Simultaneous utilization of glucose and xylose by metabolically engineered Pseudomonas putida for the production of 3-hydroxypropionic acid.

Pseudomonas putida,a robust candidate for lignocellulosicbiomass-based biorefineries, encounters challenges in metabolizing xylose. In this study, Weimberg pathway was introduced intoP. putidaEM42 under a xylose-inducible promoter, resulting in slow cell growth (0.05 h-1) on xylose.Through adaptive laboratory evolution, an evolved strain exhibited highly enhanced growth on xylose (0.36 h-1), comparable to that on glucose (0.39 h-1). Whole genome sequencing identified four mutations, with two key mutations located inPP3380andPP2219. Reverse-engineered strain 8EM42_Xyl, harboring these two mutations, showed enhanced growth on xylose but co-utilizing glucose and xylose at a rate of 0.3 g/L/h. Furthermore, 8EM42_Xyl was employed for 3-hydroxypropionic acid (3HP) production from glucose and xylose by expressing malonyl-CoA reductase and acetyl-CoA carboxylase, yielding 29 g/L in fed-batch fermentation. Moreover, the engineered strain exhibited promising performance in 3HP production from empty palm fruit bunch hydrolysate, demonstrating its potential as a promising cell factory forbiorefineries.

Photothermal Lithography for Realizing a Stretchable Multilayer Electronic Circuit Using a Laser.

Photolithography is a well-established fabrication method for realizing multilayer electronic circuits. However, it is challenging to adopt photolithography to fabricate intrinsically stretchable multilayer electronic circuits fully composed of an elastomeric matrix, due to the opacity of thick stretchable nanocomposite conductors. Here, we present photothermal lithography that can pattern elastomeric conductors and via holes using pulsed lasers. The photothermal-patterned stretchable nanocomposite conductor exhibits 3 times higher conductivity (5940 S cm-1) and 5 orders of magnitude lower resistance change (R/R0 = 40) under a 30% strained 5000th cyclic stretch, compared to those of a screen-printed conductor, based on the percolation network formed by spatial heating of the laser. In addition, a 50 µm sized stretchable via holes can be patterned on the passivation without material ablation and electrical degradation of the bottom conductor. By repeatedly patterning the conductor and via holes, highly conductive and durable multilayer circuits can be stacked with layer-by-layer material integration. Finally, a stretchable wireless pressure sensor and passive matrix LED array are demonstrated, thus showing the potential for a stretchable multilayer electronic circuit with durability, high density, and multifunctionality.

Selective Cell-Cell Adhesion Regulation via Cyclic Mechanical Deformation Induced by Ultrafast Nanovibrations.

The adoption of dynamic mechanomodulation to regulate cellular behavior is an alternative to the use of chemical drugs, allowing spatiotemporal control. However, cell-selective targeting of mechanical stimuli is challenging due to the lack of strategies with which to convert macroscopic mechanical movements to different cellular responses. Here, we designed a nanoscale vibrating surface that controls cell behavior via selective repetitive cell deformation based on a poroelastic cell model. The vibrating indentations induce repetitive water redistribution in the cells with water redistribution rates faster than the vibrating rate; however, in the opposite case, cells perceive the vibrations as a one-time stimulus. The selective regulation of cell-cell adhesion through adjusting the frequency of nanovibration was demonstrated by suppression of cadherin expression in smooth muscle cells (fast water redistribution rate) with no change in vascular endothelial cells (slow water redistribution rate). This technique may provide a new strategy for cell-type-specific mechanical stimulation.

Lifetime-configurable soft robots via photodegradable silicone elastomer composites.

Developing soft robots that can control their own life cycle and degrade on-demand while maintaining hyperelasticity is a notable research challenge. On-demand degradable soft robots, which conserve their original functionality during operation and rapidly degrade under specific external stimulation, present the opportunity to self-direct the disappearance of temporary robots. This study proposes soft robots and materials that exhibit excellent mechanical stretchability and can degrade under ultraviolet light by mixing a fluoride-generating diphenyliodonium hexafluorophosphate with a silicone resin. Spectroscopic analysis revealed the mechanism of Si─O─Si backbone cleavage using fluoride ion (F-) and thermal analysis indicated accelerated decomposition at elevated temperatures. In addition, we demonstrated a robotics application by fabricating electronics integrated gaiting robot and a fully closed-loop trigger disintegration robot for autonomous, application-oriented functionalities. This study provides a simple yet novel strategy for designing life cycle mimicking soft robotics that can be applied to reduce soft robotics waste, explore hazardous areas, and ensure hardware security with on-demand destructive material platforms.

