Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap.

Fluorescence resonance energy transfer (FRET) is a highly useful tool to investigate biomolecular interactions and dynamics in single-molecule spectroscopy and nanoscopy. However, the use of spectrally overlapping dye pairs results in various artifact signals that prevent accurate determination of FRET values. In this paper, an algorithmic method of spectral unmixing was devised to extract FRET values of spectrally overlapping dye pairs at the single molecule level. Application of this method allows the determination of both the donor-acceptor composition and the FRET efficiency of the samples labelled with spectrally overlapping dye pairs.

CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d-glutamate auxotrophic E. coli. This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (-)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.

Selective Dissolution-Derived Nanoporous Design of Impurity-Free Bi2 Te3 Alloys with High Thermoelectric Performance.

Thermoelectric technology, which has been receiving attention as a sustainable energy source, has limited applications because of its relatively low conversion efficiency. To broaden their application scope, thermoelectric materials require a high dimensionless figure of merit (ZT). Porous structuring of a thermoelectric material is a promising approach to enhance ZT by reducing its thermal conductivity. However, nanopores do not form in thermoelectric materials in a straightforward manner; impurities are also likely to be present in thermoelectric materials. Here, a simple but effective way to synthesize impurity-free nanoporous Bi0.4 Sb1.6 Te3 via the use of nanoporous raw powder, which is scalably formed by the selective dissolution of KCl after collision between Bi0.4 Sb1.6 Te3 and KCl powders, is proposed. This approach creates abundant nanopores, which effectively scatter phonons, thereby reducing the lattice thermal conductivity by 33% from 0.55 to 0.37 W m-1 K-1 . Benefitting from the optimized porous structure, porous Bi0.4 Sb1.6 Te3 achieves a high ZT of 1.41 in the temperature range of 333-373 K, and an excellent average ZT of 1.34 over a wide temperature range of 298-473 K. This study provides a facile and scalable method for developing high thermoelectric performance Bi2 Te3 -based alloys that can be further applied to other thermoelectric materials.

Alcoholic Solvent-Mediated Excited-State Proton Transfer Dynamics of a Novel Dihydroxynaphthalene Dye.

The excited-state proton transfer (ESPT) reaction is an important primary photochemical process because it is closely related to photophysical properties. Although ESPT research in aqueous solutions is predominant, alcoholic solvent-mediated ESPT studies are also significant in terms of photoacid-based reactions. Especially, the research for dihydroxynaphthalenes (DHNs) has been largely neglected due to the challenging data interpretation of two hydroxyl groups. A novel fluorescent dye, resveratrone, synthesized by light irradiation of resveratrol, which is famous for its antioxidant properties, can be regarded as a type of DHN, and it has distinctive optical properties, including high quantum yield, a large two-photon absorption coefficient, a large Stokes shift, and very high biocompatibility. In this study, we investigate the overall kinetics of the optical properties of resveratrone and find evidence for alcoholic solvent-mediated ESPT involvement in the radiative properties of resveratrone with a large Stokes shift. Our investigation provides an opportunity to revisit the overlooked photophysical properties of intriguing photoacid behavior and the large Stokes shift of the dihydroxynaphthalene dye.

Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

Electron Attachment to the (O2···CO2) van der Waals Complex Results in a Monomeric Anion (O2-CO2)-, a Possible Form of CO4.

We found that electron attachment to the van der Waals complex (O2···CO2) turns the weak intermolecular bond into a pseudochemical bond of significant strength. The resulting monomeric molecular anion (O2-CO2)- may be a form of CO4-, the gaseous anionic species suspected to be present in Earth's ionosphere whose chemical characteristics have not been comprehensively identified since its existence was first predicted by Conway in 1962. The measured vertical detachment energy of CO4- is very large (4.56 ± 0.05 eV), while the known electron affinity of its component species is much smaller (0.448 eV, O2) or even negative (-0.6 eV, CO2). These characteristics are correctly borne out by theoretical calculations that show that electron attachment transforms the van der Waals complex to a single contiguous molecular anion, with the formation of a pseudochemical bond between O2 and CO2 through an extended π-orbital system.

Anionic Activation of CO2 via (Mn-CO2)- Complex on Magic-Numbered Anionic Coinage Metal Clusters Mn- (M = Cu, Ag, Au).

