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Tau oligomers play critical roles in tau pathology and are responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization has become an important therapeutic strategy to treat tauopathies, including Alzheimer’s disease. However, progress has been slow because detecting tau oligomers in the cellular context is difficult. Working toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened libraries with FDA-approved and passed phase I drugs and identified levosimendan as a potent anti-tau agent that inhibits tau oligomerization. 14C-isotope labeling of levosimendan revealed that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan disassembles tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents methylene blue and LMTM failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued cognitive declines induced by tauopathy in the TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

Supplementary Figure Legends 1-7 from Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

mp3 file (6.8 MB). In the inaugural edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with Matthew Meyerson about his paper, which describes the identification of the DDR2 kinase as a therapeutic target in squamous cell lung cancer.

Supplementary Table 1 from Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

Supplementary Figures 1-7 from Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

Tau oligomers play critical roles in tau pathology, responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization becomes an important therapeutic strategy to treat tauopathies including Alzheimer’s disease. However, progress has been slow due to difficulties of detecting tau oligomers in cellular context. Toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened FDA-approved & Passed PhaseI drug library, and identified levosimendan as a potent anti-tau agent inhibiting tau oligomerization. 14C-isotope labeling of levosimendan identified that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan was able to disassemble tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents, methylene blue and LMTM, failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued tauopathy-induced cognitive declines in TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

Article 81, Paragraph 2 of the Trade Union And Labor Relations Adjustment Act stipulates compulsory membership provisions, such as the union shop agreement, as one of the unfair labor practices. However, union shop agreement entered into by a dominant trade union is considered to be valid as an exception. According to interpretation of the above clause, union shop agreement might apply to the act of a new employee who directly establishes a new trade union or joins another trade union without first joining and withdrawing from the dominant trade union. In this case the dispute was over the issue of whether the dismissal of a new employee based on the union shop agreement was an unfair dismissal or not, where the employee had joined another trade union without first joining and withdrawing from the dominant trade union. The trial court decided that the firing was not an unfair dismissal because the union shop agreement justified it. On the contrary, the appellate court decided that it was an unfair dismissal. The Supreme Court ruled that it was an unfair dismissal based on a limited interpretation where it was found that the union shop agreement can only be applied to a new employee who has not joined any trade union. It appears th Supreme Court’s decision is rational based on the reasons belows. 1) It is a global trend to limit the effectiveness of the union shop agreement and it also corresponds to the development of the history of trade unions. 2) It is necessary to protect employee’s right to work and right to choose the traded union by controlling employer’s dismissal. 3) Employer’s positive right to organize is guaranteed by the Constitution and union’s positive right to organize should be treated equally among plural trade unions. I hope that based on this ruling, the meaning of the right to unite, especially passive right to organize, will be contemplated and debated deeply. It is necessary, in my view, to regulate the union shop agreement as an unfair labor practice altogether, because passive right to organize should be protected equally to the positive right to organize.

Significance The ubiquitin–proteasome system and autophagy are two major intracellular proteolytic pathways, and both remove misfolded and proteotoxic proteins from eukaryotic cells. This study describes the detailed regulatory pathway of proteasome degradation by autophagy for its own quality control. We discovered that a portion of inhibited proteasomes is actively sequestered into the aggresome, an insoluble fraction of the mammalian cell. The aggresome functions as a triage point for proteasome recovery and autophagic degradation. This mainly distinguishes proteasome quality control in mammals from that in other organisms. STUB1/CHIP E3 Ub ligase has a critical role in targeting inhibited proteasomes into the aggresome. These results provide strong insights into protein catabolism in various pathological conditions originating from impaired proteasomes., The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.

This paper presents a new surface modification strategy to develop a poly(ethylene terephthalate) (PET)-based membrane having a hydrophilic surface, high nutrient ion permeability, sufficient mechanical strength, and organic fouling resistance, using an anthracene (ANT)-attached polyethylene glycol (PEG) surface modification agent (SMA) synthesized in this work. During the modification process, the ANT parts of the SMAs poke through and anchor to the surface of a commercial PET woven fabric via physical interactions and mechanical locking. The PEG chain parts coat the surface in the brush and arch forms, which generates a hydration layer on the fabric surface. The consequently obtained surface property and unique structure of the modified PET-based membrane result in higher nitrate ion permeability, organic fouling resistance, and microalgae production compared to those of the unmodified one. These are also affected by the molecular weight of the PEG and the number density of the anchored SMAs. The study demonstrates that this new surface modification method has the potential to allow the development of a desirable PET-based membrane for the efficient massive production of marine microalgae.

