and excellent selectivity. The proposed aggregation-induced emission (AIE) concept solves the problem of the fluorescence of traditional fluorescent molecules becoming weak or quenched in high concentration or aggregated state conditions. Schiff base fluorescent probes have the advantages of simple synthesis, low toxicity, and easy design. They are often used for the detection of various substances. In this review we cover late developments in Schiff base compounds with AIE characteristics working as fluorescence sensors.

Kinase-Catalyzed Crosslinking and Immunoprecipitation (K-CLIP) to Explore Kinase-Substrate Pairs.

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).

ATP hydrolysis tunes specificity of a AAA+ protease.

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. Invitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.

CBLC inhibits the proliferation and metastasis of breast cancer cells via ubiquitination and degradation of CTTN

The E3 ubiquitin ligase is an important regulator of cell signaling and proteostasis and is tightly controlled in many diseases, including cancer. Our study aimed to investigate the biological role of the E3 ubiquitin ligase CBLC in breast cancer and elucidate the specific mechanistic network underlying CBLC-mediated target substrate degradation, cell proliferation and metastasis. Here, we showed that CBLC expression was higher in breast cancer tissues and cells than that in normal tissues and cells. Higher expression of CBLC predicted a better prognosis for breast cancer patients. CBLC inhibited the proliferation, migration and invasion of breast cancer cells. Co-IP and immunofluorescence co-localization assays demonstrated that CBLC interacted with CTTN in the cytoplasm. CBLC promoted the degradation of CTTN through the ubiquitin-proteasome pathway without affecting its mRNA level. The inhibitory effect of CBLC on breast cancer cell proliferation, migration and invasion could partly be reversed by CTTN. Taken together, our study clarified the biological role of CBLC as a tumor suppressor and discovered its functional substrate, providing a molecular basis for CBLC/CTTN as a potential therapeutic target in breast cancer.

Surface Morphology-Enhanced Delivery of Bioinspired Eco-Friendly Microcapsules.

We report the development of novel mineralized protein microcapsules to address critical challenges in the environmental impact and performance of consumer, pharmaceutical, agrochemical, cosmetic, and paint products. We designed environment-friendly capsules composed of proteins and biominerals as an alternative to solid microplastic particles or core-shell capsules made of nonbiodegradable synthetic polymeric resins. We synthesized mineralized capsule surface morphologies to mimic the features of natural pollens, which dramatically improved the deposition of high value-added fragrance chemicals on target substrates in realistic application conditions. A mechanistic model accurately captures the observed enhanced deposition behavior and shows how surface features generate an adhesive torque that resists shear detachment. Mineralized protein capsule performance is shown to depend both on material selection that determines van der Waals attraction and on capsule-substrate energy landscapes as parameterized by a geometric taxonomy for surface morphologies. These findings have broad implications for engineering multifunctional environmentally friendly delivery systems.

The regulatory roles of the E3 ubiquitin ligase NEDD4 family in DNA damage response.

E3 ubiquitin ligases, an important part of ubiquitin proteasome system, catalyze the covalent binding of ubiquitin to target substrates, which plays a role in protein ubiquitination and regulates different biological process. DNA damage response (DDR) is induced in response to DNA damage to maintain genome integrity and stability, and this process has crucial significance to a series of cell activities such as differentiation, apoptosis, cell cycle. The NEDD4 family, belonging to HECT E3 ubiquitin ligases, is reported as regulators that participate in the DDR process by recognizing different substrates. In this review, we summarize recent researches on NEDD4 family members in the DDR and discuss the roles of NEDD4 family members in the cascade reactions induced by DNA damage. This review may contribute to the further study of pathophysiology for certain diseases and pharmacology for targeted drugs.