Improving hydroxyapatite coating ability on biodegradable metal through laser-induced hydrothermal coating in liquid precursor: Application in orthopedic implants.

During the past decade, there has been extensive research toward the possibility of exploring magnesium and its alloys as biocompatible and biodegradable materials for implantable applications. Its practical medical application, however, has been limited to specific areas owing to rapid corrosion in the initial stage and the consequent complications. Surface coatings can significantly reduce the initial corrosion of Mg alloys, and several studies have been carried out to improve the adhesion strength of the coating to the surfaces of the alloys. The composition of hydroxyapatite (HAp) is very similar to that of bone tissue; it is one of the most commonly used coating materials for bone-related implants owing to favorable osseointegration post-implantation. In this study, HAp was coated on Mg using nanosecond laser coating, combining the advantages of chemical and physical treatments. Photothermal heat generated in the liquid precursor by the laser improved the adhesion of the coating through the precipitation and growth of HAp at the localized nanosecond laser focal area and increased the corrosion resistance and cell adhesion of Mg. The physical, crystallographic, and chemical bondings were analyzed to explore the mechanism through which the surface adhesion between Mg and the HAp coating layer increased. The applicability of the coating to Mg screws used for clinical devices and improvement in its corrosion property were confirmed. The liquid environment-based laser surface coating technique offers a simple and quick process that does not require any chemical ligands, and therefore, overcomes a potential obstacle in its clinical use.

Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties.

BACKGROUND: Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. METHODS: A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. RESULTS: Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. CONCLUSIONS: A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

Characterization of an Entner-Doudoroff pathway-activated Escherichia coli.

BACKGROUND: Escherichia coli have both the Embden-Meyerhof-Parnas pathway (EMPP) and Entner-Doudoroff pathway (EDP) for glucose breakdown, while the EDP primarily remains inactive for glucose metabolism. However, EDP is a more favorable route than EMPP for the production of certain products. RESULTS: EDP was activated by deleting the pfkAB genes in conjunction with subsequent adaptive laboratory evolution (ALE). The evolved strains acquired mutations in transcriptional regulatory genes for glycolytic process (crp, galR, and gntR) and in glycolysis-related genes (gnd, ptsG, and talB). The genotypic, transcriptomic and phenotypic analyses of those mutations deepen our understanding of their beneficial effects on cellulosic biomass bio-conversion. On top of these scientific understandings, we further engineered the strain to produce higher level of lycopene and 3-hydroxypropionic acid. CONCLUSIONS: These results indicate that the E. coli strain has innate capability to use EDP in lieu of EMPP for glucose metabolism, and this versatility can be harnessed to further engineer E. coli for specific biotechnological applications.

Optimization of the clinically approved mg-Zn alloy system through the addition of ca.

BACKGROUND: Although several studies on the Mg-Zn-Ca system have focused on alloy compositions that are restricted to solid solutions, the influence of the solid solution component of Ca on Mg-Zn alloys is unknown. Therefore, to broaden its utility in orthopedic applications, studies on the influence of the addition of Ca on the microstructural, mechanical, and corrosion properties of Mg-Zn alloys should be conducted. In this study, an in-depth investigation of the effect of Ca on the mechanical and bio-corrosion characteristics of the Mg-Zn alloy was performed for the optimization of a clinically approved Mg alloy system comprising Ca and Zn. METHODS: The Mg alloy was fabricated by gravitational melting of high purity Mg, Ca, and Zn metal grains under an Ar gas environment. The surface and cross-section were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to analyze their crystallographic structures. Electrochemical and immersion tests in Hank's balanced salt solution were used to analyze their corrosion resistance. Tensile testing was performed with universal testing equipment to investigate the impact of Ca addition. The examination of cytotoxicity for biometric determination was in line with the ISO10993 standard. RESULTS: In this study, the 0.1% Ca alloy had significantly retarded grain growth due to the formation of the tiny and well-dispersed Ca2Mg6Zn3 phase. In addition, the yield strength and elongation of the 0.1% Ca alloy were more than 50% greater than the 2% Zn alloy. The limited cell viability of the 0.3% Ca alloy could be attributed to its high corrosion rate, whereas the 0.1% Ca alloy demonstrated cell viability of greater than 80% during the entire experimental period. CONCLUSION: The effect of the addition of Ca on the microstructure, mechanical, and corrosion characteristics of Mg-Zn alloys was analyzed in this work. The findings imply that the Mg-Zn alloy system could be optimized by adding a small amount of Ca, improving mechanical properties while maintaining corrosion rate, thus opening the door to a wide range of applications in orthopedic surgery.

Rapid Synthesis of Multifunctional Apatite via the Laser-Induced Hydrothermal Process.

