K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

Standoff Detection and Identification of Liquid Chemicals on a Reflective Substrate Using a Wavelength-Tunable Quantum Cascade Laser.

Standoff chemical detection and identification techniques are necessary for ensuring safe exposure to dangerous substances. Molecular fingerprints of unknown chemicals can be measured using wavelength-tunable quantum cascade lasers operating in long-wavelength infrared. In this work, we present a method that can identify liquid chemicals on a reflective substrate via diffuse reflection spectra measurement from 50 cm away and multiple nonlinear regression analysis. Experimental measurements and numerical analyses were conducted for different chemical surface densities and angles of light incidence using diethyl phthalate (DEP) and dimethyl methylphosphonate (DMMP). Candidate substances can be classified using a deep learning model to reduce analysis time.

Giant Nonlinear Circular Dichroism from Intersubband Polaritonic Metasurfaces.

Nonlinear metasurfaces are advancing into a new paradigm of "flat nonlinear optics" owing to the ability to engineer local nonlinear responses in subwavelength-thin films. Recently, attempts have been made to expand the design space of nonlinear metasurfaces through nonlinear chiral responses. However, the development of metasurfaces that display both giant nonlinear circular dichroism and significantly large nonlinear optical response is still an unresolved challenge. Herein, we propose a method that induces giant nonlinear responses with near-unity circular dichroism using polaritonic metasurfaces with optical modes in chiral plasmonic nanocavities coupled with intersubband transitions in semiconductor heterostructures designed to have giant second and third order nonlinear responses. A stark contrast between effective nonlinear susceptibility elements for the two spin states of circularly polarized pump beams was seen in the hybrid structure. Experimentally, near-unity nonlinear circular dichroism and conversion efficiencies beyond 10-4% for second- and third-harmonic generation were achieved simultaneously in a single chip.

[A Case of Ramucirumab-Related Small Intestinal Perforation in Gastric Cancer].

A 54-year-old man underwent laparoscopic distal gastrectomy with D2 lymph node dissection and ante-colic Roux-en-Y reconstruction for gastric cancer. The histopathological diagnosis was pT2N3aM0, pStage ⅢA, HER2 negative. After 8 courses of S-1 plus oxaliplatin as adjuvant chemotherapy, he was diagnosed as peritoneal dissemination and treated with ramucirumab(RAM)plus paclitaxel(PTX). On the 12th day of course 10, he visited to our hospital with abdominal pain. CT showed free air and massive ascites. Emergent surgery was performed under the diagnosis of gastrointestinal perforation. A small intestinal perforation in front of the jejunal limb near gastric-jejunal anastomosis was identified and there was no peritoneal dissemination. We performed partial resection of remnant stomach and jejunal limb by linear stapler and reconstruction by end to side gastric-jejunal anastomosis. Because the gastric and intestinal wall were quite fragile and RAM impaired wound healing as adverse event, we feared about leakage, but he had no major postoperative complications and discharged on the 33th day after surgery. After 24 courses of nivolumab as third-line chemotherapy, the peritoneal dissemination disappeared. He has been alive without recurrence for about 1 year since then.

Visible-Light-Driven C4-Selective Alkylation of Pyridinium Derivatives with Alkyl Bromides.

Reported herein is a general strategy for the photochemical cross-coupling between N-amidopyridinium salts and various alkyl bromides under photocatalyst-free conditions, granting facile access to various C4-alkylated pyridines. This approach exploits the intriguing photochemical activity of electron donor-acceptor (EDA) complexes between N-amidopyridinium salts and bromide, which provides a photoactive handle capable of generating silyl radicals and driving the alkylation process. The robustness of this protocol was further demonstrated by the late-stage functionalization of complex compounds under mild and metal-free conditions.

An Improved Method for Bacterial Immunofluorescence Staining To Eliminate Antibody Exclusion from the Fixed Nucleoid.