Given the immense challenge of excessive accumulation of carbon dioxide (CO2) in the earth's atmosphere, an extensive search is under way to convert atmospheric CO2 to compounds of more utility. With CO2 being thermodynamically extremely stable, activation of CO2 is the first and most important step toward its chemical conversion. Building upon our earlier model for the anionic activation of CO2 with azabenzene and inspired by the work of others on metal atom-CO2 complexes, we investigated the possibility of anionic activation of CO2 on small anionic metal clusters, which would have implications for catalytic conversion of CO2 on metal surfaces with atomic-scale structural irregularities. We carried out theoretical calculations using density functional theory to examine small anionic metal clusters of Cu, Ag, and Au to check whether they form a complex with CO2, with the sign of CO2 being chemically activated. We found that a class of anionic metal clusters Mn- with 1, 2, and 6 atoms consistently produced the activated complex (Mn-CO2)- for all three metals. There exists a strong interaction between the CO2 moiety and Mn- via a partially covalent M-C bond with a full delocalization of the electronic charge, as a result of electron transfer from the HOMO of Mn- to the LUMO of CO2 as in metal-CO2 π-backbonding. We examined the interaction of frontier orbitals from the viewpoints of the orbital geometry and orbital energetics and found that the above magic numbers are consistent with both aspects.

Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption.

Acetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. Four genomic mutations were found in the evolved strain through whole-genome sequencing, and two major mutations (in cspC and patZ) mainly contributed to efficient utilization of acetate and tolerance to acetate. Transcriptomic reprogramming was examined by analyzing the genome-wide transcriptome with different carbon sources. The evolved strain showed high levels of intracellular ATP by upregulation of genes involved in NADH and ATP biosynthesis, which facilitated the production of enhanced green fluorescent protein, mevalonate, and n-butanol using acetate alone. This new strain, given its high acetate tolerance and high ATP levels, has potential as a starting host for cell factories targeting the production of acetyl-CoA-derived products from acetate or of products requiring high ATP levels.

Asymmetric Host Molecule Bearing Pyridine Core for Highly Efficient Blue Thermally Activated Delayed Fluorescence OLEDs.

In this study, two host materials, pCzBzbCz and pCzPybCz, are synthesized to achieve a high efficiency and long lifetime of blue thermally activated delayed fluorescence organic light-emitting diodes (TADF-OLEDs). The molecular design strategy involves the introduction of a pyridine group into the core structure of pCzPybCz as an electron-withdrawing unit, and an electron-donating phenyl group into the structure of pCzBzbCz. These host materials demonstrate good thermal stability and high triplet energy (T1 =3.07 eV for pCzBzbCz and 3.06 eV for pCzPybCz) for the fabrication of blue TADF-OLEDs. In particular, pCzPybCz-based OLED devices demonstrate an external quantum efficiency (EQE) of 22.7 % and an operational lifetime of 24 h (LT90 , time to attain 90 % of initial luminance) at an initial luminance of 1000 cd m-2 . This superior lifetime could be explained by the C-N bond dissociation energy (BDE) in the host molecular structure. Furthermore, a mixed-host system using the electron-deficient 2,4-bis(dibenzo[b,d]furan-2-yl)-6-phenyl-1,3,5-triazine (DDBFT) is proposed to inhibit the formation of the anion state of our host materials. In short, the device operational lifetime is further improved by applying DDBFT. The carbazole-based asymmetric host molecule containing a pyridine core realizes a high-efficiency blue TADF-OLED showing a positive effect on the operating lifetime, and can provide useful strategies for designing new host materials.

Selective thiolation and photoswitching mechanism of Cy5 studied by time-dependent density functional theory.

Cy5 is one of the most widely used organic dyes with a photoswitching property. It can be reversibly photoconverted to the dark state through thiolation with primary thiols. Although photoswitching of Cy5 has been widely used in super-resolution nanoscopy, its thiolation mechanism remains unclear. We carried out time-dependent density functional theory calculations to investigate the excited state dynamics of Cy5 and observed its site-selective thiolation on both the ground and excited states. Scanning the excited state potential energy surfaces by rotating individual C-C bonds revealed structural similarity between the twisted form of Cy5 and the Cy5 subunit in the thiolated Cy5, which suggests that the dark state formation is strongly associated with the torsional motion on the excited state.

Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution.

Lysozyme is widely used as a model protein in studies of structure-function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.

Highly Crystalline Perovskite-Based Photovoltaics via Two-Dimensional Liquid Cage Annealing Strategy.

Rendering a high crystalline perovskite film is integral to achieve superior performance of perovskite solar cells (PSCs). Here, we established a two-dimensional liquid cage annealing system, a unique methodology for remarkable enhancement in perovskite crystallinity. During thermal annealing for crystallization, wet-perovskite films were suffocated by perfluorodecalin with distinctively low polarity, nontoxic, and chemically inert characteristics. This annealing strategy facilitated enlargement of perovskite grain and diminution in the number of trap states. The simulation results, annealing time, and temperature experiments supported that the prolonged diffusion length of precursor ions attributed to the increase of perovskite grains. Consequently, without any complicated handling, the performance of perovskite photovoltaics was remarkably improved, and the monolithic grains which directly connected the lower and upper electrode attenuated hysteresis.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor.

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain.

Understanding the underlying principles for the target-specific nuclease activity of CRISPR/Cas9 is a prerequisite to minimize its off-target DNA cleavage for genome engineering applications. Here, we show that the noncatalytic REC2 domain of Cas9 nuclease plays a crucial role in off-target discrimination. Using single-molecule fluorescence methods, we investigate conformational dynamics of the non-target strand (NTS) of DNA interacting with Cas9 and find that REC2 regulates the NTS rearrangement for cleavage reaction with the help of positively charged residues on its surface. This mechanistic model for the target specificity of Cas9 provides molecular insights for the rational approach to Cas9 engineering for highly specific genome editing.

Molecular and biochemical characterization of a novel isoprene synthase from Metrosideros polymorpha.

BACKGROUND: Isoprene is a five-carbon chemical that is an important starting material for the synthesis of rubber, elastomers, and medicines. Although many plants produce huge amounts of isoprene, it is very difficult to obtain isoprene directly from plants because of its high volatility and increasing environmental regulations. Over the last decade, microorganisms have emerged as a promising alternative host for efficient and sustainable bioisoprene production. Isoprene synthase (IspS) has received much attention for the conversion of isoprene from dimethylallyl diphosphate (DMAPP). Herein, we isolated a highly expressible novel IspS gene from Metrosideros polymorpha (MpIspS), which was cloned and expressed in Escherichia coli, using a plant cDNA library and characterized its molecular and biochemical properties. RESULTS: The signal sequence deleted MpIspS was cloned and expressed in E. coli as a 65-kDa monomer. The maximal activity of the purified MpIspS was observed at pH 6.0 and 55 °C in the presence of 5 mM Mn2+. The Km, kcat, and kcat/Km for DMAPP as a substrate were 8.11 mM, 21 min- 1, and 2.59 mM- 1 min- 1, respectively. MpIspS was expressed along with the exogenous mevalonate pathway to produce isoprene in E. coli. The engineered cells produced isoprene concentrations of up to 23.3 mg/L using glycerol as the main carbon source. CONCLUSION: MpIspS was expressed in large amounts in E. coli, which led to increased enzymatic activity and resulted in isoprene production in vivo. These results demonstrate a new IspS enzyme that is useful as a key biocatalyst for bioisoprene production in engineered microbes.

A Ru-Pt alloy electrode to suppress leakage currents of dynamic random-access memory capacitors.

Rutile TiO2, a high temperature phase, has attracted interest as a capacitor dielectric in dynamic random-access memories (DRAMs). Despite its high dielectric constant of >80, large leakage currents caused by a low Schottky barrier height at the TiO2/electrode interface have hindered the use of rutile TiO2 as a commercial DRAM capacitor. Here, we propose a new Ru-Pt alloy electrode to increase the height of the Schottky barrier. The Ru-Pt mixed layer was grown by atomic layer deposition. The atomic ratio of Ru/Pt varied in the entire range from 100 at.% Ru to 100 at.% Pt. Rutile TiO2 films were inductively formed only on the Ru-Pt layer containing ≤43 at.% Pt, while anatase TiO2 films with a relatively low dielectric constant (∼40) were formed at Pt compositions > 63 at.%. The Ru-Pt (40-50 at.%) layer also attained an increase in work function of ∼0.3-0.4 eV, leading to an improvement in the leakage currents of the TiO2/Ru-Pt capacitor. These findings suggested that a Ru-Pt layer could serve as a promising electrode for next-generation DRAM capacitors.

Palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation in the plasma membrane of RAW 264.7 cells.

We previously reported that palmitate induces receptor-interacting protein (RIP)1-dependent necrosis in RAW 264.7 macrophage cells. In response to death receptor stimuli, RIP1 is reported to activate RIP3, which causes the phosphorylation and translocation of mixed-lineage kinase domain-like (MLKL) protein to the plasma membrane, subsequent pore formation in the plasma membrane, and necrotic cell death. In the current study, we investigated the role of MLKL in palmitate-induced, RIP1/RIP3-dependent necrotic cell death in RAW 264.7 cells. The down-regulation of RIP1 or RIP3 by siRNA transfection protected the cells from palmitate-induced cell death. In addition, MLKL was phosphorylated at the serine residue and translocated to the plasma membrane in palmitate-treated cells. In these cells, MLKL was observed as aggregate dots on the plasma membrane. The findings also show that palmitate induced the formation of pores with varied shapes and sizes, and an increase in propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release. Furthermore, the down-regulation of MLKL by siRNA transfection significantly decreased palmitate-induced PI uptake and LDH release, resulting in protection against palmitate-induced necrotic cell death. The findings reported here indicate that palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation of RAW 264.7 cells in the plasma membrane, which could provide a new mechanism to explain the link between elevated levels of free fatty acids (FFAs), palmitate in particular, and macrophage death.

Leucine-induced localization of Leucyl-tRNA synthetase in lysosome membrane.

Leucyl-tRNA synthetase (LRS) plays major roles in providing leucine-tRNA and activating mechanistic target of rapamycin complex 1 (mTORC1) through intracellular leucine sensing. mTORC1 activated by amino acids affects the influence on physiology functions including cell proliferation, protein synthesis and autophagy in various organisms. Biochemical results demonstrating leucine sensing have been published, but visual results are lacking. Therefore, we observed the location of LRS with and without leucine using stimulated emission depletion (STED) microscopy one of the super-resolution microscopy and transmission electron microscopy (TEM). This revealed that LRS was translocated to the lysosome on addition of leucine. The translocation was inhibited by treatment with compound BC-LI-0186, disrupting the interaction between RagD and LRS. Immuno-TEM revealed a clear decrease in LRS translocation to the lysosome on addition of the inhibitor. This direct visualization of leucine sensing and LRS translocation to the lysosome was related to mTORC1 activation. To study the relationship between mTORC1 activation and LRS translocation, we monitored the change in autophagy for each condition using TEM and CLSM. The results showed a decrease in autophagy on addition of leucine, demonstrating crosstalk between leucine sensing, LRS translocation, RagD interaction, and mTORC1 activation.

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol.

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products.

Efficient micro-cavity top emission OLED with optimized Mg:Ag ratio cathode.

Micro-cavity top-emitting organic light emitting diodes (TEOLEDs) are now receiving prominence as a technology for the active matrix display applications. The semi-transparent metal cathode plays the crucial role in realizing TEOLEDs structure. Here, we report the optimization results on Mg:Ag ratio as the semitransparent cathode deposited by vacuum thermal evaporation. The optimized Mg:Ag cathode with 1:10 ratio (wt %) shows a sheet resistance value as low as 5.2 Ω/□, an average transmittance of 49.7%, reflectance of 41.4%, and absorbance of 8.9% over the visible spectral region (400~700 nm). The fabricated red TEOLEDs device implemented using LiF (1nm)/Mg:Ag (1:10) cathode shows the voltage value of 4.17 V at a current density of 10.00 mA/cm2, and current efficiencies variation from 55.3 to 50.1 cd/A over the brightness range 2,000 - 12,000 cd/m2. The electroluminescence (EL) spectrum displays the light emission at 608 nm wavelength with a half width of 29.5 nm. The narrow half-width of red light emission is attributed to the micro-cavity effects due to the semitransparent cathode.