High-Throughput Two-Photon 3D Printing Enabled by Holographic Multi-Foci High-Speed Scanning.

The emerging two-photon polymerization (TPP) technique enables high-resolution printing of complex 3D structures, revolutionizing micro/nano additive manufacturing. Various fast scanning and parallel processing strategies have been proposed to promote its efficiency. However, obtaining large numbers of uniform focal spots for parallel high-speed scanning remains challenging, which hampers the realization of higher throughput. We report a TPP printing platform that combines galvanometric mirrors and liquid crystal on silicon spatial light modulator (LCoS-SLM). By setting the target light field at LCoS-SLM's diffraction center, sufficient energy is acquired to support simultaneous polymerization of over 400 foci. With fast scanning, the maximum printing speed achieves 1.49 × 108 voxels s-1, surpassing the existing scanning-based TPP methods while maintaining high printing resolution and flexibility. To demonstrate the processing capability, functional 3D microstructure arrays are rapidly fabricated and applied in micro-optics and micro-object manipulation. Our method may expand the prospects of TPP in large-scale micro/nanomanufacturing.

Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-µM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.

Light-triggered multi-joint microactuator fabricated by two-in-one femtosecond laser writing.

Inspired by the flexible joints of humans, actuators containing soft joints have been developed for various applications, including soft grippers, artificial muscles, and wearable devices. However, integrating multiple microjoints into soft robots at the micrometer scale to achieve multi-deformation modalities remains challenging. Here, we propose a two-in-one femtosecond laser writing strategy to fabricate microjoints composed of hydrogel and metal nanoparticles, and develop multi-joint microactuators with multi-deformation modalities (>10), requiring short response time (30 ms) and low actuation power (<10 mW) to achieve deformation. Besides, independent joint deformation control and linkage of multi-joint deformation, including co-planar and spatial linkage, enables the microactuator to reconstruct a variety of complex human-like modalities. Finally, as a proof of concept, the collection of multiple microcargos at different locations is achieved by a double-joint micro robotic arm. Our microactuators with multiple modalities will bring many potential application opportunities in microcargo collection, microfluid operation, and cell manipulation.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Direct laser writing structural color for reversible encryption and decryption in different mediums.

Structural color (SC) has enormous potential for improving the visualization and identification of functional micro/nano structures for information encryption and intelligent sensing. Nevertheless, achieving the direct writing of SCs at the micro/nano scale and the change of color in response to external stimuli simultaneously is rather challenging. To this end, we directly printed woodpile structures (WSs) utilizing femtosecond laser two-photon polymerization (fs-TPP), which demonstrated obvious SCs under an optical microscope. After that, we achieved the change of SCs by transferring WSs between different mediums. Furthermore, the influence of laser power, structural parameters, and mediums on the SCs was systematically investigated, and the mechanism of SCs using the finite-difference time-domain (FDTD) method was further explored. Finally, we realized the reversible encryption and decryption of certain information. This finding holds broad application prospects in smart sensing, anti-counterfeiting tags, and advanced photonic devices.

Survival processing advantage demonstrated with virtual reality-based survival environment: A promising tool for survival processing research.

Many studies have been conducted to demonstrate the survival processing advantage (SPA) as an evolutionary-oriented memory effect. But few studies were conducted to demonstrate this effect in real-life or simulated survival environments. This study tested whether the SPA could be replicated in a survival virtual reality environment (VRE). In Experiment 1, the SPAs were measured in VREs (survival grasslands, survival battlefield, nonsurvival moving) in which Experiment 1A used the standard long instructions and Experiment 1B used the modified short instructions. In Experiment 2, the SPAs were measured again with the scenarios corresponding to the VREs used in Experiment 1A. All experiments demonstrated typical SPAs, suggesting that the survival VRE is a reliable tool in designing and delivering a survival situation. The potential problems of applying survival VRE to survival processing research are discussed at the end.

4D Direct Laser Writing of Submerged Structural Colors at the Microscale.