Immunofluorescence (IF) is widely used to study the cellular localization and organization of proteins. However, steps such as fixation and permeabilization may affect cell morphology and/or introduce artifacts. For bacterial cells, commonly used permeabilization methods for IF include treatment with lysozyme. Here, we demonstrate two potential pitfalls in IF due to specific permeabilization methods: flattening or disruption of the cells caused by lysozyme treatment and inaccessibility of the antibody to the fixed nucleoid region. To solve these issues, we propose an improved IF method for bacterial cells, which includes the combined treatment with 70% ethanol, lysozyme, and DNase I. Treatment with 70% ethanol before the lysozyme permeabilization can better preserve the three-dimensional shape of the cell, and treatment with DNase I after the lysozyme permeabilization can eliminate the inaccessibility of the antibody to the nucleoid region. We further demonstrate that the DNase I treatment does not affect the preservation of the DNA-associated structure or organization of proteins. Finally, the method is also compatible with applications in which IF needs to be combined with RNA fluorescence in situ hybridization.

Alignment-tolerant single-shot digital holographic microscopy based on computer-controlled telecentricity.

An alignment-tolerant telecentric digital holographic microscopy (AT-T-DHM) system based on computer-controlled telecentricity is proposed. It consists of a three-step process-optical recording, computational compensation, and retrieving processes. With a tube-lens-based two-beam interferometer, phase information of the object is recorded on the hologram, where another optical quadratic phase error (O-QPE) due to the misalignment of the tube lens happens to be added. In the computational compensation process, this phase error can be estimated, by which the O-QPE is balanced out from the recorded hologram. Then, only the phase information of the object can be retrieved from the O-QPE-compensated hologram. This computational compensation process makes the proposed system virtually operate in a telecentric imaging mode, which enables implementing a practical AT-T-DHM. Wave-optical analysis and experiments with a test object confirm the feasibility of the proposed system.

Toward Single-Cell Single-Molecule Pull-Down.

Single-molecule pull-down (SiMPull) can capture native protein complexes directly from cell lysates for analysis of complex composition and activities at the single-molecule level. Although SiMPull requires many fewer cells compared to conventional pull-down assays, all studies so far have been performed using lysates from many cells. In principle, extending SiMPull to the single-cell level will allow the investigation of cell-to-cell variations on the stoichiometry and activities of biomolecular complexes. We developed a protocol to lyse bacterial cells in situ and capture the released proteins on the imaging surface using antibodies. The use of lysozymes delayed the protein release until after the flow has ceased, and the use of a 10-µm spacer reduces the capture radius within which ∼70% of target proteins can be captured to below 30 µm. Proteins thus captured can be unambiguously assigned to the originating cell. The developed platform should be compatible with high-throughput protein analysis and protein-protein interaction analysis at the single-cell level through single-molecule imaging.

Closha: bioinformatics workflow system for the analysis of massive sequencing data.

BACKGROUND: While next-generation sequencing (NGS) costs have fallen in recent years, the cost and complexity of computation remain substantial obstacles to the use of NGS in bio-medical care and genomic research. The rapidly increasing amounts of data available from the new high-throughput methods have made data processing infeasible without automated pipelines. The integration of data and analytic resources into workflow systems provides a solution to the problem by simplifying the task of data analysis. RESULTS: To address this challenge, we developed a cloud-based workflow management system, Closha, to provide fast and cost-effective analysis of massive genomic data. We implemented complex workflows making optimal use of high-performance computing clusters. Closha allows users to create multi-step analyses using drag and drop functionality and to modify the parameters of pipeline tools. Users can also import the Galaxy pipelines into Closha. Closha is a hybrid system that enables users to use both analysis programs providing traditional tools and MapReduce-based big data analysis programs simultaneously in a single pipeline. Thus, the execution of analytics algorithms can be parallelized, speeding up the whole process. We also developed a high-speed data transmission solution, KoDS, to transmit a large amount of data at a fast rate. KoDS has a file transfer speed of up to 10 times that of normal FTP and HTTP. The computer hardware for Closha is 660 CPU cores and 800 TB of disk storage, enabling 500 jobs to run at the same time. CONCLUSIONS: Closha is a scalable, cost-effective, and publicly available web service for large-scale genomic data analysis. Closha supports the reliable and highly scalable execution of sequencing analysis workflows in a fully automated manner. Closha provides a user-friendly interface to all genomic scientists to try to derive accurate results from NGS platform data. The Closha cloud server is freely available for use from http://closha.kobic.re.kr/ .

