Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Single-shot optical imaging with spectrum circuit bridging timescales in high-speed photography.

Single-shot optical imaging based on ultrashort lasers has revealed nonrepetitive processes in subnanosecond timescales beyond the recording range of conventional high-speed cameras. However, nanosecond photography without sacrificing short exposure time and image quality is still missing because of the gap in recordable timescales between ultrafast optical imaging and high-speed electronic cameras. Here, we demonstrate nanosecond photography and ultrawide time-range high-speed photography using a spectrum circuit that produces interval-tunable pulse trains while keeping short pulse durations. We capture a shock wave propagating through a biological cell with a 1.5-ns frame interval and 44-ps exposure time while suppressing image blur. Furthermore, we observe femtosecond laser processing over multiple timescales (25-ps, 2.0-ns, and 1-ms frame intervals), showing that the plasma generated at the picosecond timescale affects subsequent shock wave formation at the nanosecond timescale. Our technique contributes to accumulating data of various fast processes for analysis and to analyzing multi-timescale phenomena as a series of physical processes.

An in vitro model of temporal enhancement of epithelium barrier permeability by low-energy shock waves without contrast agents.

One of the commonly used techniques for drug delivery is to temporarily increase the permeability of tissue barriers. Acoustic energies such as ultrasound and shock waves are known to modulate tissue permeability. Recently, it was found that shock waves modulate the blood-brain barrier in the rat brain without injection of contrast agents such as microbubbles. This finding implies that modulation of other tissue barriers by shock wave exposure without contrast agents may be possible. To examine whether the modulation is also possible with other tissue barriers, we here investigated whether shock waves would modulate an in vitro tissue barrier model consisting of epithelial cells cultured on culture inserts. The permeability of the epithelium sheets evaluated by trans-epithelial electrical resistance (TEER) was increased following shock waves at a peak pressure of 11 MPa. The increased permeability recovered within 2 h. This enhancement was realized with one-shot low-energy shock waves having an acoustic energy of 0.013 mJ/mm2. Monitoring the peak pressures in every exposure revealed that the minimum peak pressure required for the enhancement is 2.9 MPa. These results indicate that shock wave exposure has the potential to temporarily increase the permeability of epithelium barriers to enhance drug delivery without contrast agents. Graphical abstract Enhancements of epithelial barrier permeability were evaluated by trans-epithelial electrical resistance (TEER) before and after shock wave exposures.

Nonlinear photoacoustic waves for light guiding to deep tissue sites.

Light scattering by tissues limits performance in biological sensing or stimulation. Here we present a photoacoustic technique that increases light transmittance by one order of magnitude and enables light localization in deep tissue. Laser-induced nonlinear acoustic waves are utilized to produce a high refractive index contrast in scattering medium without high-intensity pressure. The size of guiding area is around 60 µm, which is equivalent or smaller than the diameter of multimode fibers. To show potential use in biomedical fields, we performed light guiding and imaging of fluorescence, through swine tissues with thickness more than 1 mm.

Low-energy shock waves evoke intracellular Ca2+ increases independently of sonoporation.

Low-energy shock waves (LESWs) accelerate the healing of a broad range of tissue injuries, including angiogenesis and bone fractures. In cells, LESW irradiations enhance gene expression and protein synthesis. One probable mechanism underlying the enhancements is mechanosensing. Shock waves also can induce sonoporation. Thus, sonoporation is another probable mechanism underlying the enhancements. It remains elusive whether LESWs require sonoporation to evoke cellular responses. An intracellular Ca2+ increase was evoked with LESW irradiations in endothelial cells. The minimum acoustic energy required for sufficient evocation was 1.7 µJ/mm2. With the same acoustic energy, sonoporation, by which calcein and propidium iodide would become permeated, was not observed. It was found that intracellular Ca2+ increases evoked by LESW irradiations do not require sonoporation. In the intracellular Ca2+ increase, actin cytoskeletons and stretch-activated Ca2+ channels were involved; however, microtubules were not. In addition, with Ca2+ influx through the Ca2+ channels, the Ca2+ release through the PLC-IP3-IP3R cascade contributed to the intracellular Ca2+ increase. These results demonstrate that LESW irradiations can evoke cellular responses independently of sonoporation. Rather, LESW irradiations evoke cellular responses through mechanosensing.

High-throughput separation of cells by dielectrophoresis enhanced with 3D gradient AC electric field.

