A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane.

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

An Implementation of Trust Chain Framework with Hierarchical Content Identifier Mechanism by Using Blockchain Technology.

Advances in information technology (IT) and operation technology (OT) accelerate the development of manufacturing systems (MS) consisting of integrated circuits (ICs), modules, and systems, toward Industry 4.0. However, the existing MS does not support comprehensive identity forensics for the whole system, limiting its ability to adapt to equipment authentication difficulties. Furthermore, the development of trust imposed during their crosswise collaborations with suppliers and other manufacturers in the supply chain is poorly maintained. In this paper, a trust chain framework with a comprehensive identification mechanism is implemented for the designed MS system, which is based and created on the private blockchain in conjunction with decentralized database systems to boost the flexibility, traceability, and identification of the IC-module-system. Practical implementations are developed using a functional prototype. First, the decentralized application (DApp) and the smart contracts are proposed for constructing the new trust chain under the proposed comprehensive identification mechanism by using blockchain technology. In addition, the blockchain addresses of IC, module, and system are automatically registered to InterPlanetary File System (IPFS), individually. In addition, their corresponding hierarchical CID (content identifier) values are organized by using Merkle DAG (Directed Acyclic Graph), which is employed via the hierarchical content identifier mechanism (HCIDM) proposed in this paper. Based on insights obtained from this analysis, the trust chain based on HCIDM can be applied to any MS system, for example, this trust chain could be used to prevent the counterfeit modules and ICs employed in the monitoring system of a semiconductor factory environment. The evaluation results show that the proposed scheme could work in practice under the much lower costs, compared to the public blockchain, with a total cost of 0.002094 Ether. Finally, this research is developed an innovation trust chain mechanism that could be provided the system-level security for any MS toward Industrial 4.0 in order to meet the requirements of both manufacturing innovation and product innovation in Sustainable Development Goals (SDGs).

Blue fluorescent amino acid for biological spectroscopy and microscopy.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Microscopic nucleation and propagation rates of an alanine-based α-helix.

An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2788-2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants - a long-sought goal in the study of the helix-coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.

Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single-Molecule Spectroscopy.

Because of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 µs-1 at high pH to approximately 80 µs-1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

Reducing side effects of hiding sensitive itemsets in privacy preserving data mining.

Data mining is traditionally adopted to retrieve and analyze knowledge from large amounts of data. Private or confidential data may be sanitized or suppressed before it is shared or published in public. Privacy preserving data mining (PPDM) has thus become an important issue in recent years. The most general way of PPDM is to sanitize the database to hide the sensitive information. In this paper, a novel hiding-missing-artificial utility (HMAU) algorithm is proposed to hide sensitive itemsets through transaction deletion. The transaction with the maximal ratio of sensitive to nonsensitive one is thus selected to be entirely deleted. Three side effects of hiding failures, missing itemsets, and artificial itemsets are considered to evaluate whether the transactions are required to be deleted for hiding sensitive itemsets. Three weights are also assigned as the importance to three factors, which can be set according to the requirement of users. Experiments are then conducted to show the performance of the proposed algorithm in execution time, number of deleted transactions, and number of side effects.

Efficiently hiding sensitive itemsets with transaction deletion based on genetic algorithms.

Data mining is used to mine meaningful and useful information or knowledge from a very large database. Some secure or private information can be discovered by data mining techniques, thus resulting in an inherent risk of threats to privacy. Privacy-preserving data mining (PPDM) has thus arisen in recent years to sanitize the original database for hiding sensitive information, which can be concerned as an NP-hard problem in sanitization process. In this paper, a compact prelarge GA-based (cpGA2DT) algorithm to delete transactions for hiding sensitive itemsets is thus proposed. It solves the limitations of the evolutionary process by adopting both the compact GA-based (cGA) mechanism and the prelarge concept. A flexible fitness function with three adjustable weights is thus designed to find the appropriate transactions to be deleted in order to hide sensitive itemsets with minimal side effects of hiding failure, missing cost, and artificial cost. Experiments are conducted to show the performance of the proposed cpGA2DT algorithm compared to the simple GA-based (sGA2DT) algorithm and the greedy approach in terms of execution time and three side effects.

A GA-based approach to hide sensitive high utility itemsets.