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

Ionic Liquid Designed for PEDOT:PSS Conductivity Enhancement.

Poly-3,4-ethylenedioxythiophene:polystyrenesulfonate (PEDOT:PSS) is a water-processable conducting polymer with promise for use in transparent flexible electrodes and thermoelectric devices, but its conductivity is not satisfactory. Its low conductivity is attributed to the formation of hydrophilic/insulating PSS outer layers encapsulating the conducting/hydrophobic p-doped PEDOT cores. Recently a significant conductivity enhancement has been achieved by adding ionic liquid (IL). It is believed that ion exchange between PEDOT:PSS and IL components helps PEDOT to decouple from PSS and to grow into large-scale conducting domains, but the exact mechanism is still under debate. Here we show through free energy calculations using density functional theory on a minimal model that the most efficient IL pairs are the least tightly bound ones with the lowest binding energies, which would lead to the most efficient ion exchange with PEDOT:PSS. This spontaneous ion exchange followed by nanophase segregation between PEDOT and PSS, with formation of a π-stacked PEDOT aggregate decorated by IL anions, is also supported by molecular dynamics performed on larger PEDOT:PSS models in solution. We also show that the most efficient IL anions would sustain the highest amount of charge carriers uniformly distributed along the PEDOT backbone to further enhance the conductivity, providing that they remain in the PEDOT domain after the ion exchange. Hence, our design principle is that the high-performance IL should induce not only an efficient ion exchange with PEDOT:PSS to improve the PEDOT morphology (to increase mobility) but also a uniform high-level p-doping of PEDOT (to enhance intrinsic conductivity). Based on this principle, a promising (electron-withdrawing, but bulky, soft, and hydrophobic) new IL pair is proposed.

Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population.

Sub-nanometer pore formation in single-molecule-thick polyurea molecular-sieving membrane: a computational study.

A polymeric network of 1-(4-tritylphenyl)urea (TPU) built via layer-by-layer cross-linking polymerization has been proposed to be an excellent mesh equipped with single-molecule-thick pores (i.e., cyclic poly-TPU rings), which can sieve glucose (∼0.7 nm) out of its mixture with urea for hemodialysis applications. Monte Carlo search for the lowest-energy conformation of various sizes of poly-TPU rings unravels the origin of narrow pore size distribution, which is around the sizes of dimer and trimer rings (0.3-0.8 nm). Flexible rings larger than the dimer and trimer rings, in particular tetramer rings, prefer a twisted conformation in the shape of the infinity symbol (∞, which looks like two dimer rings joined together) locked by a hydrogen bond between diphenylurea linker groups facing each other. Translocation energy profiles across these TPU rings reveal their urea-versus-glucose sieving mechanism: glucose is either too large (to enter dimers and twisted tetramers) or too perfectly fit (to exit trimers), leaving only a dimer-sized free space in the ring, whereas smaller-sized urea and water pass through these effective dimer-sized rings (bare dimers, twisted tetramers, and glucose-filled trimers) without encountering a substantial energy barrier or trap.

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria.IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins.

Programming of Plant Leaf Senescence with Temporal and Inter-Organellar Coordination of Transcriptome in Arabidopsis.

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

Single-shot digital holographic microscopy with a modified lateral-shearing interferometer based on computational telecentricity.

