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CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.

Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. Rocaglates perturb eIF4A activity by imparting a gain-of-function activity to eIF4A and mediating clamping to RNA. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as identify responsive tumor types. Here, we report on the results of a positive selection, ORFeome screen aimed at identifying cDNAs capable of conferring resistance to rocaglates. Two of the most potent modifiers of rocaglate response identified were the transcription factors FOXP3 and NR1I3, both of which have been implicated in ABCB1 regulation—the gene encoding P-glycoprotein (Pgp). Pgp has previously been implicated in conferring resistance to silvestrol, a naturally occurring rocaglate, and we show here that this extends to additional synthetic rocaglate derivatives. In addition, FOXP3 and NR1I3 impart a multi-drug resistant phenotype that is reversed upon inhibition of Pgp, suggesting a potential therapeutic combination strategy.

End-to-end (E2E) spoken language understanding (SLU) can infer semantics directly from speech signal without cascading an automatic speech recognizer (ASR) with a natural language understanding (NLU) module. However, paired utterance recordings and corresponding semantics may not always be available or sufficient to train an E2E SLU model in a real production environment. In this paper, we propose to unify a well-optimized E2E ASR encoder (speech) and a pre-trained language model encoder (language) into a transformer decoder. The unified speech-language pre-trained model (SLP) is continually enhanced on limited labeled data from a target domain by using a conditional masked language model (MLM) objective, and thus can effectively generate a sequence of intent, slot type, and slot value for given input speech in the inference. The experimental results on two public corpora show that our approach to E2E SLU is superior to the conventional cascaded method. It also outperforms the present state-of-the-art approaches to E2E SLU with much less paired data.

Covering: up to 2019Pharmacological targeting of eukaryotic mRNA translation initiation is a promising approach for cancer therapy, since several signaling pathways that are commonly deregulated during tumor progression converge on this process. The DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, is essential for translation initiation and facilitates the loading of the 43S pre-initiation complex onto mRNAs. Hippuristanol, rocaglates, and pateamine A are natural products that each target eIF4A by interfering with the helicase's RNA-binding activity in distinct manners. They exert a selective change in gene expression that results in potent anti-tumorigenic activity in pre-clinical studies. This review will provide an update on the molecular mechanisms of action of these natural products.

Paper-based surface-enhanced Raman scattering (SERS) substrates receive a great deal of attention due to low cost and high flexibility. Herein, we developed an efficient SERS substrate by gravure printing of sulfonated reduced graphene-oxide (S-RGO) thin film and inkjet printing of silver nanoparticles (AgNPs) on weighing paper successively. Malachite green (MG) and rhodamine 6G (R6G) were chosen as probe molecules to evaluate the enhanced performance of the fabricated SERS-active substrates. It was found that the S-RGO/AgNPs composite structure possessed higher enhancement ability than the pure AgNPs. The Raman enhancement factor of S-RGO/AgNPs was calculated to be as large as 109. The minimum detection limit for MG and R6G was down to 10−7 M with good linear responses (R2 = 0.9996, 0.9983) range from 10−4 M to 10−7 M. In addition, the S-RGO/AgNPs exhibited good uniformity with a relative standard deviation (RSD) of 7.90% measured by 572 points, excellent reproducibility with RSD smaller than 3.36%, and long-term stability with RSD less than 7.19%.

DExD (DDX)- and DExH (DHX)-box RNA helicases, named after their Asp-Glu-x-Asp/His motifs, are integral to almost all RNA metabolic processes in eukaryotic cells. They play myriad roles in processes ranging from transcription and mRNA-protein complex remodeling, to RNA decay and translation. This last facet, translation, is an intricate process that involves DDX/DHX helicases and presents a regulatory node that is highly targetable. Studies aimed at better understanding this family of conserved proteins have revealed insights into their structures, catalytic mechanisms, and biological roles. They have also led to the development of chemical modulators that seek to exploit their essential roles in diseases. Herein, we review the most recent insights on several general and target-specific DDX/DHX helicases in eukaryotic translation initiation.

With the emergence of printed electronics, the development of printed sensors on flexible substrates is on-going. Described herein is a flexible and lightweight chemiresistor made of a thin film composed of WO3 and poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonicacid) (PEDOT:PSS), which were printed onto a flexible polyamide substrate by using gravure technique. The optimized WO3-PEDOT:PSS sensor exhibits a low initial resistance(∼870 Ω) and good sensing response characteristics (∼1.2) towards 50 ppb NO2 gas at room temperature, with response and recovery time of 45.1 and 88.7 s, respectively. In comparison with a pure WO3 sensor, the enhanced sensing performance is attributed to the conducting channels provided by PEDOT:PSS, formation hetero-junctions at the WO3-PEDOT:PSS interfaces as well as the printed micro-scale striped structures. The printed WO3-PEDOT:PSS sensors show great potential for ultrasensitive detection of NO2 gas in a wide variety of fields where operation at room temperature, light weight and mechanical flexibility are important issues.

