Structural basis for Cas9 off-target activity.

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

A double-blind, randomized, two-part, two-period crossover study to evaluate the pharmacokinetics of caffeine versus d9-caffeine in healthy subjects.

The deuterium kinetic isotope effect has been used to affect the cytochrome P450 metabolism of the deuterated versions of substances. This study compares the pharmacokinetics of caffeine, a Generally Recognized As Safe food and beverage ingredient, versus d9-caffeine, a potential caffeine alternative, and their respective metabolites at two dose levels in 20 healthy adults. A single dose of 50 mg or 250 mg of caffeine, or a molar-equivalent dose of d9-caffeine, were orally administered in solution with blood samples collected for up to 48 h post-dose. Plasma concentrations of parent and metabolites were analyzed using validated LC-MS/MS methods. Both d9-caffeine and caffeine were rapidly absorbed; however, d9-caffeine exhibited a higher (ca. 29%-43%) Cmax and 4-5-fold higher AUClast than caffeine, and lower Cmax, lower AUClast, and a 5-10-fold reduction in the relative exposure to the active metabolites of caffeine. Results were consistent in normal and rapid metabolizers, and both substances were well tolerated.

An Intelligent Virtual-Reality System With Multi-Model Sensing for Cue-Elicited Craving in Patients With Methamphetamine Use Disorder.

Methamphetamine abuse is getting worse amongst the younger population. While there is methadone or buprenorphine harm-reduction treatment for heroin addicts, there is no drug treatment for addicts with methamphetamine use disorder (MUD). Recently, non-medication treatment, such as the cue-elicited craving method integrated with biofeedback, has been widely used. Further, virtual reality (VR) is proposed to simulate an immersive virtual environment for cue-elicited craving in therapy. In this study, we developed a VR system equipped with flavor simulation for the purpose of inducing cravings for MUD patients in therapy. The VR system was integrated with multi-model sensors, such as an electrocardiogram (ECG), galvanic skin response (GSR) and eye tracking to measure various physiological responses from MUD patients in the virtual environment. The goal of the study was to validate the effectiveness of the proposed VR system in inducing the craving of MUD patients via the physiological data. Clinical trials were performed with 20 MUD patients and 11 healthy subjects. VR stimulation was applied to each subject and the physiological data was measured at the time of pre-VR stimulation and post-VR stimulation. A variety of features were extracted from the raw data of heart rate variability (HRV), GSR and eye tracking. The results of statistical analysis found that quite a few features of HRV, GSR and eye tracking had significant differences between pre-VR stimulation and post-VR stimulation in MUD patients but not in healthy subjects. Also, the data of post-VR stimulation showed a significant difference between MUD patients and healthy subjects. Correlation analysis was made and several features between HRV and GSR were found to be correlated. Further, several machine learning methods were applied and showed that the classification accuracy between MUD and healthy subjects at post-VR stimulation attained to 89.8%. In conclusion, the proposed VR system was validated to effectively induce the drug craving in MUD patients.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Facile Generation of Thermally Activated Delayed Fluorescence and Fabrication of Highly Efficient Non-Doped OLEDs Based on Triazine Derivatives.

A series of donor-acceptor-donor triazine-based molecules with thermally activated delayed fluorescence (TADF) properties were synthesized to obtain highly efficient blue-emitting OLEDs with non-doped emitting layers (EMLs). The targeted molecules use a triazine core as the electron acceptor, and a benzene ring as the conjugated linker with different electron donors to alternate the energy level of the HOMO to further tune the emission color. The introduction of long alkyl chains on the triazine core inhibits the unwanted intermolecular D-D/A-A-type π-π interactions, resulting in the intermolecular D-A charge transfer. The weak aggregation-caused quenching (ACQ) effect caused by the suppressed intermolecular D-D/A-A-type π-π interaction further enhances the emission. The crowded molecular structure allows the electron donor and acceptor to be nearly orthogonal, thereby reducing the energy gap between triplet and singlet excited states (ΔEST ). As a result, blue-emitting devices with TH-2DMAC and TH-2DPAC non-doped EMLs showed satisfactory efficiencies of 12.8 % and 15.8 %, respectively, which is one of the highest external quantum efficiency (EQEs) reported for blue TADF emitters (λpeak <475 nm), demonstrating that our tailored molecular designs are promising strategies to endow OLEDs with excellent electroluminescent performances.

Function of Tetrabutylammonium on High-Efficiency Ruthenium Sensitizers for Both Outdoor and Indoor DSC Application.