A facile approach towards Wrinkle-Free transfer of 2D-MoS2 films via hydrophilic Si3N4 substrate

Two-dimensional (2D) transition-metal dichalcogenides (TMDs) have been widely explored as next-generation semiconductor materials owing to their attractive electrical and optical properties. Currently, high-performance 2D-TMD-based electronic devices are obtained by transferring growth substrates onto target substrates. In practice, however, transfer-induced wrinkles and structural deformation on the target substrate pose a significant challenge. Herein, we successfully demonstrated a wrinkle-free transfer method that could facilely control the wrinkles of MoS2 thin films on hydrophilic Si3N4 substrates. Owing to the high wettability of the Si3N4 substrate, the residual water droplets between the MoS2 thin film and the target substrate were effectively removed, minimizing unexpected wrinkles and structural deformation. By simply adjusting the wetting properties of the target substrate, the device performance improved to a field-effect mobility of ∼ 0.756 cm2/(V·s), which is six times higher than that of the SiO2 substrate. Furthermore, the standard deviation of the electrical characteristics of the 50 2D-field-effect transistors was reduced by the Si3N4 substrate. In addition, the intrinsic characteristics of the MoS2 FET were evaluated by suppressing the effect of charge trapping through pulsed I-V measurements. Facilely-controlled wrinkles in MoS2 thin films can be used in various electronic devices, sensors, optoelectronic devices, and neuromorphic devices.

Reconfigurable Acoustic Absorber Comprising Flexible Tubular Resonators for Broadband Sound Absorption

Typical acoustic metamaterial absorbers rely on relatively rigid materials for acoustic resonances, which, however, poses a challenge in reshaping and reconfiguring them. Here, we demonstrate soft reconfigurable acoustic absorbers, which are realized by the use of flexible tubular resonators. Owing to the structural compliance, the tube absorbers promote intriguing acoustic-structure coupling and exhibit hybrid resonances of acoustic and structural modes, which are characterized by Rabi splitting in the strong-coupling regime and Fano-like resonance in the weak-coupling regime. Also, the soft walls of the flexible resonators induce decreased resonance frequencies for low-frequency absorption by reducing the effective sound speed inside the tube. Moreover, we find that these hybrid resonances are critically influenced by the structural loss of the flexible resonators. While elucidating the role of the material rigidity and structural loss in acoustic resonances, this work allows us to design versatile broadband absorbers that can be reconfigured depending on the topology of a target substrate.

Discovery of Graphene-Water Membrane Structure: Toward High-Quality Graphene Process.

It is widely accepted that solid‐state membranes are indispensable media for the graphene process, particularly transfer procedures. But these membranes inevitably bring contaminations and residues to the transferred graphene and consequently compromise the material quality. This study reports a newly observed free‐standing graphene‐water membrane structure, which replaces the conventional solid‐state supporting media with liquid film to sustain the graphene integrity and continuity. Experimental observation, theoretical model, and molecular dynamics simulations consistently indicate that the high surface tension of pure water and its large contact angle with graphene are essential factors for forming such a membrane structure. More interestingly, water surface tension ensures the flatness of graphene layers and renders high transfer quality on many types of target substrates. This report enriches the understanding of the interactions on reduced dimensional material while rendering an alternative approach for scalable layered material processing with ensured quality for advanced manufacturing.

One-Step Patterning of Organic Semiconductors on Gold Electrodes via Capillary-Bridge Manipulation.

Patterning organic semiconductors directly on Au electrodes possesses the advantages of eliminating metal atomic penetration effect, compatibility with fine lithography processes, and feasibility of work function modification on electrodes, and it is therefore of value in the commercial manufacturability application of optoelectronic devices with low cost, large scale, and high efficiency. Solution processing, is relatively inexpensive and is scalable to large areas. Nonetheless, conventional solution processing approaches have trade-offs among controllable morphology, regular alignment, precise position, and ordered molecular packing arising from the uncontrollable dewetting kinetics. Here, one-step patterning of 1D polymer nanowire arrays directly on Au source-drain electrodes with precise position, controlled orientation, regulated distribution, and tunable width size were realized by employing a capillary-bridge manipulation method to guide the processes of liquid dewetting and nanowire assembly. Organic field-effect transistors (OFETs) with mobility of 10.1 cm2 V-1 s-1 and on/off current ratio of 1.9 × 104 were fabricated. Moreover, we verified generality of our method by patterning different solution-processable optoelectronic materials, including small molecules, quantum dot (QD) nanoparticles, and metal-halide perovskites, into ordered structures directly on the target substrate. The work provides a novel insight into efficient manufacturing the regular aligned and precisely positioned 1D organic semiconductors directly on the channel region of prefabricated Au electrodes in one step and facilitates their applications in high-performance electronic devices.

