Erratum to: Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in.

[This corrects the article DOI: 10.1093/biomethods/bpaa006.].

Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-directed repair process that requires donor constructs as repair templates. Therefore, it is desirable to generate a series of donor templates efficiently and cost-effectively. In this study, we developed a new strategy that combines (i) Gibson assembly reaction, (ii) a linker pair composed of eight in silico screened restriction enzyme sites, and (iii) a hierarchical framework, to remarkably improve the efficiency of producing donor constructs for common genes as well as for the genes containing unbalanced guanine-cytosine content and requiring a selectable marker. Furthermore, the approach provides the ability of inserting additional elements into the donor templates, such as single guide RNA recognition sites that have been reported to enhance the efficiency of homology-directed repair. Conclusively, our modularized process is simple, fast, and cost-effective for making donor constructs and benefits the application of CRISPR knock-in methods.

Learn to segment single cells with deep distance estimator and deep cell detector.

Single cell segmentation is a critical and challenging step in cell imaging analysis. Traditional processing methods require time and labor to manually fine-tune parameters and lack parameter transferability between different situations. Recently, deep convolutional neural networks (CNN) treat segmentation as a pixel-wise classification problem and have become a general and efficient method for image segmentation. However, cell imaging data often possesses characteristics that adversely affect segmentation accuracy: absence of established training datasets, few pixels on cell boundaries, and ubiquitous blurry features. We developed a strategy that combines strengths of CNN and traditional watershed algorithm. First, we trained a CNN to learn Euclidean distance transform (EDT) of the mask corresponding to the input images (deep distance estimator). Next, we trained a faster R-CNN (Region with CNN) to detect individual cells in the EDT image (deep cell detector). Then, the watershed algorithm performed the final segmentation using the outputs of previous two steps. Tests on a library of fluorescence, phase contrast and differential interference contrast (DIC) images showed that both the combined method and various forms of the pixel-wise classification algorithm achieved similar pixel-wise accuracy. However, the combined method achieved significantly higher cell count accuracy than the pixel-wise classification algorithm did, with the latter performing poorly when separating connected cells, especially those connected by blurry boundaries. This difference is most obvious when applied to noisy images of densely packed cells. Furthermore, both deep distance estimator and deep cell detector converge fast and are easy to train.

A Vision toward Ultimate Optical Out-Coupling for Organic Light-Emitting Diode Displays: 3D Pixel Configuration.

Despite stringent power consumption requirements in many applications, over years organic light-emitting diode (OLED) displays still suffer unsatisfactory energy efficiency due to poor light extraction. Approaches have been reported for OLED light out-coupling, but they in general are not applicable for OLED displays due to difficulties in display image quality and fabrication complexity and compatibility. Thus to date, an effective and feasible light extraction technique that can boost efficiencies and yet keep image quality is still lacking and remains a great challenge. Here, a highly effective and scalable extraction-enhancing OLED display pixel structure is proposed based on embedding the OLED inside a three-dimensional reflective concave structure covered with a patterned high-index filler. It can couple as much internal emission as possible into the filler region and then redirect otherwise confined light for out-coupling. Comprehensive multi-scale optical simulation validates that ultimately high light extraction efficiency approaching ≈80% and excellent viewing characteristics are simultaneously achievable with optimized structures using highly transparent top electrodes. This scheme is scalable and wavelength insensitive, and generally applicable to all red, green, and blue pixels in high-resolution full-color displays. Results of this work are believed to shed light on the development of future generations of advanced OLED displays.

Pathway crosstalk enables cells to interpret TGF-ß duration.

The detection and transmission of the temporal quality of intracellular and extracellular signals is an essential cellular mechanism. It remains largely unexplored how cells interpret the duration information of a stimulus. In this paper, we performed an integrated quantitative and computational analysis on TGF-ß induced activation of SNAIL1, a key transcription factor that regulates several subsequent cell fate decisions such as apoptosis and epithelial-to-mesenchymal transition. We demonstrate that crosstalk among multiple TGF-ß activated pathways forms a relay from SMAD to GLI1 that initializes and maintains SNAILl expression, respectively. SNAIL1 functions as a key integrator of information from TGF-ß signaling distributed through upstream divergent pathways. The intertwined network serves as a temporal checkpoint, so that cells can generate a transient or sustained expression of SNAIL1 depending on TGF-ß duration. Furthermore, we observed that TGF-ß treatment leads to an unexpected accumulation of GSK3 molecules in an enzymatically active tyrosine phosphorylation form in Golgi apparatus and ER, followed by accumulation of GSK3 molecules in an enzymatically inhibitive serine phosphorylation in the nucleus. Subsequent model analysis and inhibition experiments revealed that the initial localized increase of GSK3 enzymatic activity couples to the positive feedback loop of the substrate Gli1 to form a network motif with multi-objective functions. That is, the motif is robust against stochastic fluctuations, and has a narrow distribution of response time that is insensitive to initial conditions. Specifically for TGF-ß signaling, the motif ensures a smooth relay from SMAD to GLI1 on regulating SNAIL1 expression.

