Hsing-Yu Hsu

Bacteria detection utilizing electrical conductivity

Real-time and specific detection of single bacterium remains a fundamental challenge and draws very much attention. Using test patterns composed of interdigitated Au-electrode arrays modified with antibody, the specific and quantitative detection of the electrical conductivity of a single Escherichia coli (E. coli, JM109) has been carried out in this work. The key is to ensure low background current of the antibody-modified test patterns before bacteria detection (<0.7pA in this case) and minimize the residual moisture or hydration after E. coli immobilization, such as via the use of 1-min bake at 50 degrees C prior to electrical measurement. This method holds great potential for future application in the real-time, specific, and quantitative bacterium detection down to a single bacterium cell.

c-Jun-mediated anticancer mechanisms of tylophorine

Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb Tylophora indica. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein 1 (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun N-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation activating transcription factor (ATF) site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: (i) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus c-Jun protein translation. To the best of our knowledge, this report is the first to demonstrate the involvement of c-Jun in the anticancer activity of tylophorine and the release of c-Jun translation from a global translational blockade via the PI3K_PDK1_eEF2 signaling cascade.

Synthesis and Biological Evaluation of Tylophorine-Derived Dibenzoquinolines as Orally Active Agents-Exploration of the Role of Tylophorine E Ring on Biological Activity.

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.

Isolation and biological activities of phenanthroindolizidine and septicine alkaloids from the Formosan Tylophora ovata.

An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC₅₀ values ranging from 84 nM to 20.6 μM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI₅₀ values ranging from 4 nM to 24.2 μM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure–activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5–9).

Cytotoxic cardenolide glycosides from the root of Reevesia formosana.

Bioassay-guided fractionation of the root tissue of Reevesia formosana led to isolation of 13 cardenolide glycosides, reevesiosides A-I and epi-reevesiosides F-I. Their structures were determined by means of spectroscopic analysis and single-crystal X-ray diffraction was performed using reevesioside A. Reevesioside A, reevesioside F, and epi-reevesioside F displayed especially potent cytotoxicity against the MCF-7 and NCI-H460 cancer cell lines, with IC(50) values of 63±2 and 19±1, 72±8 and 20±0, and 34±6 and 10±1 nM, respectively. Identification of the sugar constituents and unusual 18,20-epoxide cardenolide glycosides are described herein. Cardiac glycosides were previously unknown in the Sterculiaceae family.

Identification of anti-viral activity of the cardenolides, Na+/K+-ATPase inhibitors, against porcine transmissible gastroenteritis virus

ABSTRACT A series of naturally occurring cardenolides that exhibit potent anti‐transmissible gastroenteritis virus (TGEV) activity in swine testicular (ST) cells has been identified. In an immunofluorescence assay, these cardenolides were found to diminish the expressions of TGEV nucleocapsid and spike protein, which was used as an indication for viral replication; block TGEV infection induced apoptosis and cytopathic effects; and impart the same trend of inhibitory activity against Na+/K+‐ATPase as for anti‐TGEV activity. The viral titer inhibition was found to take place in a dose‐dependent manner. Knocking down expression of Na+/K+‐ATPase, the cellular receptor of cardenolides, in ST cells was found to significantly impair the susceptibility of ST cells to TGEV infectivity. Thus, we have identified Na+/K+‐ATPase as an anti‐viral drug target and its antagonists, cardenolides, a novel class of anti‐ TGEV agents. Graphical abstract Figure. No Caption available. HighlightsCardenolides were identified as a novel class of anti‐TGEV agents.Cardenolides diminished TGEV replication/viral titers in a dose dependent manner.Cardenolides blocked TGEV infection induced apoptosis and cytopathic effect.These cardenolides imparted the same trend of inhibitory activity for Na+/K+‐ATPase.Na+/K+‐ATPase was identified as an anti‐viral drug target.

Design, Synthesis, and Evaluation of Thiazolidine-2,4-dione Derivatives as a Novel Class of Glutaminase Inhibitors

Humans have two glutaminase genes, GLS (GLS1) and GLS2, each of which has two alternative transcripts: the kidney isoform (KGA) and glutaminase C (GAC) for GLS, and the liver isoform (LGA) and glutaminase B (GAB) for GLS2. Initial hit compound (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (2), a thiazolidine-2,4-dione, was obtained from a high throughput screening of 40 000 compounds against KGA. Subsequently, a series of thiazolidine-2,4-dione derivatives was synthesized. Most of these were found to inhibit KGA and GAC with comparable activities, were less potent inhibitors of GAB, and were moderately selective for GLS1 over GLS2. The relationships between chemical structure, activity, and selectivity were investigated. The lead compounds obtained were found to (1) offer in vitro cellular activities for inhibiting cell growth, clonogenicity, and cellular glutamate production, (2) exhibit high concentrations of exposure in plasma by a pharmacokinetic study, and (3) reduce the tumor size of xenografted human pancreatic AsPC-1 carcinoma cells in mice.

Targeting Coronaviral Replication and Cellular JAK2 Mediated Dominant NF-κB Activation for Comprehensive and Ultimate Inhibition of Coronaviral Activity

Tylophorine-based compounds exert broad spectral, potent inhibition of coronaviruses. NF-κB activation is a common pro-inflammatory response of host cells to viral infection. The aims of this study were to (i) find an effective combination treatment for coronaviral infections through targeting of the virus per se and cellular NF-κB activity; and (ii) to study the underling mechanisms. We found that tylophorine-based compounds target the TGEV viral RNA and effectively inhibit TGEV replication. NF-κB inhibition also leads to anti-TGEV replication. NF-κB activation induced by TGEV infection was found to be associated with two convergent pathways, IKK-2_IκBα/p65 and JAK2 mediated p65 phosphorylation, in swine testicular cells. JAK2 inhibition either by CYT387 (a JAK family inhibitor) or by silencing JAK2-expression revealed a dominant JAK2 mediated p65 phosphorylation pathway for NF-κB activation and resulted in NF-κB inhibition, which overrode the IκBα regulation via the IKK-2. Finally, tylophorine-based compounds work cooperatively with CYT387 to impart comprehensive anti-TGEV activities. The combination treatment, wherein a tylophorine compound targets TGEV and a JAK2 inhibitor blocks the alternative dominant NF-κB activation mediated by JAK2, is more effective and comprehensive than either one alone and constitutes a feasible approach for the treatment of SARS-CoV or MERS-CoV.

The cardenolide ouabain suppresses coronaviral replication via augmenting a Na+/K+-ATPase-dependent PI3K_PDK1 axis signaling

&NA; Cardenolides are plant‐derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti‐viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti‐transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na+/K+‐ATPase. Herein, we further explore the potential signaling cascades associated with this anti‐TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV‐induced production of IL‐6 in a dose dependent manner, with 50% inhibitory concentrations of 37 nM and 23 nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV‐infected ST cells, and ouabain imparted a degree of anti‐TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na+/K+‐ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti‐viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti‐viral applications for the cardenolides in the future. Graphical abstract Figure. No caption available. HighlightsOuabain eliminated TGEV titers and inhibited viral replication.Ouabain diminished TGEV induced IL‐6 production.Ouabain enhanced PI3K or PDK1 activation induced by TGEV via Na+/K+‐ATPase.PI3K or PDK1 inhibition antagonized the anti‐TGEV activity of ouabain.Ouabain augmented the PI3K_PDK1 axis signaling that inhibited TGEV activity.