Molecular-Weight-Dependent Interplay of Brittle-to-Ductile Transition in High-Strain-Rate Cold Spray Deposition of Glassy Polymers.

Based on the cold spray technique, the solvent-free and solid-state deposition of glassy polymers is envisioned. Adiabatic inelastic deformation mechanisms in the cold spray technique are studied through high-velocity collisions (<1000 m/s) of polystyrene microparticles against stationary target substrates of polystyrene and silicon. During extreme collisions, a brittle-to-ductile transition occurs, leading to either fracture- or shear-dominant inelastic deformation of the colliding microparticles. Due to the nonlinear interplay between the adiabatic shearing and the thermal softening of polystyrene, the plastic shear flow becomes the dominant deformation channel over brittle fragmentation when increasing the rigidity of the target substrate. High molecular weights (>20 kDa) are essential to hinder the evolution of brittle fracture and promote shear-induced heating beyond the glass transition temperature of polystyrene. However, an excessively high molecular weight (∼100 kDa) reduces the adhesion of the microparticles to the substrate due to insufficient wetting of the softened polystyrene. Due to the two competing viscoelastic effects, proper selection of molecular weight becomes critical for the cold spray technique of glassy polymers.

Modeling and computational characterization of a Xanthomonas sp. Hypothetical protein identifies a remote ortholog of Burkholderia lethal factor 1

Burkholderia Lethal Factor 1 (BLF1) is a deamidase first characterized in Burkholderia pseudomallei. This enzyme inhibits cellular protein synthesis by deamidating a glutamine residue to a glutamic acid in its target protein, the eukaryotic translation initiation factor 4 A (eIF4A). In this work, we present the characterization of a hypothetical protein from Xanthomonas sp. Leaf131 as the first report of a BLF1 family ortholog outside of the Burkholderia genus. Although standard sequence similarity searches such as BLAST were not able to detect the homology between the Xanthomonas sp. Leaf131 hypothetical protein sequence and BLF1, our computed structure model for the Xanthomonas sp. hypothetical protein revealed structural similarities with an RMSD of 2.7 Å/164 Cα atoms and a TM-score of 0.72 when superposed. Structural comparisons of the Xanthomonas model structure against BLF1 and Escherichia coli cytotoxic necrotizing factor 1 (CNF1) revealed that the conserved signature LXGC motif and putative catalytic residues are structurally aligned thus signifying a level of functional or mechanistic similarity. Protein-protein docking analysis and molecular dynamics simulations also demonstrated that eIF4A could still be a possible target substrate for deamidation by XLF1 as it is for BLF1. We therefore propose that this Xanthomonas hypothetical protein be renamed as Xanthomonas Lethal Factor 1 (XLF1). Our work also provides further evidence of the utility of programs such as AlphaFold in bridging the computational function annotation transfer gap despite very low sequence identities of under 20%. Communicated by Ramaswamy H. Sarma

CFD Simulation Using ANSYS FLUENT of Jet Nozzle of Ethanol at Temperature of 360 K

Spray coating technology is an advanced manufacturing process to break the liquid down into small droplets and disperse these droplets uniformly on the target substrate. The nozzle used in this fabrication process rapidly disintegrates the liquid jet at a direction of motion governed mainly by the prevailing airflow pattern in the spray region environment. According to Navier–Stokes energy equation, the fluid and environmental temperature capable of supplying additional energy to the droplet imparted the ejected liquid jet from the nozzles. With a view to understanding the liquid droplet behaviour at elevated temperatures, this study investigates the jet velocity, pressure, and temperature of ethanol at its evaporation temperature of 360 K using commercial CFD software (ANSYS FLUENT 2020). A numerical simulation of a simple nozzle system with a cone angle of 56° and an outlet radius of 0.1966 mm was modelled at 1 bar inlet pressure to study the effect of the environment temperature on the liquid jet. The result from the temperature profile showed that the liquid jet exhibited turbulence or a swirling effect at elevated temperature as the liquid absorbed thermal energy. The liquid jet temperature increased to the maximum temperature of +3K at a liquid jet distance of 10 cm when compared with normal environment temperature. This numerical study showed that environmental conditions such as temperature can impact the liquid jet quality and properties, which are difficult to observe during experimental work. It is expected that the finding from this work would benefit the industries, leading to better spray nozzle design for future coating applications.