Urothelial Carcinomas of the Urinary Bladder With Plasmacytoid or Rhabdoid Features and Tendency of Epithelial-Mesenchymal Transition in 3 Dogs.

Plasmacytoid and rhabdoid variants of urothelial carcinomas (UCs) of the urinary bladder have been described in humans with plasma cell-like or rhabdoid cellular appearance and aggressive clinical outcome. Canine UC of the bladder is generally classified as papillary/nonpapillary and infiltrating/noninfiltrating with limited information regarding other histological patterns. We report 3 cases of UC of the urinary bladder showing a unique discohesive cellular morphology with malignant behavior resembling the human plasmacytoid and rhabdoid variants of UC, which may raise some difficulties in diagnosis. Epithelial-mesenchymal transition and reduced E-cadherin expression were revealed by immunohistochemistry in 2 cases, possibly explaining the discohesive and invasive behavior of the tumor cells. The findings broaden the morphological spectrum as well as the distinct clinical features of canine UC of the urinary bladder.

AlGaInP Red LEDs with Hollow Hemispherical Polystyrene Arrays.

A hollow hemispherical polystyrene (HHPS) was fabricated to reduce total internal reflection in AlGaInP-based LEDs. At an injection current of 350 mA, the external quantum efficiencies of LED-I, LED-II, LED-III, and LED-IV are 20.92%, 24.65%, 27.28%, and 33.77% and the wall-plug efficiencies are 17.11%, 20%, 22.5%, and 27.33%, respectively. The enhanced performance is attributed to the light output power enhancement through the surface roughness, microlens-liked PS hemisphere, and scatter-liked HHPS array. In this paper, the rigorous coupled wave analysis (RCWA) numerical method was also conducted to demonstrate the HHPS array effectively enlarge the effective light cone.

Phosphorylation potential of Drosophila E-Cadherin intracellular domain is essential for development and adherens junction biosynthetic dynamics regulation.

Phosphorylation of a highly conserved serine cluster in the intracellular domain of E-Cadherin is essential for binding to ß-Catenin in vitro In cultured cells, phosphorylation of specific serine residues within the cluster is also required for regulation of adherens junction (AJ) stability and dynamics. However, much less is known about how such phosphorylation of E-Cadherin regulates AJ formation and dynamics in vivo In this report, we generated an extensive array of Drosophila E-Cadherin (DE-Cad) endogenous knock-in alleles that carry mutations targeting this highly conserved serine cluster. Analyses of these mutations suggest that the overall phosphorylation potential, rather than the potential site-specific phosphorylation, of the serine cluster enhances the recruitment of ß-Catenin by DE-Cad in vivo Moreover, phosphorylation potential of the serine cluster only moderately increases the level of ß-Catenin in AJs and is in fact dispensable for AJ formation in vivo Nonetheless, phosphorylation-dependent recruitment of ß-Catenin is essential for development, probably by enhancing the interactions between DE-Cad and α-Catenin. In addition, several phospho-mutations dramatically reduced the biosynthetic turnover rate of DE-Cad during apical-basal polarization, and such biosynthetically stable DE-Cad mutants specifically rescued the polarity defects in embryonic epithelia lacking the polarity proteins Stardust and Crumbs.

Analyses of optical out-coupling of organic light-emitting devices having micromesh indium tin oxide and conducting polymer as composite transparent electrode.

We report the characterization and analyses of organic light-emitting devices (OLEDs) using microstructured composite transparent electrodes consisting of the high-index ITO (indium tin oxide) micromesh and the low-index conducting polymer PEDOT: PSS [poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)], that are fabricated by the facile and convenient microsphere lithography and are useful for enhancing light extraction. The rigorous electromagnetic simulation based on the three-dimensional finite-difference time-domain (FDTD) method was conducted to study optical properties and mechanisms in such devices. It provides a different but consistent viewpoint/insight of how this microstructured electrode enhances optical out-coupling of OLEDs, compared to that provided by ray optics simulation in previous works. Both experimental and simulation studies indicate such a microstructured electrode effectively enhances coupling of internal radiation into the substrate, compared to devices with the typical planar ITO electrode. By combining this internal extraction structure and the external extraction scheme (e.g. by attaching extraction lens) to further extract radiation into the substrate, a rather high external quantum efficiency of 46.8% was achieved with green phosphorescent OLEDs, clearly manifesting its high potential.

A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia.

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting.

Enhancing Optical Out-Coupling of Organic Light-Emitting Devices with Nanostructured Composite Electrodes Consisting of Indium Tin Oxide Nanomesh and Conducting Polymer.

A nanostructured composite electrode consisting of a high-index indium-tin-oxide nanomesh and low-index high-conductivity conducting polymer effectively enhances coupling of internal radiation of organic light-emitting devices into their substrates. When combining this internal extraction structure and the external extraction scheme, a very high external quantum efficiency of nearly 62% is achieved with a green phosphorescent device.

The protein O-glucosyltransferase Rumi modifies eyes shut to promote rhabdomere separation in Drosophila.

The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.

Plasmonic ITO-free polymer solar cell.