Biomimetic stimuli-responsive structure colors (SCs) can improve the visualization and identification in the micro functional structure field such as information encryption/decryption and smart actuators. However, it is still challenging to develop the ability to 4D print arbitrary submerged colorful patterns with stimuli-responsive materials at the microscale. Herein, a hydrogel photoresist with feature resolution (98 nm) for the fabrication of 4D microscopic SCs by the femtosecond direct laser writing method is developed. The 4D printed woodpile SCs are grouped as pixel palettes with various laser parameters and they spanned almost the entire color space. The coloring mechanism of diffraction gratings is not only investigated by optics microscopy and spectroscopy but also supported by simulation. Moreover, the 4D printed hydrogel-integrated amphichromatic fish constructions and pixelated painting can visually discolor reversibly by regulating the solution pH. This finding promises an ideal coloring method for sensors, anti-counterfeiting labels, and transformable photonic devices.

On-Demand Maneuver of Millirobots with Reprogrammable Motility by a Hard-Magnetic Coating.

Millirobots that can be actuated and accurately steered by external magnetic fields, are highly desirable for bioengineering and wearable devices. However, existing designs of millirobots are limited by their specific material composition, hindering their wider application due to a lack of scalability. Here, we present a method for the generation of heterogeneous magnetic millirobots based on magnetic coatings. The coatings, composed of hard-magnetic CrO2 particles dispersed in an adhesive solution, impart magnetic actuation to diverse substrates with planar sheets or 3D structures. Millirobots constructed from the coatings can be readily reprogrammed with intricate magnetization profiles using laser localized heating, enabling reconfigurable shape changes under magnetic actuation. Using this approach, we demonstrate on-demand maneuvering capability of reconfiguring locomotion involving crawling, overturning and rolling with a single millirobot. Various functions, including the ability to catch a fast-moving ball, object transportation, and targeted assembly, have been achieved. This adhesive strategy facilitates the design of millirobots and may open avenues to the creation of complex millirobots for broad applications.

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.

Light-mediated multi-target protein degradation using arylazopyrazole photoswitchable PROTACs (AP-PROTACs).

Light-activable spatiotemporal control of PROTAC-induced protein degradation was achieved with novel arylazopyrazole photoswitchable PROTACs (AP-PROTACs). The use of a promiscuous kinase inhibitor in the design enables this unique photoswitchable PROTAC to selectively degrade four protein kinases together with on/off optical control using different wavelengths of light.

The effect of immersive virtual reality-based attentional bias modification on anxiety mitigation in healthy population.

The present study compared an immersive virtual reality-based attentional bias modification (immersive VR-ABM) with the desktop version of the VR-ABM and an immersive virtual reality-based game (immersive VR-game) to examine the possible effect of the immersive presence on self-reported emotional reactions to a stressful task. One hundred and twenty participants were randomly assigned into three groups, and each group received a three-turn induction-intervention training. Anxiety symptoms were assessed at pre-induction, post-induction, and post-training. Results showed that virtual reality-based anxiety was induced and alleviated successfully in all three groups, but only the immersive VR-ABM group showed an accumulated effect on self-reported anxiety across sessions. The attentional bias based on probe latencies indicated no significant change in either the immersive or desktop VR-ABM groups. The present findings support the hypothesized VR-ABM's effect on self-reported anxiety at the immersive presence. The practical implications of using immersive VR-ABM are discussed for obtaining ecological validity.

Ultrafast Laser-Ablated Bioinspired Hydrogel-Based Porous Gating System for Sustained Drug Release.

Gating systems have been extensively researched in energy harvesting, lab-on-chip applications, and so forth. However, the controlled drug delivery system with artificial hydrogel-based porous gating systems (HPGSs) is rarely reported. Herein, a biomimetic HPGS with a pH-responsive hydrogel as the valve and polydimethylsiloxane as the frame is fabricated by in situ femtosecond laser microdrilling and subsequent ultraviolet exposure. The proposed HPGS loaded with doxorubicin hydrochloride (DOX) is stable under physiological conditions, has a low drug leakage rate, and can achieve sustained drug release in a low pH environment. The experimental results show that the drug release is mainly controlled by non-Fickian diffusion, which renders the dynamic speed control of molecular transport possible. Moreover, the HPGS can also be prepared into an antitumor microcapsule. The results of in vitro cell experiments demonstrate that DOX@HPGS can release drugs and achieve terrific therapeutic efficacy in the elimination of HeLa cells in the acidic environments around tumor cells. This functional HPGS is envisioned to be an ideal pH-response carrier for sustained drug release treatment of digestive diseases such as inflammatory bowel disease and gastrointestinal cancer.