Underwater Multispectral Laser Serial Imager for Spectral Differentiation of Macroalgal and Coral Substrates

The advancement of innovative underwater remote sensing detection and imaging methods, such as continuous wave laser line scan or pulsed laser (i.e., LiDAR—Light Detection and Ranging) imaging approaches can provide novel solutions for studying biological substrates and manmade objects/surfaces often encountered in underwater coastal environments. Such instruments can be used shipboard or coupled with proven and available deployment platforms as AUVs (Autonomous Underwater Vehicles). With the right planning, large areas can be surveyed, and more extreme and difficult-to-reach environments can be studied. A prime example, and representing a certain navigational challenge, is the under ice in the Arctic/Antarctic or winter/polar environments or deep underwater survey. Among many marine biological substrates, numerous species of macroalgae can be found worldwide in shallow down to 70+ m (clear water) coastal habitats and are essential ecosystem service providers through the habitat they provide for other species, the potential food resource value, and carbon sink they represent. Similarly, corals also provide important ecosystem services through their structure and diversity, are found to harbor increased local diversity, and are equally valid targets as “keystone” species. Hence, we expand current underwater remote sensing methods to combine macroalgal and coral surveys via the development of a multispectral laser serial imager designed for classification via spectral response. By using multiple continuous wave laser wavelength sources to scan and illuminate recreated benthic environments composed of macroalgae and coral, we show how elastic (i.e., reflectance) and inelastic (i.e., fluorescence) spectral responses can potentially be used to differentiate algal color groups and certain coral genus. Experimentally, three laser diodes (450 nm, 490 nm, 520 nm) are sequentially used in conjunction with up to 5 emission filters (450 nm, 490 nm, 520 nm, 580 nm, 685 nm) to acquire images generated by laser line scan pattern via high-speed galvanometric mirrors. Placed directly adjacent to a large saltwater imaging tank fitted with optical viewports, the optical system records target substrate spectral response using a photomultiplier preceded by a filter and is synchronously digitized to the scan rate by a high sample rate Analog-to-Digital Converter (ADC). Acquired images are normalized to correct for imager optical effects allowing for fluorescence intensity-based pixel segmentation via intensity thresholding. Overall, the multispectral laser serial imaging technique shows that the resulting high resolution data can be used for detection and classification of benthic substrates by their spectral response. These methods highlight a path towards eventual pixel-wise spectral response analysis for spectral differentiation.

Vitamin C-reduced graphene oxide improves the performance and stability of multimodal neural microelectrodes

SummaryNanocarbons are often employed as coatings for neural electrodes to enhance surface area. However, processing and integrating them into microfabrication flows requires complex and harmful chemical and heating conditions. This article presents a safe, scalable, cost-effective method to produce reduced graphene oxide (rGO) coatings using vitamin C (VC) as the reducing agent. We spray coat GO + VC mixtures onto target substrates, and then heat samples for 15 min at 150°C. The resulting rGO films have conductivities of ∼44 S cm−1, and are easily integrated into an ad hoc microfabrication flow. The rGO/Au microelectrodes show ∼8x lower impedance and ∼400x higher capacitance than bare Au, resulting in significantly enhanced charge storage and injection capacity. We subsequently use rGO/Au arrays to detect dopamine in vitro, and to map cortical activity intraoperatively over rat whisker barrel cortex, demonstrating that conductive VC-rGO coatings improve the performance and stability of multimodal microelectrodes for different applications.