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.

Microalgal biomass productivity of semi-permeable membrane photobioreactors (SPM-PBRs) highly depends on diffusion of nutrient ions from surrounding environment through SPMs. The objective of this study was to enhance microalgal biomass productivity in floating photobioreactors (PBRs) with semi-permeable membranes (SPMs) grafted with 4-hydroxyphenethyl bromide (4-HPB) by increasing ion permeabilities and reducing biofouling of SPMs. In this study, we investigated the reduction of biofouling and improvement of biomass productivity by grafting 4-HPB onto SPMs which could decrease hydrophobicity. The degree of alkylation could be increased by up to 4.9%, with water contact angles on alkylated SPM surfaces decreased from 102.5° to 58.2°, indicating decreased surface hydrophobicity. Ion and water permeabilities of SPMs were increased in alkylated SPMs by 148% and 200%, respectively, compared to non-treated SPM control. Moreover, biomass productivity of PBRs with alkylated SPMs was 75% (740 mg m−2 day−1) higher than that of the control. Biofouling was also remarkably reduced by up to 40% in 4-HPB grafted SPMs after cultivation of microalgae. These results suggest that decreasing hydrophobicity of SPMs by grafting 4-HPB could enhance biomass productivity in SPM-PBRs through improving ion permeability and reducing biofouling.

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies., Multiple-hit model for tau aggregation, where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process.

Small molecule modulators of mitochondrial function have been attracted much attention in recent years due to their potential therapeutic applications for neurodegenerative diseases. The mitochondrial translocator protein (TSPO) is a promising target for such compounds, given its involvement in the formation of the mitochondrial permeability transition pore in response to mitochondrial stress. In this study, we performed a ligand-based pharmacophore design and virtual screening, and identified a potent hit compound, 7 (VH34) as a TSPO ligand. After validating its biological activity against amyloid-β (Aβ) induced mitochondrial dysfunction and in acute and transgenic Alzheimer’s disease (AD) model mice, we developed a library of analogs, and we found two most active compounds, 31 and 44, which restored the mitochondrial membrane potential, ATP production, and cell viability under Aβ-induced mitochondrial toxicity. These compounds recovered learning and memory function in acute AD model mice with improved pharmacokinetic properties.

Tau oligomers play critical roles in tau pathology, responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization becomes an important therapeutic strategy to treat tauopathies including Alzheimer’s disease, however progress has been slow due to difficulties of detecting tau oligomers in cellular context. Toward tau-targeted drug discovery, our group have developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened 1,018 compounds in FDA-approved & Passed Phase I drug library, and identified levosimendan as a potent anti-tau agent inhibiting tau oligomerization. 14C-isotope labeling of levosimendan identified that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan was able to disassemble tau oligomers into monomers, and rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents, methylene blue (MB) and LMTM, failed to protect neurons from tau-mediated toxicity, generating high-molecular weight tau oligomers. The administration of levosimendan also suppressed tau pathology in the brain, preventing cognitive declines in TauP301L-BiFC transgenic mice. Although careful validation is required, here we present the potential of levosimendan as a disease modifying therapy for tauopathies targeting tau oligomerization.

Overexpression of GRP78 in a variety of cancers such as glioblastoma, leukemia, lung, prostate, breast, gastric, and colon makes it a prime target for anticancer drug development. Present study reports GRP78-based design of novel anticancer agents using in-silico methods. As a first step toward the work, the interactions between GRP78 and 15 known ligands were modeled by docking simulation. The docked complex, GRP78-13, superior to other compounds with respect to its experimental activity and energy descriptors, was deduced into a structure-based pharmacophore. This hypothesis was applied as a screening filter to Asinex and Chemdiv databases. Finally, 23 hits were tested in vitro. Among these, VH1019 and VH1011 induced a concentration-dependent strong broad antiproliferative effect in glioma (U87-MG), breast cancer (MCF-7), and prostate cancer (DU-145) cell lines as compared to nontumorigenic control, neonatal foreskin fibroblast (HFF-1). These compounds showed preferential growth inhibition of cancer cells over normal cells. The acetohydrazide derivative VH1019 was identified as a potential new chemotype for GRP78 inhibitors with an IC50 of 12.7 μM in MCF-7.