We propose a novel, high-performance dielectrophoretic (DEP) cell-separation flow chamber with a parallel-plate channel geometry. The flow chamber, consisting of a planar electrode on the top and an interdigitated-pair electrode array at the bottom, was developed to facilitate the separation of cells by creating a nonuniform AC electric field throughout the volume of the flow chamber. The operation and performance of the device were evaluated using live and dead human epithermal breast (MCF10A) cells. The separation dynamics of the cell suspension in the flow chamber was also investigated by numerically simulating the trajectories of individual cells. A theoretical model to describe the dynamic cell behavior under the action of DEP, including dipole-dipole interparticle, viscous, and gravitational forces, was developed. The results demonstrated that the live cells traveling through the flow chamber congregated into sites where the electric field gradient was minimal, in the middle of the flow stream slightly above the centerlines of the grounded electrodes at the bottom. Meanwhile, the dead cells were trapped on the edges of the high-voltage electrodes at the bottom. Cells were thus successfully separated with a remarkably high separation ratio (∼98%) at the appropriately tuned field frequency and applied voltage. The numerically predicted behavior and spatial distribution of the cells during separation also showed good agreement with those observed experimentally.

Enhancement of continuous-flow separation of viable/nonviable yeast cells using a nonuniform alternating current electric field with complex spatial distribution.

The variability in cell response to AC electric fields is selective enough to separate not only the cell types but also the activation states of similar cells. In this work, we use dielectrophoresis (DEP), which exploits the differences in the dielectric properties of cells, to separate nonviable and viable cells. A parallel-plate DEP device consisting of a bottom face with an array of micro-fabricated interdigitated electrodes and a top face with a plane electrode was proposed to facilitate the separation of cells by creating a nonuniform electric field throughout the flow channel. The operation and performance of the device were evaluated using live and dead yeast cells as model biological particles. Further, numerical simulations were conducted for the cell suspensions flowing in a channel with a nonuniform AC electric field, modeled on the basis of the equation of motion of particles, to characterize the separation efficiency by changing the frequency of applied AC voltage. Results demonstrated that dead cells traveling through the channel were focused onto a site around the minimum electric field gradient in the middle of the flow stream, while live cells were trapped on the bottom face. Cells were thus successfully separated under the appropriately tuned frequency of 1 MHz. Predictions showed good agreement with the observation. The proposed DEP device provides a new approach to, for instance, hematological analysis or the separation of different cancer cells for application in circulating tumor cell identification.

Visible-Light-Induced Morphological Changes of Giant Vesicles by Photoisomerization of a Ruthenium Aqua Complex.

Visible- and red-light responsive vesicles were prepared by incorporating a ruthenium aqua complex having two alkyl chains on tridentate and asymmetrical bidentate ligands (proximal-2: [Ru(C10 tpy)(C10 pyqu)OH2 ](2+) , C10 tpy=4'-decyloxy-2,2';6',2"-terpyridine, C10 pyqu=2-[2'-(6'-decyloxy)-pyridyl]quinoline). The ruthenium complex of proximal-2 with closed alkyl chain geometry and a cylinder-like molecular shape exhibited photoisomerization to distal-2 with an open alkyl chain geometry and a cone-like shape, both in an aqueous solution and in vesicle dispersions. We observed that light irradiation of giant vesicles containing proximal-2 induced diverse morphological changes.

Design for sequentially timed all-optical mapping photography with optimum temporal performance.

A recently developed ultrafast burst imaging method known as sequentially timed all-optical mapping photography (STAMP) [Nat. Photonics8, 695 (2014)10.1038/nphoton.2014.163] has been shown effective for studying a diverse range of complex ultrafast phenomena. Its all-optical image separation circumvents mechanical and electronic restrictions that traditional burst imaging methods have long struggled with, hence realizing ultrafast, continuous, burst-type image recording at a fame rate far beyond what is achievable with conventional methods. In this Letter, considering various design parameters and limiting factors, we present an optimum design for STAMP in terms of temporal properties including exposure time and frame rate. Specifically, we first derive master equations that can be used to predict the temporal performance of a STAMP system and then analyze them to realize optimum conditions. This Letter serves as a general guideline for the camera parameters of a STAMP system with optimum temporal performance that is expected to be of use for tackling problems in science that are previously unsolvable with conventional imagers.

Limited damage of tissue mimic caused by a collapsing bubble under low-frequency ultrasound exposure.