The function of tetrabutyl ammonium ions (TBA+) in a sensitizer used in dye-sensitized solar cells (DSC) is contradictory. TBA+ can reduce unwanted charge-recombination by protecting the TiO2 surface and reduce dye aggregation, enhancing the photovoltaic performance. It will also compete with the dye-loading on the TiO2 film, decreasing the short-circuit current density of the cell. Three ruthenium sensitizers (DYE III, DUY11, and DUY12 containing two H+, one H+/one TBA+, and two TBA+, respectively) were prepared to systematically investigate the function of TBA+ in a dye for DSC under both standard sunlight and indoor illumination. The optical properties and frontier orbital energy level of the sensitizers are not influenced significantly by the number of TBA+. Under the standard 1 sun illumination, DSCs based on DUY11 (containing one H+ and one TBA+) achieved the highest power conversion efficiency (PCE) of 11.47%. Overall, optimized DSCs sensitized by the three ruthenium dyes all have the PCE over 10%, which is higher than that (9.95%) of N719-dyed cell fabricated at the same conditions. Under the illumination of a light emitting diode (LED), DSCs sensitized by DUY11 also have the highest efficiency of 19%. Furthermore, DUY12 with two TBA+ exhibits superior photovoltaic performance compared to a DYE III (containing two H+ in the anchoring ligands)-dyed cell; although these two dyes have similar photovoltaic performance under standard 1 sun lighting. The important function of TBA+ in reducing the charge recombination (by protecting TiO2 surface and avoiding dye aggregation) of a DSC under indoor lighting (when small number of electrons were excited by weak light) is also revealed.

Further emission efficiency improvement of a commercial-quality light-emitting diode through surface plasmon coupling.

It is usually believed that surface plasmon (SP) coupling is practically useful only for improving the performance of a light-emitting diode (LED) with a low intrinsic internal quantum efficiency (IQE). In this Letter, we demonstrate that the performance of a commercial-quality blue LED with a high IQE (>80%) can still be significantly improved through SP coupling based on a surface Ag nanoparticle (NP) structure. The performance improvement of such an LED is achieved by increasing the Mg doping concentration in its p-AlGaN electron blocking layer to enhance the hole injection efficiency such that the p-GaN layer thickness can be significantly reduced without sacrificing its electrical property. In this situation, the distance between surface Ag NPs and quantum wells is decreased and hence SP coupling strength is increased. By reducing the distance between the surface Ag NPs and the top quantum well to 66 nm, the IQE can be increased to almost 90% (an ∼11% enhancement) and the electroluminescence intensity can be enhanced by ∼24%.

Efficiency enhancement of light color conversion through surface plasmon coupling.

The efficiency enhancement of light color conversion from blue quantum well (QW) emission into red quantum dot (QD) emission through surface plasmon (SP) coupling by coating CdSe/ZnS QDs on the top of an InGaN/GaN QW light-emitting diode (LED) is demonstrated. Ag nanoparticles (NPs) are fabricated within a transparent conductive Ga-doped ZnO interlayer to induce localized surface plasmon (LSP) resonance for simultaneously coupling with the QWs and QDs. Such a coupling process generates three enhancement effects, including QW emission, QD absorption at the QW emission wavelength, and QD emission, leading to an overall enhancement effect of QD emission intensity. An Ag NP geometry for inducing an LSP resonance peak around the middle between the QW and QD emission wavelengths results in the optimized condition for maximizing QD emission enhancement. Internal quantum efficiency and photoluminescence (PL) decay time measurements are performed to show consistent results with LED performance characterizations, even though the QD absorption of PL excitation laser may mix with the SP-induced QD absorption enhancement effect in PL measurement.

Coupling of a light-emitting diode with surface plasmon polariton or localized surface plasmon induced on surface silver gratings of different geometries.

A metal grating on top of a light-emitting diode (LED) with a designed grating period for compensating the momentum mismatch can enhance the surface plasmon polariton (SPP) coupling effect with the quantum wells (QWs) to improve LED performance. Here, we demonstrate the experimental results showing that the induced localized surface plasmon (LSP) resonance on such a metal grating can dominate the QW coupling effect for improving LED performance, particularly when grating ridge height is large. The finding is illustrated by fabricating Ag gratings on single-QW, green-emitting LEDs of different p-type thicknesses with varied grating ridge height and width such that the distance between the grating ridge tip and the QW can be controlled. Reflection spectra of the Ag grating structures are measured and simulated to identify the SPP or LSP resonance behaviors at the QW emission wavelength. The measured results of LED performances show that in the LED samples under study, both SPP and LSP couplings can lead to significant enhancements of internal quantum efficiency and electroluminescence intensity. At the designated QW emission wavelength, with a grating period theoretically designed for momentum matching, the LSP coupling effect is stronger, when compared with SPP coupling.