Functional Shape-Morphing Microarchitectures Fabricated by Dynamic Holographically Shifted Femtosecond Multifoci.

Functional microdevices based on responsive hydrogel show great promise in targeted delivery and biomedical analysis. Among state-of-the-art techniques for manufacturing hydrogel-based microarchitectures, femtosecond laser two-photon polymerization distinguishes itself by high designability and precision, but the point-by-point writing scheme requires mechanical apparatuses to support focus scanning. In this work, by predesigning holograms combined with lens phase modulation, multiple femtosecond laser spots are holographically generated and shifted for prototyping of three-dimensional shape-morphing structures without any moving equipment in the construction process. The microcage array is rapidly fabricated for high-performance target capturing enabled by switching environmental pH. Moreover, the built scaffolds can serve as arrayed analytical platforms for observing cell behaviors in normal or changeable living spaces or revealing the anticancer effects of loaded drugs. The proposed approach opens a new path for facile and flexible manufacturing of hydrogel-based functional microstructures with great versatility in micro-object manipulation.

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured ECs. Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Quasi-phase-matching-division multiplexing holography in a three-dimensional nonlinear photonic crystal.

Nonlinear holography has recently emerged as a novel tool to reconstruct the encoded information at a new wavelength, which has important applications in optical display and optical encryption. However, this scheme still struggles with low conversion efficiency and ineffective multiplexing. In this work, we demonstrate a quasi-phase-matching (QPM) -division multiplexing holography in a three-dimensional (3D) nonlinear photonic crystal (NPC). 3D NPC works as a nonlinear hologram, in which multiple images are distributed into different Ewald spheres in reciprocal space. The reciprocal vectors locating in a given Ewald sphere are capable of fulfilling the complete QPM conditions for the high-efficiency reconstruction of the target image at the second-harmonic (SH) wave. One can easily switch the reconstructed SH images by changing the QPM condition. The multiplexing capacity is scalable with the period number of 3D NPC. Our work provides a promising strategy to achieve highly efficient nonlinear multiplexing holography for high-security and high-density storage of optical information.

A Suite of Activity-Based Probes To Dissect the KLK Activome in Drug-Resistant Prostate Cancer.

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

Magnetically driven rotary microfilter fabricated by two-photon polymerization for multimode filtering of particles.

In this Letter, a magnetically driven rotary microfilter that enables switching the modes of filtering and passing is fabricated in microfluidic devices via two-photon polymerization using a femtosecond laser for selective filtering of particles. The high-quality integration of a microfilter is ensured by accurately formulating the magnetic photoresist and optimizing the processing parameters. By changing the direction of the external magnetic field, the fabricated microfilter can be remotely manipulated to rotate by desired angles, thereby achieving the "filtering" or "passing" mode on demand. Taking advantage of this property, the magnetically rotary microfilter realizes multi-mode filtering functions such as capturing 8 µm particles/passing the 2.5 µm particles and passing both particles. More importantly, the responsive characteristic increases the reusability of the microchip. The lab-on-chip devices integrated with remotely rotary microfilters by the femtosecond laser two-photon polymerization with the functional photoresist will offer extensive applications in chemical and biological studies.

Persistent 2c-3e σ-bonded heteronuclear radical cations centered on S/Se and P/As atoms.

The two-center three-electron (2c-3e) bonded species are important in chemical and biological science. Reported isolable 2c-3e σ-bonded species are usually constructed in homoatomic radicals. The one-electron oxidation of main-group heteronuclear species Nap(SPh)(P(Mes)2) (1), Nap(SePh)(P(Mes)2) (2), Nap(SPh)(As(Mes)2) (3) and Nap(SePh)(As(Mes)2) (4) produced persistent radical cations 1˙+-4˙+ in solution. Large couplings of heteroatoms in EPR spectra of 1˙+-4˙+, shorter bond distances and bigger Wiberg bond orders of Ch-Pn in 1˙+-4˙+ than those in 1-4 in DFT calculations indicate large amounts of spin densities over heteroatoms and the formation of 2c-3e σ-bonds between chalcogen and pnicogen atoms. This work provides evidence of 2c-3e σ-bonds constructed between main-group heteronuclears and rare examples of radical cations involving three-electron σ-bonds between S/Se and P/As atoms.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT).

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (K i=38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.