Lipid extraction is a critical step in the downstream processing of biodiesel production from microalgae. Solvent extraction using mixtures of non-polar and polar solvents is one of the most well-known processes for this purpose. Hexane is the most common solvent of choice for large-scale lipid extractions due to its technical and economic advantages, especially its high selectivity toward lipids and low cost. In this study, extractions using mixtures of hexane and polar solvents were evaluated for their performance in order to develop a more efficient method for large-scale lipid extraction from microalgae. The combination of hexane and methanol resulted in the highest fatty acid methyl ester (FAME) yield for lipids from Tetraselmis sp. The effects of extraction conditions, including proportions of methanol to hexane, ratios of total solvent volume to dry biomass, and extraction time, on extraction yields were evaluated to determine optimum conditions providing higher lipid and FAME yields. The optimal conditions were as follows: proportion of hexane to methanol of 1:1, ratio of total solvent volume to dry biomass of 10 mL/g, and extraction time of 120 min. Finally, the selected solvent mixture and optimal conditions were applied to larger scale extraction experiments with scale-up factors of 10, 50, and 100. FAME yields of large-scale extractions were almost completely consistent with increasing scale-up factors. The results of this study suggest that a hexane and methanol mixture is a promising solvent for large-scale lipid extraction from microalgae.

In this study, hypersaline media were used for ocean cultivation of the marine microalga Tetraselmis sp. KCTC12432BP for enhanced biomass and fatty acid (FA) productivity. Hypersaline media (55, 80, and 105 PSU) were prepared without sterilization by addition of NaCl to seawater obtained from Incheon, Korea. The highest biomass productivity was obtained at 55 PSU (0.16 g L−1 day−1) followed by 80 PSU (0.15 g L−1 day−1). Although the specific growth rate of Tetraselmis decreased at salinities higher than 55 PSU, prevention of contamination led to higher biomass productivity at 80 PSU than at 30 PSU (0.03 g L−1 day−1). FA content of algal biomass increased as salinity increased to 80 PSU, above which it declined, and FA productivity was highest at 80 PSU. Ocean cultivation of Tetraselmis was performed using 50-L tubular module photobioreactors and 2.5-kL square basic ponds, closed- and open-type ocean culture systems, respectively. Culturing microalgae in hypersaline medium (80 PSU) improved biomass productivities by 89 and 152% in closed and open cultures, respectively, compared with cultures with regular salinity. FA productivity was greatly improved by 369% in the closed cultures. The efficacy of salinity shift and N-deficiency to enhance FA productivity was also investigated. Lowering salinity to 30 PSU with N-starvation following cultivation at 80 PSU improved FA productivity by 19% in comparison with single-stage culture without N-deficiency at 30 PSU. The results show that salinity manipulation could be an effective strategy to improve biomass and FA productivity in ocean cultivation of Tetraselmis sp.

Present work aimed to introduce non-peptidic ABAD loop D (LD) hot spots mimetics as ABAD-Aβ inhibitors. A full length atomistic model of ABAD-Aβ complex was built as a scaffold to launch the lead design and its topology later verified by crosschecking the computational mutagenesis results with that of in vitro data. Thereafter, the interactions of prime Aβ-binding LD residues- Tyr101, Thr108, and Thr110 were translated into specific pharmacophore features and this hypothesis subsequently used as a virtual screen query. ELISA-based screening of 20 hits identified two promising lead candidates, VC15 and VC19 with an IC50 of 4.4±0.3 and 9.6±0.1 μM respectively. They productively reversed Aβ-induced mitochondrial dysfunctions such as mitochondrial membrane potential loss (JC-1 assay), toxicity (MTT assay), and ATP reduction (ATP assay) in addition to increased cell viabilities. This is the first reporting of LD hot spots-centric in silico scheme to discover novel compounds with promising ABAD-Aβ inhibitory potential. These chemotypes are proposed for further structural optimization to derive novel Alzheimer's disease (AD) therapeutics. This article is protected by copyright. All rights reserved.

Enhanced cell migration in the course of wound healing is required to repair damaged skin. We investigated the effects of rice callus extracts on the migration of skin keratinocytes. Rice callus extracts were obtained by using three different methods: pressurized hot water, crude ethanol, and liquid-liquid extractions. The extract obtained by using crude ethanol extraction was more effective in the migration of skin cells than that obtained by pressurized hot water extraction (PHWE). The crude ethanol extract (CEE) was further partitioned by performing liquid-liquid extraction. As phenolics are inhibitory compounds affecting cell migration, we analyzed the total phenolic content of the rice callus extracts. The level of phenolics in the n-hexane partitioned extract (n-HPE) of CEE was lower than that in all other partitioned extracts. The n-HPE was most effective in enhancing cell migration. We analyzed wound healingrelated factors including platelet derived growth factor B (PDGF-B), transforming growth factor beta 1 (TGF-β1), heparin-binding epidermal growth factor (HB-EGF), and fibroblast growth factor 2 (FGF-2) after the treatment of n-HPE. Most of the expressions of cell migration-related growth factors increased, but HB-EGF dramatically increased (6.5-fold) in keratinocytes treated with n-HPE. The results indicate that n-HPE contains more stimulating growth factors or proteins and less cell migration inhibiting factors than the other tested extracts; thus, n-HPE treatment produced the greatest enhancement of cell migration.

Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by memory loss and cognitive impairment. As this disease is becoming a serious global health issue, development of disease modifying therapeutics is urgently required. AD is characterized by deposits of two protein, amyloid β and tau. Although amyloid β-based therapeutics have been extensively investigated so far, tau has also received great attention as one of promising molecular targets for AD. In this review, a variety of tau-directed strategies to rescue tau-mediated neurotoxicity will be reviewed especially focusing on small molecules. Subsequently, recent patents published from 2014 to 2018 that integrate efforts to develop tau-directed small molecules for the treatment of AD will be reviewed.

Objective: The aim of this study was to compare the effectiveness of five intracanal agitation techniques on the penetration of irrigant and sealer into dentinal tubules. Background data: Intracanal agitation techniques could promote chemomechanical debridement and the sealing of root canals during endodontic treatment. However, there is limited evidence for the agitation effect of Nd:YAP laser. Materials and methods: Human maxillary premolars with single straight canals (N = 60) were prepared with ProTaper Next® NiTi rotary files (Dentsply Maillefer) up to X4 (ISO 40 size). Rhodamine B-labeled sodium hypochlorite was used for final irrigation together with the conventional syringe (control), sonic, ultrasonic, Nd:YAP laser, or V-Clean™ endodontic agitation system. All canals were obturated with gutta-percha and fluorescein isothiocyanate-labeled AH Plus sealer. Transverse sections were obtained at 2, 5, and 8 mm from the apex and observed under confocal laser scanning microscopy. Maximum penetration depth and penetration percentage of both irrigant and sealer were recorded. Kruskal-Wallis and Mann-Whitney tests were performed for multiple comparisons. The Spearman coefficient was calculated to confirm correlations between irrigant and sealer penetration. Results: Laser agitation attained the most irrigant and sealer penetration depth and penetration percentage (p < 0.05). Sonic and ultrasonic agitation attained significantly more penetration percentage than the control group at 2 mm from the apex (p < 0.05). Patterns of irrigant and sealer penetration correlated significantly for all agitation techniques (p < 0.001). Conclusions: Nd:YAP laser was superior to other agitation techniques in dentinal tubule penetration of irrigant and sealer at one or more sectioned levels from the apex.

Ras proteins are members of a large family of GTPase enzymes that are commonly mutated in cancer where they act as dominant oncogenes. We previously developed an irreversible guanosine-derived inhibitor, SML-8-73-1, of mutant G12C RAS that forms a covalent bond with cysteine 12. Here we report exploration of the structure–activity relationships (SAR) of hydrolytically stable analogues of SML-8-73-1 as covalent G12C KRAS inhibitors. We report the discovery of difluoromethylene bisphosphonate analogues such as compound 11, which, despite exhibiting reduced efficiency as covalent G12C KRAS inhibitors, remove the liability of the hydrolytic instability of the diphosphate moiety present in SML-8-73-1 and provide the foundation for development of prodrugs to facilitate cellular uptake. The SAR and crystallographic results reaffirm the exquisite molecular recognition that exists in the diphosphate region of RAS for guanosine nucleotides which must be considered in the design of nucleotide-competitive inhibitors.

Deregulation of EGFR is involved in the development of many cancers. The inhibition of EGFR kinase activity has been clinically validated as a promising approach for the treatment of non-small-cell lung cancer (NSCLC). However, all NSCLC patients who initially benefited from first-generation EGFR inhibitors eventually develop drug resistance. A point mutation at the gatekeeper position, T790M in EGFR kinase domain accounts for more than 50% of acquired resistance. Therefore, second- and third-generation EGFR inhibitors have been developed to overcome the resistance conferred by the gatekeeper mutation. This review has highlighted recent advances in overcoming acquired resistance for the development of each generation of EGFR inhibitors along with their potential issues, and urgent quest for the development of new generation of EGFR inhibitors.

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Cav3.1 and Cav3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.