Dependencies of surface plasmon coupling effects on the p-GaN thickness of a thin-p-type light-emitting diode.

The high performance of a light-emitting diode (LED) with the total p-type thickness as small as 38 nm is demonstrated. By increasing the Mg doping concentration in the p-AlGaN electron blocking layer through an Mg pre-flow process, the hole injection efficiency can be significantly enhanced. Based on this technique, the high LED performance can be maintained when the p-type layer thickness is significantly reduced. Then, the surface plasmon coupling effects, including the enhancement of internal quantum efficiency, increase in output intensity, reduction of efficiency droop, and increase of modulation bandwidth, among the thin p-type LED samples of different p-type thicknesses that are compared. These advantageous effects are stronger as the p-type layer becomes thinner. However, the dependencies of these effects on p-type layer thickness are different. With a circular mesa size of 10 µm in radius, through surface plasmon coupling, we achieve the record-high modulation bandwidth of 625.6 MHz among c-plane GaN-based LEDs.

Combinatorial Microenvironments Impose a Continuum of Cellular Responses to a Single Pathway-Targeted Anti-cancer Compound.

Tumor microenvironments are a driver of resistance to anti-cancer drugs. Dissecting cell-microenvironment interactions into tractable units of study presents a challenge. Here, we assess the impact of hundreds of tumor-inspired microenvironments, in parallel, on lapatinib responses in four cancer cell lines. Combinations of ECM and soluble factors were printed on stiffness-tunable substrata to generate a collection of controlled microenvironments in which to explore cell-based functional responses. Proliferation, HER2 protein expression and phosphorylation, and morphology were measured in single cells. Using dimension reduction and linear modeling, the effects of microenvironment constituents were identified and then validated empirically. Each of the cell lines exhibits unique microenvironment-response patterns. Fibronectin, type IV collagen, and matrix rigidity are significant regulators of lapatinib resistance in HER2-amplified breast cancer cells. Small-molecule inhibitors were identified that could attenuate microenvironment-imposed resistance. Thus, we demonstrate a strategy to identify resistance- and sensitivity-driving microenvironments to improve the efficacy of anti-cancer therapeutics.

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells.

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter.

Differential changes in the pharmacokinetics of statins in collagen-induced arthritis rats.

The elevated systemic levels of cytokines in rheumatoid arthritis (RA) can change the expression of metabolic enzymes and transporters. Given that statins are lipid-lowering agents frequently used in RA patients with concurrent cardiovascular diseases, the objective of the present study was to investigate the impacts of RA on the pharmacokinetics of statins of different disposition properties in rats with collagen-induced arthritis (CIA). The expression of metabolic enzymes and transporters in tissues of CIA rats were analyzed by RT-qPCR. Statins were given to CIA rats and controls through different routes, respectively. Blood samples were collected and analyzed by UPLC/MS/MS. Isolated microsomes and hepatocytes were used to determine the metabolic and uptake clearance of statins. The results showed that, compared with controls, the mRNA levels of intestinal Cyp3a1 and hepatic Cyp2c6, Cyp2c7, Cyp3a1, Oatp1a1, Oatp1b2, Oatp1a4, and Mrp2 were markedly decreased in the CIA rats. The maximal metabolic activities of Cyp2c and Cyp3a were reduced in liver microsomes of CIA rats. When given orally or injected through hepatic portal vein, the systemic levels of fluvastatin, simvastatin, and atorvastatin, but not of rosuvastatin and pravastatin, were increased in CIA rats. The metabolic clearance of simvastatin and hepatic uptake clearance of fluvastatin and atorvastatin were decreased in CIA rats. These findings suggest that the changes in the expression of enzymes and/or transporters in CIA rats differentially affect the pharmacokinetics of statins.

Effect of the CF3 Substituents on the Charge-Transfer Kinetics of High-Efficiency Cyclometalated Ruthenium Sensitizers.