A GA-based privacy preserving utility mining method is proposed to find appropriate transactions to be inserted into the database for hiding sensitive high utility itemsets. It maintains the low information loss while providing information to the data demanders and protects the high-risk information in the database. A flexible evaluation function with three factors is designed in the proposed approach to evaluate whether the processed transactions are required to be inserted. Three different weights are, respectively, assigned to the three factors according to users. Moreover, the downward closure property and the prelarge concept are adopted in the proposed approach to reduce the cost of rescanning database, thus speeding up the evaluation process of chromosomes.

Using VIPT-jump to distinguish between different folding mechanisms: application to BBL and a Trpzip.

Protein folding involves a large number of sequential molecular steps or conformational substates. Thus, experimental characterization of the underlying folding energy landscape for any given protein is difficult. Herein, we present a new method that can be used to determine the major characteristics of the folding energy landscape in question, e.g., to distinguish between activated and barrierless downhill folding scenarios. This method is based on the idea that the conformational relaxation kinetics of different folding mechanisms at a given final condition will show different dependences on the initial condition. We show, using both simulation and experiment, that it is possible to differentiate between disparate kinetic folding models by comparing temperature jump (T-jump) relaxation traces obtained with a fixed final temperature and varied initial temperatures, which effectively varies the initial potential (VIP) of the system of interest. We apply this method (hereafter refer to as VIPT-jump) to two model systems, tryptophan zipper (Trpzip)-2c and BBL, and our results show that BBL exhibits characteristics of barrierless downhill folding, whereas Trpzip-2c folding encounters a free energy barrier. In addition, using the T-jump data of BBL we are able to provide, via Langevin dynamics simulations, a realistic estimate of its conformational diffusion coefficient.

Video-Assisted Thoracoscopic Surgery in Community-Acquired Thoracic Empyema: Analysis of Risk Factors for Mortality.

Background: Thoracic empyema is a disease with high mortality and morbidity. Video-assisted thoracoscopic surgery (VATS) is recommended to treat advanced stage empyema. The purpose of this study was to explore risk factors associated with post-surgery mortality for community-acquired empyema. Patients and Methods: We retrospectively reviewed 440 patients who received VATS for community-acquired empyema, higher than stage 2, in a tertiary medical center in Taiwan. Patients' age, comorbidities, pleural effusion analysis, and post-surgery outcome were compiled. Cox regression model for survival was applied to identify risk factors of 90-day death after surgery. Results: Fifty-three patients (12.05%) had died within 90 days post-surgery. The risk factors of mortality were advanced age (hazard ratio [HR], 1.027; 95% confidence interval [CI], 1.001-1.052), chronic kidney disease (HR, 5.322; 95% CI, 2.635-10.746), cancer (HR, 6.038; 95% CI, 2.737-13.321), pleural effusion pH ≤7 (HR, 2.61; 95% CI, 1.344-5.069), pleural effusion protein ≤4 (HR, 2.021; 95% CI, 1.035-3.947), and late surgery (HR, 3.014; 95% CI, 1.595-5.696). The 90-day mortality in the early surgery group versus the late group was 6.85% versus 26.05%. The increased mortality risk from late surgery was observed in most subgroups, except for patients who were female, had chronic renal disease, and had coronary artery disease. Conclusions: Patients who are elderly, have chronic kidney disease, cancer history, low pleural effusion pH, low pleural effusion protein, and late surgery are associated with post-surgery mortality for community-acquired advanced empyema. Early VATS surgery for advanced empyema or treatment failure of chest tube drainage appears to beneficial and is recommended.

Photoisomerization of a maleonitrile-type salen Schiff base and its application in fine-tuning infinite coordination polymers.

Strategically designed salen ligand 2,3-bis[4-(di-p-tolylamino)-2-hydroxybenzylideneamino]maleonitrile (1), which has pronounced excited-state charge-transfer properties, shows a previously unrecognized form of photoisomerization. On electronic excitation (denoted by an asterisk), 1Z*-->1E isomerization takes place by rotation about the C2--C3 bond, which takes on single-bond character due to the charge-transfer reaction. The isomerization takes place nonadiabatically from the excited-state (1Z) to the ground-state (1E) potential-energy surface in the singlet manifold; 1Z and 1E are neither thermally inconvertible at ambient temperature (25-30 degrees C), nor does photoinduced reverse 1E*-->1Z (or 1Z*) isomerization occur. Isomers 1Z and 1E show very different coordination chemistry towards a Zn(II) precursor. More prominent coordination chemistry is evidenced by a derivative of 1 bearing a carboxyl group, namely, N,N'-dicyanoethenebis(salicylideneimine)dicarboxylic acid (2). Applying 2Z and its photoinduced isomer 2E as building blocks, we then demonstrate remarkable differences in morphology (sphere- and needlelike nanostructure, respectively) of their infinite coordination polymers with Zn(II).