Synthetic biomaterials are used to overcome the limited quantity of human-derived biomaterials and to impart additional biofunctionality. Although numerous synthetic processes have been developed using various phases and methods, currently commonly used processes have some issues, such as a long process time and difficulties with extensive size control and high-concentration metal ion substitution to achieve additional functionality. Herein, we introduce a rapid synthesis method using a laser-induced hydrothermal process. Based on the thermal interaction between the laser pulses and titanium, which was used as a thermal reservoir, hydroxyapatite particles ranging from nanometer to micrometer scale could be synthesized in seconds. Further, this method enabled selective metal ion substitution into the apatite matrix with a controllable concentration. We calculated the maximum temperature achieved by laser irradiation at the surface of the thermal reservoir based on the validation of three simplification assumptions. Subsequent linear regression analysis showed that laser-induced hydrothermal synthesis follows an Arrhenius chemical reaction. Hydroxyapatite and Mg2+-, Sr2+-, and Zn2+-substituted apatite powders promoted bone cell attachment and proliferation ability due to ion release from the hydroxyapatite and the selective ion-substituted apatite powders, which had a low crystallinity and relatively high solubility. Laser-induced hydrothermal synthesis is expected to become a powerful ceramic material synthesis technology.

Development of Organic/Inorganic Hybrid Materials for Fully Degradable Reactive Oxygen Species-Releasing Stents for Antirestenosis.

Despite innovative advances in stent technology, restenosis remains a crucial issue for the clinical implantation of stents. Reactive oxygen species (ROS) are known to potentially accelerate re-endothelialization and lower the risk of restenosis by selectively controlling endothelial cells and smooth muscle cells. Recently, several studies have been conducted to develop biodegradable polymeric stents. As biodegradable polymers are not electrically conductive, double metallic layers are required to constitute a galvanic couple for ROS generation. Here, we report a new biodegradable hybrid material composed of a biodegradable polymer substrate and double anodic/cathodic metallic layers for enhancing re-endothelialization and suppressing restenosis. Pure Zn and Mg films (3 µm thick) were deposited onto poly-l-lactic acid (PLLA) substrates by DC magnetron sputtering, and a long-term immersion test using biodegradable hybrid materials was performed in phosphate-buffered solution (PBS) for 2 weeks. The concentrations of superoxide anions and hydrogen peroxide generated by the corrosion of biodegradable metallic films were monitored every 1 or 2 days. Both superoxide anions and hydrogen peroxide were seamlessly generated even after the complete consumption of the anodic Mg layer. It was confirmed that the superoxide anions and hydrogen peroxide were formed not only by the galvanic corrosion between the anode and cathode layers but also by the corrosion of a single Mg or Zn layer. The corrosion products of the Mg and Zn films in PBS were phosphate, oxide, or chloride of the biodegradable metals. Thus, it is concluded that ROS generation by the corrosion of PLLA-based hybrid materials can be sustained until the exhaustion of the cathode metal layer.

On/off switchable physical stimuli regulate the future direction of adherent cellular fate.

The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.

Regulation of cell locomotion by nanosecond-laser-induced hydroxyapatite patterning.

Hydroxyapatite, an essential mineral in human bones composed mainly of calcium and phosphorus, is widely used to coat bone graft and implant surfaces for enhanced biocompatibility and bone formation. For a strong implant-bone bond, the bone-forming cells must not only adhere to the implant surface but also move to the surface requiring bone formation. However, strong adhesion tends to inhibit cell migration on the surface of hydroxyapatite. Herein, a cell migration highway pattern that can promote cell migration was prepared using a nanosecond laser on hydroxyapatite coating. The developed surface promoted bone-forming cell movement compared with the unpatterned hydroxyapatite surface, and the cell adhesion and movement speed could be controlled by adjusting the pattern width. Live-cell microscopy, cell tracking, and serum protein analysis revealed the fundamental principle of this phenomenon. These findings are applicable to hydroxyapatite-coated biomaterials and can be implemented easily by laser patterning without complicated processes. The cell migration highway can promote and control cell movement while maintaining the existing advantages of hydroxyapatite coatings. Furthermore, it can be applied to the surface treatment of not only implant materials directly bonded to bone but also various implanted biomaterials implanted that require cell movement control.

Durable and Fatigue-Resistant Soft Peripheral Neuroprosthetics for In Vivo Bidirectional Signaling.