Six thiocyanate-free complexes, DUY1-DUY6, were synthesized, and their application in a dye-sensitized solar cell was studied to explore the effect of the CF3 substituent positioned in the ancillary ligand and the structure of the anchoring ligand on the physicochemical properties, charge-transfer kinetics, and photovoltaic properties of ruthenium sensitizers. When the electron-withdrawing groups were installed on the cyclometalating ligands and their π conjugation of the ancillary ligand was extended, the frontier orbital energy levels of the ruthenium complex appeared to be sufficient for effective electron injection and dye regeneration, at the same time having high light-harvesting ability. Two electron-withdrawing CF3 groups meta to the cyclometalated position reduce the electron density at the metal center less seriously than o-CF3 and p-CF3 groups. The sensitizers containing a m-CF3 group also reveal a more favorable distribution of ß lowest unoccupied spin orbital for interaction between the oxidized dyes and the iodide ion, which promotes dye regeneration. The absorption profiles of DUY1-DUY4 adsorbed a TiO2 film extended to longer wavelength compared to those in an N,N-dimethylformamide solution, especially DUY1 and DUY2 dyes, which have λmax red shifts of up to 30 nm. The DUY2-dyed cell exhibited the highest efficiency of 9.03%, while the power conversion efficiencies of DUY1-, DUY3-, DUY4-, and N719-based devices were 7.40%, 7.01%, 8.92%, and 8.63%, respectively. DUY5 and DUY6 (the side products of DUY3 and DUY4) without anchoring groups have very weak physical adsorption on a TiO2 anode. The corresponding cells exhibit very low efficiency (<0.1%), although both dyes have high light-harvesting ability and proper frontier orbital energy levels.

Regularly patterned multi-section GaN nanorod arrays grown with a pulsed growth technique.

The growth of regularly patterned multi-section GaN nanorod (NR) arrays based on a pulsed growth technique with metalorganic chemical vapor deposition is demonstrated. Such an NR with multiple sections of different cross-sectional sizes is formed by tapering a uniform cross section to another through stepwise decreasing of the Ga supply duration to reduce the size of the catalytic Ga droplet. Contrast line structures are observed in either a scanning electron microscopy or transmission electron microscopy image of an NR. Such a contrast line-marker corresponds to a thin Ga-rich layer formed at the beginning of GaN precipitation of a pulsed growth cycle and illustrates the boundary between two successive growth cycles in pulsed growth. By analyzing the geometry variation of the contrast line-markers, the morphology evolution in the growth of a multi-section NR, including a tapering process, can be traced. Such a morphology variation is controlled by the size of the catalytic Ga droplet and its coverage range on the slant facets at the top of an NR. The comparison of emission spectra between single-, two-, and three-section GaN NRs with sidewall InGaN/GaN quantum wells indicates that a multi-section NR can lead to a significantly broader sidewall emission spectrum.

Evaluating the blue-shift behaviors of the surface plasmon coupling of an embedded light emitter with a surface Ag nanoparticle by adding a dielectric interlayer or coating.

The surface plasmon (SP) coupling behaviors of an embedded light emitter or radiating dipole in GaN with a surface Ag nanoparticle (NP) in four structures of different added dielectric geometries, including an extended dielectric interlayer (DI) and a DI of a finite width between the Ag NP and GaN, a dielectric coating on the Ag NP, and no dielectric addition, are numerically compared. Either an added DI or dielectric coating can lead to the blue shift of localized surface plasmon (LSP) dipole resonance peak or the spectral peak of radiated power enhancement ratio with respect to that of the structure without dielectric addition. A smaller dielectric refractive-index or a larger dielectric thickness results in a larger blue-shift range. Under the condition of the same dielectric refractive-index and thickness, the structure of a DI with a finite width leads to the largest blue-shift range, followed by the structure of an extended DI and then the structure of a dielectric coating. In a practical application, for a given emission wavelength of a blue-emitting quantum well, the emission enhancement effect through SP coupling depends on the LSP resonance strength at this wavelength. Our study also shows that although the LSP resonance peak can be blue-shifted by reducing the size of a surface Ag NP, its SP coupling strength is dramatically reduced. Adding a DI or dielectric coating is a more practical approach for shifting the major LSP resonance mode of a surface Ag NP from the green into blue range.

Multi-mechanism efficiency enhancement in growing Ga-doped ZnO as the transparent conductor on a light-emitting diode.