32-Channel transmit beamformer with high timing resolution for high-frequency ultrasound imaging systems.

This paper presents a 32-channel high timing resolution transmit-beamforming circuit for use in high-frequency ultrasound imaging systems. Conventional transmit-beamforming circuits are typically implemented using field-programmable gate array (FPGA) chips. However, it is difficult for FPGAs to provide high timing resolution to meet the beamforming requirements of high-frequency ultrasound imaging systems. The proposed transmit-beamforming design can generate stable and suitable delays to excite 32-channel array transducer elements without variations in the process, voltage, and temperature. In addition, the proposed low-complexity architecture can maintain the duty cycle of long prorogation signals with low hardware cost to meet the timing requirements of a large channel number array transducer. The proposed designed transmit beamformer uses 0.18-µm CMOS technology for a 30-MHz high-frequency linear array, and the simulation results show that the proposed transmit-beamforming application-specific integrated circuit can achieve a maximum time delay of 619.5 ns with a time resolution of 617 ps.

Future trends and guidance for the triple bottom line and sustainability: a data driven bibliometric analysis.

This study conducts a comprehensive literature review of articles on the triple bottom line (TBL) published from January 1997 to September 2018 to provide significant insights and support to guide further discussion. There were three booms in TBL publications, occurring in 2003, 2011, and 2015, and many articles attempt to address the issue of sustainability by employing the TBL. This literature analysis includes 720, 132, and 58 articles from the Web of Science (WOS), Inspec, and Scopus databases, respectively, and reveals the gaps in existing research. To discover the barriers and points of overlap, these articles are categorized into six aspects of the TBL: economic, environmental, social, operations, technology, and engineering. Examining the top 3 journals in terms of published articles on each aspect reveals the research trends and gaps. The findings provide solid evidence confirming the argument that the TBL as currently defined is insufficient to cover the entire concept of sustainability. The social and engineering aspects still require more discussion to support the linkage of the TBL and to reinforce its theoretical basis. Additionally, to discover the gaps in the data sources, theories applied, methods adopted, and types of contributions, this article summarizes 82 highly cited articles covering each aspect. This article offers theoretical insights by identifying the top contributing countries, institutions, authors, keyword networks, and authorship networks to encourage scholars to push the current discussion further forward, and it provides practical insights to bridge the gap between theory and practice for enhancing the efficiency and effectiveness of improvements.

Dual excited-state intramolecular proton transfer reaction in 3-hydroxy-2-(pyridin-2-yl)-4H-chromen-4-one.

The synthesis, characterization and fundamental of the dual excited-state proton-transfer properties of 3-hydroxy-2-(pyridin-2-yl)-4H-chromen-4-one (1a) are reported. In the electronic ground state, there exist two competitive hydrogen bonding (HB) isomers for 1a. Conformer 1a(O) reveals a five-membered ring HB structure between O-H and carbonyl oxygen, while conformer 1a(N) possesses a six-membered ring HB formation between O-H and pyridyl nitrogen. In a single crystal, the X-ray crystallography unveils an exclusive formation of conformer 1a(N). In solution such as CH(2)Cl(2), 1a(O) and 1a(N) are in equilibrium, and their respective absorption chromophores are significantly different due to different degrees of hydrogen-bond induced pi electron delocalization. Upon excitation, both conformers 1a(O) and 1a(N) undergo excited-state intramolecular proton transfer (ESIPT) reaction. Following ESIPT, 1a(O) gives rise to a tautomer emission maximized at 534 nm in CH(2)Cl(2). Conversely, due to dominant radiationless quenching processes the tautomer emission for 1a(N) cannot be obtained with a steady-state manner but can be resolved from time-resolved fluorescence. Time resolved fluorescence estimates an equilibrium constant of 27 +/- 5 in favor of 1a(N) in CH(2)Cl(2). Ultrafast ESIPT also takes place for the unique 1a(N) form in the crystal. Due to the prohibition of quenching processes in the solid state, bright tautomer emission maximized at 540 nm is resolved for 1a(N) (Phi(f) approximately 0.3). The interplay between two HB conformers with on(1a(O))/off(1a(N)) character in tautomer emission may find future applications such as the recognition of organic Lewis acid/base in organic solvents.