Single-shot digital holographic microscopy (SS-DHM) with a modified lateral-shearing interferometer (MLSI) based on computational telecentricity is proposed. The proposed system is composed of three-step processes such as optical recording, digital compensation and numerical reconstruction processes. In the 1st step, the object beam is optically recorded with the MLSI, where a tube lens is set to be located at the slightly shorter distance than its focal length from the objective lens. Then, another phase factor due to the deviated locating of the tube lens from its focal length is additionally generated, which is called an additional quadratic phase factor (AQPF). However, in the 2nd step, this AQPF can be balanced out with the computer-generated version of the AQPF. In the 3rd step, the three-dimensional (3-D) object can be finally reconstructed from this AQPF-compensated hologram. Thus, by combined use of the optical recording and digital compensation processes of the AQPF, the proposed system can be made virtually operate in a so-called computational telecentricity, which enables us to implement a MLSI-based SS-DHM system. Wave-optical analysis and successful experiments with actual 3-D objects confirm the feasibility of the proposed system in the practical application fields.

Ultraviolet photodetector using pn junction formed by transferrable hollow n-TiO2 nano-spheres monolayer.

We report an ultraviolet (UV) photodetector with a universally transferable monolayer film with ordered hollow TiO2 spheres on p-GaN. After forming a TiO2 monolayer film by unidirectional rubbing of hollow TiO2 spheres on a polydimethylsiloxane (PDMS) supporting plate, we used a 5% polyvinyl alcohol (PVA) aqueous solution to transfer the film onto the target substrate. The PVA/TiO2 monolayer film was detached from the PDMS film and transferred to the p-GaN/Al2O3 substrate. To investigate the effects of crystallized phases of the TiO2 hollow spheres, anatase and rutile TiO2 sphere monolayers prepared by combining template synthesis and thermal treatment. The responsiveness of the UV photodetectors using anatase and rutile hollow n-TiO2 monolayer/p-GaN was 0.203 A/W at 312 nm and 0.093 A/W at 327 nm, respectively.

Global transcription network incorporating distal regulator binding reveals selective cooperation of cancer drivers and risk genes.

Global network modeling of distal regulatory interactions is essential in understanding the overall architecture of gene expression programs. Here, we developed a Bayesian probabilistic model and computational method for global causal network construction with breast cancer as a model. Whereas physical regulator binding was well supported by gene expression causality in general, distal elements in intragenic regions or loci distant from the target gene exhibited particularly strong functional effects. Modeling the action of long-range enhancers was critical in recovering true biological interactions with increased coverage and specificity overall and unraveling regulatory complexity underlying tumor subclasses and drug responses in particular. Transcriptional cancer drivers and risk genes were discovered based on the network analysis of somatic and genetic cancer-related DNA variants. Notably, we observed that the risk genes were functionally downstream of the cancer drivers and were selectively susceptible to network perturbation by tumorigenic changes in their upstream drivers. Furthermore, cancer risk alleles tended to increase the susceptibility of the transcription of their associated genes. These findings suggest that transcriptional cancer drivers selectively induce a combinatorial misregulation of downstream risk genes, and that genetic risk factors, mostly residing in distal regulatory regions, increase transcriptional susceptibility to upstream cancer-driving somatic changes.

Allosteric Regulation of E-Cadherin Adhesion.

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

miRseqViewer: multi-panel visualization of sequence, structure and expression for analysis of microRNA sequencing data.

SUMMARY: Deep sequencing of small RNAs has become a routine process in recent years, but no dedicated viewer is as yet available to explore the sequence features simultaneously along with secondary structure and gene expression of microRNA (miRNA). We present a highly interactive application that visualizes the sequence alignment, secondary structure and normalized read counts in synchronous multipanel windows. This helps users to easily examine the relationships between the structure of precursor and the sequences and abundance of final products and thereby will facilitate the studies on miRNA biogenesis and regulation. The project manager handles multiple samples of multiple groups. The read alignment is imported in BAM file format. Implemented features comprise sorting, zooming, highlighting, editing, filtering, saving, exporting, etc. Currently, miRseqViewer supports 84 organisms whose annotation is available at miRBase. AVAILABILITY AND IMPLEMENTATION: miRseqViewer, implemented in Java, is available at https://github.com/insoo078/mirseqviewer or at http://msv.kobic.re.kr. CONTACT: sanghyuk@ewha.ac.kr.