In this study, we investigated the bubble induced serious damage to tissue mimic exposed to 27-kHz ultrasound. The initial bubble radius ranged from 80 to 100 µm, which corresponded approximately to the experimentally-evaluated resonant radius of the given ultrasound frequency. The tissue mimic consisted of 10 wt% gelatine gel covered with cultured canine kidney epithelial cells. The collapsing bubble behaviour during the ultrasound exposure with negative peak pressures of several hundred kPa was captured by a high-speed camera system. After ultrasound exposure, a cell viability test was conducted based on microscopic bright-field images and fluorescence images for living and dead cells. In the viability test, cells played a role in indicating the damaged area. The bubble oscillations killed the cells, and on occasion detached layers of cultured cells from the gel. The damaged area was comparable or slightly larger than the initial bubble size, and smaller than the maximum bubble size. We concluded that only a small area in close proximity to the bubble could be damaged even above transient cavitation threshold.

Dielectric aggregation kinetics of cells in a uniform AC electric field.

BACKGROUND: Cell manipulation and separation technologies have potential biological and medical applications, including advanced clinical protocols such as tissue engineering. OBJECTIVE: An aggregation model was developed for a human carcinoma (HeLa) cell suspension exposed to a uniform AC electric field, in order to explore the field-induced structure formation and kinetics of cell aggregates. METHODS: The momentum equations of cells under the action of the dipole-dipole interaction were solved theoretically and the total time required to form linear string-like cluster was derived. The results were compared with those of a numerical simulation. Experiments using HeLa cells were also performed for comparison. RESULTS: The total time required to form linear string-like clusters was derived from a simple theoretical model of the cell cluster kinetics. The growth rates of the average string length of cell aggregates showed good agreement with those of the numerical simulation. In the experiment, cells were found to form massive clusters on the bottom of a chamber. The results imply that the string-like cluster grows rapidly by longitudinal attraction when the electric field is first applied and that this process slows at later times and is replaced by lateral coagulation of short strings. CONCLUSIONS: The findings presented here are expected to enable design of methods for the organization of three-dimensional (3D) cellular structures without the use of micro-fabricated substrates, such as 3D biopolymer scaffolds, to manipulate cells into spatial arrangement.

Contactless cell trapping by the use of a uniform AC electric field.

The AC electric field-driven manipulation of suspended polarizable particles has become a major technique in micro- and nano-devices. In the present study, suspensions of cultured HeLa cells in isotonic solution were used to explore the mechanisms underlying the suspension behaviors during exposure to a uniform AC electric field of strength E(rms)=1.67×10(4) V/m at frequency 1 kHz. Molecular dynamics (MD) simulations based on the Langevin equation of particle kinetics were performed to elucidate the corresponding problem. A theoretical model to compute the trajectories of individual cells under the action of electro-mechanical, viscous and gravitational forces in the suspending medium was newly developed. Numerical computations demonstrated that the suspended cells began to aggregate to form chainlike clusters along the direction of the uniform AC electric field at an earlier stage of the field application. Moreover, the predicted results were similar to the experimental results. These findings indicate that the chain-like cell clustering arises from the long-range dipole-dipole interaction of neighboring cells, but under the action of the gravitational force that likely hinders the growth of clusters in the vertical direction.

Advantageous grain boundaries in iron pnictide superconductors.

High critical temperature superconductors have zero power consumption and could be used to produce ideal electric power lines. The principal obstacle in fabricating superconducting wires and tapes is grain boundaries-the misalignment of crystalline orientations at grain boundaries, which is unavoidable for polycrystals, largely deteriorates critical current density. Here we report that high critical temperature iron pnictide superconductors have advantages over cuprates with respect to these grain boundary issues. The transport properties through well-defined bicrystal grain boundary junctions with various misorientation angles (θ(GB)) were systematically investigated for cobalt-doped BaFe(2)As(2) (BaFe(2)As(2):Co) epitaxial films fabricated on bicrystal substrates. The critical current density through bicrystal grain boundary (J(c)(BGB)) remained high (>1 MA cm(-2)) and nearly constant up to a critical angle θ(c) of ∼9°, which is substantially larger than the θ(c) of ∼5° for YBa(2)Cu(3)O(7-δ). Even at θ(GB)>θ(c), the decay of J(c)(BGB) was much slower than that of YBa(2)Cu(3)O(7-δ).

Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound.

Mechanisms of primary blast-induced traumatic brain injury: insights from shock-wave research.