Dual-cell structure digitally controlled oscillator with portability for clock generation applications.

This paper presents a dual-cell structure and cell-based design digitally controlled oscillator (DCO) for high-performance clock generation applications. The proposed dual-cell DCO not only can reduce the intrinsic delay to increase the maximum output frequency significantly but also extend the controllable range. In addition, the proposed digitally controlled delay cell uses the cascading structure to realize the wide output frequency range and high timing resolution at the same time. A DCO chip is fabricated in 0.18 µm CMOS technology, and the core area is 0.003 mm2. Measurement results show that operation range is from 169 MHz to 522 MHz and the finest delay resolution is 2.2 ps. Furthermore, because the proposed DCO is implemented with the digital standard cells, it is a portable design to migrate to different processes easily and reduce design time significantly.

New supramolecular isomers with 2D 4(4) square-grid and 3D 6(5).8 frameworks in a one-pot synthesis; reversible solvent uptake and intriguing luminescence properties.

Two supramolecular isomers of [Ni(4-bpd)(2)(NCS)(2)] (4-bpd = 1,4-bis(4-pyridyl)-2,3-diaza-1,3-butadiene) with 2D 4(4) square-grid and 3D 6(5).8 frameworks are co-crystallized in a one-pot reaction, both of which exhibit interesting luminescence properties and reversible adsorption-desorption with respect to guest solvents.

Thoracic chordoma: an unusual presentation of the spinal tumor.

Chordoma is an uncommon malignant bone tumor that originates from the remnants of the embryonic notochord. In most of the reported cases, chordomas occur at the skull base or in the sacrococcygeal spine but rarely in the thoracic spine. In this report, we describe a case of a large thoracic chordoma with posterior mediastinal extension in a 25-year-old woman who presented with left-hand anhydrosis. The imaging studies, pathology findings, and recent advances in treatment are discussed.

Enhancing immunogenicity of antigens through sustained intradermal delivery using chitosan microneedles with a patch-dissolvable design.

Reducing the dosage required for vaccination is highly desirable, particularly in cases of epidemic emergencies. This study evaluated the potential of a chitosan microneedle (MN) system with a patch-dissolvable design for low-dose immunization. This system comprises antigen-loaded chitosan MNs and a hydrophilic polyvinyl alcohol/polyvinyl pyrrolidone supporting array patch, which provides extra strength to achieve complete MN insertion and then quickly dissolves in the skin to reduce patch-induced skin irritation. After insertion, MNs could be directly implanted in the dermal layer as an intradermal (ID) depot to allow a sustained release of the model antigen ovalbumin (OVA) for up to 28 days. We found that rats immunized with MNs containing low-dose OVA (approximately 200 µg) had persistently high antibody levels for 18 weeks, which were significantly higher than those observed after an intramuscular injection of full-dose OVA (approximately 500 µg), demonstrating at least 2.5-fold dose sparing. Moreover, OVA-encapsulated chitosan MNs had superior immunogenicity to OVA plus chitosan solution, indicating that MN-based delivery and prolonged skin exposure can further enhance chitosan's adjuvanticity. Therefore, this patch-dissolvable MN system offers a needle-free, accurate, and reliable ID delivery of antigens and has potential as a sustained ID delivery device to improve vaccine efficacy and facilitate dose sparing with existing vaccines. STATEMENT OF SIGNIFICANCE: This study developed implantable chitosan microneedles (MNs) with a patch-dissolvable design for the sustained intradermal (ID) delivery of antigens and demonstrated their antigen dose-sparing potential. We found that rats immunized with chitosan MNs containing low-dose OVA had persistently high antibody levels for 18 weeks, which were significantly higher than those observed after an intramuscular injection of full-dose OVA, demonstrating at least 2.5-fold dose sparing. Our results indicate that chitosan MNs can not only serve as an efficient vaccine delivery system but also exert their promising adjuvant activity by forming an ID depot for prolonged antigen exposure and activating dendritic cells for promoting immune responses.

Oligomerization State of CXCL4 Chemokines Regulates G Protein-Coupled Receptor Activation.

CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first ß-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.