The aluminum and sliver multilayered nano-grating structure is fabricated by laser interference lithography and the intervals between nanoslits is filled with modified PEDOT:PSS. The grating structured transparent electrode functions as the anti-reflection layer which not only decreases the reflected light but also increases the absorption of the active layer. The performances of P3HT:PC61BM solar cells are studied experimentally and theoretically in detail. The field intensities of the transverse magnetic (TM) and transverse electrical (TE) waves distributed in the active layer are simulated by rigorous coupled wave analysis (RCWA). The power conversion efficiency of the plasmonic ITO-free polymer solar cell can reach 3.64% which is higher than ITO based polymer solar cell with efficiency of 3.45%.

Design and fabrication of birefringent nano-grating structure for circularly polarized light emission.

Three different nano-grating structures are designed as phase retarders that can transform linearly polarized light to circularly polarized emission for the wavelengths of 488 nm, 532 nm and 632.8 nm, respectively. Gold based nano-grating structures with various periods are fabricated by utilizing laser interference lithography. The ellipticity of all circularly polarized emission can reach around 90% such that the structure has great potential in the applications of three-dimensional (3D) display. The effects of the slit width and metal thickness modulations are simulated by rigorous coupled wave analysis (RCWA) method. Besides, the field intensity and phase of the transmitted TM and TE waves are also simulated to understand their polarization characteristics.

Hydrogen induced redox mechanism in amorphous carbon resistive random access memory.

We investigated the bipolar resistive switching characteristics of the resistive random access memory (RRAM) device with amorphous carbon layer. Applying a forming voltage, the amorphous carbon layer was carbonized to form a conjugation double bond conductive filament. We proposed a hydrogen redox model to clarify the resistive switch mechanism of high/low resistance states (HRS/LRS) in carbon RRAM. The electrical conduction mechanism of LRS is attributed to conductive sp2 carbon filament with conjugation double bonds by dehydrogenation, while the electrical conduction of HRS resulted from the formation of insulating sp3-type carbon filament through hydrogenation process.

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin.

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.

Method of compensating for pixel migration in volume holographic optical disc (VHOD).

Volume holographic optical disc (VHOD) technology is simpler than the angular multiplexing holographic system. However, disc rotation usually causes pixel migration, thus reducing signal quality. This study proposes a special geometrical arrangement to counteract pixel migration. Using paraxial approximation analysis, an optimal geometrical distance ratio, K, is calculated to compensate for pixel migration and improve image quality during disc rotation. The results of approximation analysis are confirmed by both simulation and experimental results.

Differential regulation of adherens junction dynamics during apical-basal polarization.

Adherens junctions (AJs) in epithelial cells are constantly turning over to modulate adhesion properties under various physiological and developmental contexts, but how such AJ dynamics are regulated during the apical-basal polarization of primary epithelia remains unclear. Here, we used new and genetically validated GFP markers of Drosophila E-cadherin (DE-cadherin, hereafter referred to as DE-Cad) and ß-catenin (Armadillo, Arm) to quantitatively assay the in vivo dynamics of biosynthetic turnover and membrane redistribution by fluorescence recovery after photobleaching (FRAP) assays. Our data showed that membrane DE-Cad and Arm in AJs of polarizing epithelial cells had much faster biosynthetic turnover than in polarized cells. Fast biosynthetic turnover of membrane DE-Cad is independent of actin- and dynamin-based trafficking, but is microtubule-dependent. Furthermore, Arm in AJs of polarizing cells showed a faster and diffusion-based membrane redistribution that was both quantitatively and qualitatively different from the slower and exchange-based DE-Cad membrane distribution, indicating that the association of Arm with DE-Cad is more dynamic in polarizing cells, and only becomes stable in polarized epithelial cells. Consistently, biochemical assays showed that the binding of Arm to DE-Cad is weaker in polarizing cells than in polarized cells. Our data revealed that the molecular interaction between DE-Cad and Arm is modulated during apical-basal polarization, suggesting a new mechanism that might be crucial for establishing apical-basal polarity through regulating the AJ dynamics.

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.

Regulation of unfolded protein response modulator XBP1s by acetylation and deacetylation.

XBP1 (X-box-binding protein 1) is a key modulator of the UPR (unfolded protein response), which is involved in a wide range of pathological and physiological processes. The mRNA encoding the active spliced form of XBP1 (XBP1s) is generated from the unspliced form by IRE1 (inositol-requiring enzyme 1) during the UPR. However, the post-translational modulation of XBP1s remains largely unknown. In the present study, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 (sirtuin 1) respectively. p300 increases the acetylation and protein stability of XBP1s, and enhances its transcriptional activity, whereas SIRT1 deacetylates XBP1s and inhibits its transcriptional activity. Deficiency of SIRT1 enhances XBP1s-mediated luciferase reporter activity in HEK (human embryonic kidney)-293 cells and the up-regulation of XBP1s target gene expression under ER (endoplasmic reticulum) stress in MEFs (mouse embryonic fibroblasts). Consistent with XBP1s favouring cell survival under ER stress, Sirt1-/- MEFs display a greater resistance to ER-stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels, as well as the transcriptional activity, of XBP1s. The present study provides a novel insight into the molecular mechanisms by which SIRT1 regulates UPR signalling.