Soft neuroprosthetics that monitor signals from sensory neurons and deliver motor information can potentially replace damaged nerves. However, achieving long-term stability of devices interfacing peripheral nerves is challenging, since dynamic mechanical deformations in peripheral nerves cause material degradation in devices. Here, a durable and fatigue-resistant soft neuroprosthetic device is reported for bidirectional signaling on peripheral nerves. The neuroprosthetic device is made of a nanocomposite of gold nanoshell (AuNS)-coated silver (Ag) flakes dispersed in a tough, stretchable, and self-healing polymer (SHP). The dynamic self-healing property of the nanocomposite allows the percolation network of AuNS-coated flakes to rebuild after degradation. Therefore, its degraded electrical and mechanical performance by repetitive, irregular, and intense deformations at the device-nerve interface can be spontaneously self-recovered. When the device is implanted on a rat sciatic nerve, stable bidirectional signaling is obtained for over 5 weeks. Neural signals collected from a live walking rat using these neuroprosthetics are analyzed by a deep neural network to predict the joint position precisely. This result demonstrates that durable soft neuroprosthetics can facilitate collection and analysis of large-sized in vivo data for solving challenges in neurological disorders.

Femtosecond laser induced nano-textured micropatterning to regulate cell functions on implanted biomaterials.

Posterior capsular opacification (PCO) is the most common complication of cataract surgery. PCO is due to the proliferation, migration, and epithelial-to-mesenchymal transition of the residual lens epithelial cells (LECs) within the lens capsule. As surface topography influences cellular response, we investigated the effect of modulating the dimensions of periodic nano-textured patterns on the surface of an intraocular lens material to regulate lens epithelial cell functions such as cell adhesion, migration, orientation, and proliferation. Patterned poly(HEMA) samples were prepared by a femtosecond laser microfabrication, and the behaviors of human B-3 LECs were observed on groove/ridge patterns with widths varying from 5 to 40 µm. In the presence of ridge and groove patterns, the adherent cells elongated along the direction of the patterns, and f-actin of the cells was spread to a lesser extent on the nano-textured groove surfaces. Both single and collective cell migrations were significantly inhibited in the perpendicular direction of the patterns on the nano-textured micro-patterned samples. We also fabricated the patterns on the curved surface of a commercially available intraocular lens for in vivo evaluation. In vivo results showed that a patterned IOL could help suppress the progression of PCO by inhibiting cell migration from the edge to the center of the IOL. Our reports demonstrate that nano- and microscale topographical patterns on a biomaterial surface can regulate cellular behavior when it is implanted into animals.

Biodegradable Magnesium Alloys Promote Angio-Osteogenesis to Enhance Bone Repair.

Biodegradable metallic materials represent a potential step-change technology that may revolutionize the treatment of broken bones. Implants made with biodegradable metals are significantly stronger than their polymer counterparts and fully biodegradable in vivo, removing the need for secondary surgery or long-term complications. Here, it is shown how clinically approved Mg alloy promotes improved bone repair using an integrated state of the art fetal mouse metatarsal assay coupled with in vivo preclinical studies, second harmonic generation, secretome array analysis, perfusion bioreactor, and high-resolution 3D confocal imaging of vasculature within skeletal tissue, to reveal a vascular-mediated pro-osteogenic mechanism controlling enhanced tissue regeneration. The optimized mechanical properties and corrosion rate of the Mg alloy lead to a controlled release of metallic Mg, Ca, and Zn ions at a rate that facilitates both angiogenesis and coupled osteogenesis for better bone healing, without causing adverse effects at the implantation site. The findings from this study support ongoing development and refinement of biodegradable metal systems to act as crucial portal technologies with significant potential to improve many clinical applications.

Curved Structure of Si by Improving Etching Direction Controllability in Magnetically Guided Metal-Assisted Chemical Etching.

Metal-assisted chemical etching (MACE) is widely used to fabricate micro-/nano-structured Si owing to its simplicity and cost-effectiveness. The technique of magnetically guided MACE, involving MACE with a tri-layer metal catalyst, was developed to improve etching speed as well as to adjust the etching direction using an external magnetic field. However, the controllability of the etching direction diminishes with an increase in the etching dimension, owing to the corrosion of Fe due to the etching solution; this impedes the wider application of this approach for the fabrication of complex micro Si structures. In this study, we modified a tri-layer metal catalyst (Au/Fe/Au), wherein the Fe layer was encapsulated to improve direction controllability; this improved controllability was achieved by protecting Fe against the corrosion caused by the etching solution. We demonstrated curved Si microgroove arrays via magnetically guided MACE with Fe encapsulated in the tri-layer catalyst. Furthermore, the curvature in the curved Si microarrays could be modulated via an external magnetic field, indicating that direction controllability could be maintained even for the magnetically guided MACE of bulk Si. The proposed fabrication method developed for producing curved Si microgroove arrays can be applied to electronic devices and micro-electromechanical systems.