Traumatic brain injury caused by explosive or blast events is traditionally divided into four phases: primary, secondary, tertiary, and quaternary blast injury. These phases of blast-induced traumatic brain injury (bTBI) are biomechanically distinct and can be modeled in both in vivo and in vitro systems. The primary bTBI injury phase represents the response of brain tissue to the initial blast wave. Among the four phases of bTBI, there is a remarkable paucity of information about the cause of primary bTBI. On the other hand, 30 years of research on the medical application of shockwaves (SW) has given us insight into the mechanisms of tissue and cellular damage in bTBI, including both air-mediated and underwater SW sources. From a basic physics perspective, the typical blast wave consists of a lead SW followed by supersonic flow. The resultant tissue injury includes several features observed in bTBI, such as hemorrhage, edema, pseudoaneurysm formation, vasoconstriction, and induction of apoptosis. These are well-described pathological findings within the SW literature. Acoustic impedance mismatch, penetration of tissue by shock/bubble interaction, geometry of the skull, shear stress, tensile stress, and subsequent cavitation formation, are all important factors in determining the extent of SW-induced tissue and cellular injury. Herein we describe the requirements for the adequate experimental set-up when investigating blast-induced tissue and cellular injury; review SW physics, research, and the importance of engineering validation (visualization/pressure measurement/numerical simulation); and, based upon our findings of SW-induced injury, discuss the potential underlying mechanisms of primary bTBI.

Liquid phase immunoassays utilizing magnetic markers and SQUID magnetometer.

BACKGROUND: Immunoassays are one main detection system used in the field of clinical chemistry. Recent developments of a new detection method utilizing a magnetic marker and magnetic sensor have enabled rapid and sensitive immunoassay without the need for bound/free (BF) separation. METHODS: Newly-synthesized conjugated avidin was used as the magnetic marker for quantitative analysis of human interleukin-8 (hIL-8) and immunoglobulin E (hIgE) in several media. A superconducting quantum interference device sensor detected the magnetic fields from markers fixed to antigens by the sandwich method. Magnetic signals from unbound markers were nearly zero due to Brownian rotation. RESULTS: Our magnetic immunoassay could detect four attomoles of model proteins (hIL-8, hIgE) in phosphate buffer without BF separation. Using our standard curve, the range of protein detected ranged from 40 femtomoles to 4 attomoles, and we observed a strong association between protein amounts and magnetic signals from the bound markers. The homogeneous immunoassay could also quantify three hundred cells from the fungus Candida albicans in phosphate buffer. CONCLUSIONS: The present study demonstrates the ability of magnetic markers for measuring biological targets without BF separation. This detection system has great potential for use as the next generation's analytical system.

Spatio-temporal PLC activation in parallel with intracellular Ca2+ wave propagation in mechanically stimulated single MDCK cells.

Intracellular Ca2+ transients are evoked either by the opening of Ca2+ channels on the plasma membrane or by phospholipase C (PLC) activation resulting in IP3 production. Ca2+ wave propagation is known to occur in mechanically stimulated cells; however, it remains uncertain whether and how PLC activation is involved in intracellular Ca2+ wave propagation in mechanically stimulated cells. To answer these questions, it is indispensable to clarify the spatio-temporal relations between intracellular Ca2+ wave propagation and PLC activation. Thus, we visualized both cytosolic Ca2+ and PLC activation using a real-time dual-imaging system in individual Mardin-Darby Canine Kidney (MDCK) cells. This system allowed us to simultaneously observe intracellular Ca2+ wave propagation and PLC activation in a spatio-temporal manner in a single mechanically stimulated MDCK cell. The results showed that PLC was activated not only in the mechanically stimulated region but also in other subcellular regions in parallel with intracellular Ca2+ wave propagation. These results support a model in which PLC is involved in Ca2+ signaling amplification in mechanically stimulated cells.

[Development of liquid phase immunoassay system using magnetic nanoparticles].

Biological immunoassay is a major detection system of biological materials from patient samples. Our group has been developing a highly sensitive immunoassay system using magnetic nanoparticles made from Fe3O4. Since unbound magnetic markers randomly move in solvent due to Brownian motion, there is no magnetic signal from unbound magnetic markers; therefore, the separation of bound from unbound markers (B/F separation) is not required. This advantage means that the detection time is greatly decreased in comparison with a normal method using fluorescent/enzyme reagent. In this paper, we describe the configuration of the developed system and demonstrate the performance of the detection of magnetic nanoparticles.

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons.

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus.

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5' portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg(-), Leu(-)), obtaining three good Arg(+) transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.