A novel three-dimensional (3D) nanoarchitecture consisting of hybrid graphene nanosheets (GNs)/graphene foam (GF) was fabricated on the FTO conducting substrate as a high efficient counter electrode (CE) for dye sensitized solar cells (DSSCs). The GNs with various sized such as large-sized heat-reduced graphene nanosheets (H-GNs) and small-sized laser-reduced graphene quantum dots (L-GQDs) were synthesized and used as catalytic materials incorporated into a 3D GF network, respectively. In this design, the aggregations and restacking of GNs were efficiently reduced, which is beneficial for increasing the amount of the active defective sites at the edges of graphene to the electrolyte solution. Especially, L-GQDs with smaller dimension less than 100 nm have more active defective sites at edges, providing superiority over the large-sized H-GNs in terms of electrocatalytic activity. Meanwhile, the GF network with high conductivity provides fast electron transport channels for charge injection between the GNs and FTO. The DSSC with this hybrid CE exhibited energy conversion efficiency (η) of 7.70% with an open circuit voltage (VOC), short circuit photocurrent density (JSC) and fill factor (FF) of 760 mV, 15.21 mA cm−2, and 72.0%, respectively, which is comparable to that of the conventional Pt CE (7.68%).

Reduced graphene oxide (RGO) has proven to be effective in trace gas detection at room temperature ambient conditions. However, the slow response-recovery characteristic is a major hurdle for the RGO-based gas sensors. Herein, we report a gravure-printed chemoresistor-type NO2 sensor based on sulfonated RGO (S-RGO) decorated with Ag nanoparticles (Ag-S-RGO). Large amounts of silver nanoparticles with an average particle size of 10-20 nm were uniformly assembled on flat S-RGO surfaces. The printed Ag-S-RGO sensor possesses a high sensitivity and fast response-recovery characteristic over NO2 concentrations ranging from 0.5 to 50 ppm. Upon exposure to 50 ppm NO2 at room temperature, the Ag-S-RGO sensor shows a sensitivity of 74.6%, a response time of 12 s and a recovery time of 20 s. In addition, the Ag-S-RGO sensors exhibit satisfactory flexibility with an almost constant resistance after 1000 bending cycles. The printed and high-performance Ag-S-RGO sensors described here will be a good prospect in environmental monitoring of NO2.

We define Backward Stochastic Difference Equations related to a discrete finite time single jump process. We prove the existence and uniqueness of solutions under some assumptions. A comparison theorem for these solutions is also given. Applications to the theory of nonlinear expectations are then investigated. In this paper the single jump process takes values in a general measurable space where as previous work has considered the situation where the noise is a finite state Markov chain, so the state space is finite. Refereed/Peer-reviewed

The Mycobacterium tuberculosis ATP-binding cassette transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). The structures of FHA-1 and FHA-2 were determined by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, respectively. Relative to the canonical 11-strand β-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one β-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (∼50 μM versus 500 μM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. We hypothesize that this enables tunable phosphorylation-dependent multimerization to regulate Rv1747 transporter activity.

This paper defines a g-variance and p-confidence interval for a g-expectation. We also determine the p-confidence intervals under some specific assumptions on g.

The paper shows the gravure printing technology is used as deposition method to pattern a graphene film on flexible polyimide substrates for a wireless strain sensor (WSS). According to the inductive coupling between the reader antenna and the graphene pattern (GP), a change in the GP characteristic can be detected wirelessly by a corresponding change in the complex impedance of the reader antenna. The WSS overcomes the inadequacy of the existing conventional sensors limited monitoring locations. And the WSS also exhibits a high sensitivity of 51% under a load of 100 N.

In this paper, we demonstrate gravure printing of crumpled graphene ink to obtain a highly porous pattern of interdigitated electrodes, leading to an interdigital microsupercapacitor (MSC) on a flexible polyimide substrate. During the process of synthesizing crumpled graphene ink, Mg(OH)2 nanosheets as nanospacers were inserted into graphite oxide layers, resulting in sufficient crumples in graphene nanosheets to prevent the graphene sheets from restacking to enhance the ion transport and expose the electrochemical active area with oxygen-containing groups to provide more pseudo-capacitance. The gravure-printed interdigital MSCs achieved a high energy density (1.41 mW h cm(-3) at 25 mW cm(-3)) and high power density (0.35 mW h cm(-3) at 300 mW cm(-3)), respectively. Additionally, a liquid crystal display was driven by the two serial and two parallel connected MSCs for 35 s after charging for 3 s.

We consider backward stochastic differential equations (BSDEs) related to a finite continuous time single jump process. We prove the existence and uniqueness of solutions when the coefficients satisfy Lipschitz continuity conditions. A comparison theorem for these solutions is also given. Applications to the theory of nonlinear expectations are then investigated. Refereed/Peer-reviewed

The pencil-drawn sensor is expected to enable a simple, low-cost, and reproducible paper-based sensor platform for widely deployed wireless environmental monitoring of NO2. Herein, we demonstrated a rapid prototyping of chemiresistor-type NO2 sensor by mechanical abrasion of an 8B pencil to form a stripe of uniform graphitic coating on printed silver interdigitated electrodes (IDEs). The Ag IDEs not only offer a low resistance but also provide the assembly of Ag nanoparticles into exfoliated graphene sheets for the paper-based NO2 gas sensors in order to realise much higher sensitivity and better reproducibility comparing with pencil-drawn sensors directly on weighing paper.

Smart ink-like hybrid hydrogels that simultaneously possess semi-interpenetrating network structure, strong photoluminescence and temperature sensitivity are successfully fabricated based on the crosslink of poly(acrylamide) (PAAm) in the presence of poly(N-isopropylacrylamide) (PNIPAM) and carbon nanodots (CNDs) at room temperature. The resulting hybrid hydrogels were highly photoluminescent. The photoluminescence was sensitive to external temperature stimuli and reversible. Moreover, the hybrid hydrogels were applied as fluorescent inks for patterning using gravure printing, which may open a door towards developing smart CND based thermosensitive photoluminescent markers and sensors.