O2-Mediated Te(II)-Redox Catalysis for the Cross-Dehydrogenative Coupling of Indoles

Very few elements in the periodic system can catalytically activate O2, such as in the context of cross-dehydrogenative couplings. The development of O2-activating catalysts is essential to enable new and sustainable reactivity concepts to emerge, because these catalysts also often feature specific activating interactions with the target substrates. In this context, the unprecedented Te(II)/Te(III) catalyzed dehydrogenative C3–C2 dimerization of indoles is described herein. The fact that O2 can be directly utilized as a terminal oxidant in this reaction, as well as the absence of any background reactivity without the redox-active Te catalyst, constitute very important milestones for the fields of cross-dehydrogenative couplings and tellurium catalysis.

Generalized Approach towards Secretion-Based Protein Production via Neutralization of Secretion-Preventing Cationic Substrate Residues.

Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but some proteins are not secretable even though they carry the right signal sequence. The invention of a method to secrete these non-secretable proteins would be valuable both for understanding the secretory physiology of ABC transporters and for industrial applications. Herein, we postulate that cationic “supercharged” regions within the target substrate protein block the secretion by ABC transporters. We also suggest that the secretion of such substrate proteins can be rescued by neutralizing those cationic supercharged regions via structure-preserving point mutageneses. Surface-protruding, non-structural cationic amino acids within the cationic supercharged regions were replaced by anionic or neutral hydrophilic amino acids, reducing the cationic charge density. The examples of rescued secretions we provide include the spike protein of SARS-CoV-2, glutathione-S-transferase, streptavidin, lipase, tyrosinase, cutinase, growth factors, etc. In summary, our study provides a method to predict the secretability and a tool to rescue the secretion by correcting the secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.

A new model based on the in-plane deformation for the conformal piezoelectric systems for characterization of soft tissue modulus

Mechanical assessment of the human skin is critically important for clinical diagnosis and treatment of disease. Past works developed a conformal piezoelectric system (CPS) that enables the noninvasive in vivo measurement of the elasticity modulus of the skin, and a mechanical model based on the mechanism of out-of-plane extension of the capacitor-type structures. Here, we present a new theoretical model that is established based on the mechanism of the in-plane deformation. The study on both the free-standing CPS and the CPS mounting to the polydimethylsiloxane (PDMS) target substrates verified the universality of the new model. The experimental and theoretical results consistently show that the output voltage of the first sensor approximately linearly increases with the increase of not only the applied voltage but also the modulus of the PDMS. The optimal usage mode of the CPS, in which the voltage is applied to all actuators simultaneously, is proposed for the measurement of the elastic modulus of target substrates such as skin.

Structural Integrity Preserving and Residue-Free Transfer of Large-Area Wrinkled Graphene onto Polymeric Substrates

Wrinkled graphene offers many advantageous features resulting from modifying the structural and physical properties as well as the chemical reactivity of graphene. However, its inadequate transferability to other substrates has limited its usability. This paper reports a roll-based clean transfer approach that enables the damage-free and contamination-free transfer of large-area wrinkled graphene onto polymeric substrates without compromising the integrity of wrinkle structures. The method implements the simultaneous imidazole-assisted etching and doping of chemical vapor-deposited graphene to fabricate multilayer graphene on a thermoplastic polystyrene (PS) substrate coated with a water-soluble poly(4-styrenesulfonic acid) (PSS) sacrificial layer via a roll-based transfer process. The compliant PSS layer affords the conformal contact between the PS substrate and graphene during the wrinkle formation process, enabling the controllable fabrication of graphene wrinkle structures on a large area. The water-soluble properties of PSS simplify the typically difficult separation of wrinkled graphene from the PS substrate after its transfer onto a target substrate. This improves the transferability of wrinkled graphene, rendering the transfer process solvent-free and residue-free. This work demonstrates the feasibility of the formulated method by transferring centimeter-scale wrinkled graphene onto currently used transparent flexible substrates (i.e., polyethylene terephthalate and polydimethylsiloxane). The results indicate that the transferred wrinkled graphene possesses the desirable combination of superior stretchability, optical transmittance, sheet resistance, and electromechanical stability, rendering its suitable application to transparent flexible and stretchable electronics.