The combined effects of a few mechanisms for emission efficiency enhancement produced in the overgrowth of the transparent conductor layer of Ga-doped ZnO (GaZnO) on a surface Ag-nanoparticle (NP) coated light-emitting diode (LED), including surface plasmon (SP) coupling, current spreading, light extraction, and contact resistivity reduction, are demonstrated. With a relatively higher GaZnO growth temperature (350 °C), melted Ag NPs can be used as catalyst for forming GaZnO nanoneedles (NNs) through the vapor-liquid-solid growth mode such that light extraction efficiency can be increased. Meanwhile, residual Ag NPs are buried in a simultaneously grown GaZnO layer for inducing SP coupling. With a relatively lower GaZnO growth temperature (250 °C), all the Ag NPs are preserved for generating a stronger SP coupling effect. By using a thin annealed GaZnO interlayer on p-GaN before Ag NP fabrication, the contact resistivity at the GaZnO/p-GaN interface and hence the overall device resistance can be reduced. Although the use of this interlayer blue-shifts the localized surface plasmon resonance peak of the fabricated Ag NPs from the quantum well emission wavelength of the current study (535 nm) such that the SP coupling effect becomes weaker, it is useful for enhancing the SP coupling effect in an LED with a shorter emission wavelength.

Multi-section core-shell InGaN/GaN quantum-well nanorod light-emitting diode array.

The growth of a two-section, core-shell, InGaN/GaN quantum-well (QW) nanorod- (NR-) array light-emitting diode device based on a pulsed growth technique with metalorganic chemical vapor deposition is demonstrated. A two-section n-GaN NR is grown through a tapering process for forming two uniform NR sections of different cross-sectional sizes. The cathodoluminescence (CL), photoluminescence (PL), and electrolumines-cence (EL) characterization results of the two-section NR structure are compared with those of a single-section NR sample, which is prepared under the similar condition to that for the first uniform NR section of the two-section sample. All the CL, PL, and EL spectra of the two-section sample (peaked between 520 and 525 nm) are red-shifted from those of the single-section sample (peaked around 490 nm) by >30 nm in wavelength. Also, the emitted spectral widths of the two-section sample become significantly larger than their counterparts of the single-section sample. The PL spectral full-width at half-maximum increases from ~37 to ~61 nm. Such variations are attributed to the higher indium incorporation in the sidewall QWs of the two-section sample due to the stronger strain relaxation in an NR section of a smaller cross-sectional size and the more constituent atom supply from the larger gap volume between neighboring NRs.

Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors.

Stiffness is a biophysical property of the extracellular matrix that modulates cellular functions, including proliferation, invasion, and differentiation, and it also may affect therapeutic responses. Therapeutic durability in cancer treatments remains a problem for both chemotherapies and pathway-targeted drugs, but the reasons for this are not well understood. Tumor progression is accompanied by changes in the biophysical properties of the tissue, and we asked whether matrix rigidity modulated the sensitive versus resistant states in HER2-amplified breast cancer cell responses to the HER2-targeted kinase inhibitor lapatinib. The antiproliferative effect of lapatinib was inversely proportional to the elastic modulus of the adhesive substrata. Down-regulation of the mechanosensitive transcription coactivators YAP and TAZ, either by siRNA or with the small-molecule YAP/TEAD inhibitor verteporfin, eliminated modulus-dependent lapatinib resistance. Reduction of YAP in vivo in mice also slowed the growth of implanted HER2-amplified tumors, showing a trend of increasing sensitivity to lapatinib as YAP decreased. Thus we address the role of stiffness in resistance to and efficacy of a HER2 pathway-targeted therapeutic via the mechanotransduction arm of the Hippo pathway.

Thermally induced variations of strain condition and emission behavior in flat and bendable light-emitting diodes on different substrates.

The emission behaviors of four light-emitting diodes (LEDs) of different substrate structures, including a lateral LED grown on sapphire, a vertical LED wafer-bonded onto Si (111), a bendable LED Ag-epoxied onto a flat metal, and another bendable LED Ag-epoxied onto a metal of a curved surface, under different duty cycles of current injection are compared. Their different variation trends of emission behavior with injection duty cycle are attributed to the different thermally-induced strain conditions in the epitaxial layers, which are controlled by their substrate structures, in increasing injection duty cycle or current level. The results of Raman scattering measurements during LED operation show that a stronger tensile strain is generated under heating for reducing the quantum-confined Stark effect and hence increasing emission efficiency when the epitaxial layer is not tightly bonded onto a hard substrate. Such a behavior is particularly stronger when the epitaxial layer is bent.