Novel 12N-substituted matrinanes as potential anti-coxsackievirus agents

A series of novel 12N-substituted matrinane derivatives were synthesized and evaluated for their activities against coxsackievirus type B3 (CVB3) taking compound 1 as the lead. SAR analysis indicated that the introduction of a suitable heteroaromatic ring on the 12N-atom might be beneficial for the activity. Among them, compound 8a exhibited the highest potency against all CVB serotypes as well as CVA16 with IC₅₀ values ranging from 2.02 μM to 7.41 μM, indicating a broad-spectrum anti-coxsackieviruse effect. Furthermore, compound 8a demonstrated a good safety profile in vivo. Thus, we consider 12N-substituted matrinanes to be a promising family of anti-coxsackievirus agents, and compound 8a to be a promising drug candidate in the treatment of various diseases related to coxsackievirus infection.     

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.     

Locally Addressable Electrochemical Patterning Technique (LAEPT) applied to poly(L-lysine)-graft-poly(ethylene glycol) adlayers on titanium and silicon oxide surfaces

The protein-resistant polycationic graft polymer, poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), was uniformly adsorbed onto a homogenous titanium surface and subsequently subjected to a direct current (dc) voltage. Under the influence of an ascending cathodic and anodic potential, there was a steady and gradual loss of PLL-g-PEG from the conductive titanium surface while no desorption was observed on the insulating silicon oxide substrates. We have implemented this difference in the electrochemical response of PLL-g-PEG on conductive titanium and insulating silicon oxide regions as a biosensing platform for the controlled surface functionalization of the titanium areas while maintaining a protein-resistant background on the silicon oxide regions. A silicon-based substrate was micropatterned into alternating stripes of conductive titanium and insulating silicon oxide with subsequent PLL-g-PEG adsorption onto its surfaces. The surface modified substrate was then subjected to +1800 mV (referenced to the silver electrode). It was observed that the potentiostatic action removed the PLL-g-PEG from the titanium stripes without inducing any polyelectrolyte loss from the silicon oxide regions. Time-of-flight secondary ions mass spectroscopy and fluorescence microscopy qualitatively confirmed the PLL-g-PEG retention on the silicon oxide stripes and its absence on the titanium region. This method, known as "Locally Addressable Electrochemical Patterning Technique" (LAEPT), offers great prospects for biomedical and biosensing applications. In an attempt to elucidate the desorption mechanism of PLL-g-PEG in the presence of an electric field on titanium surface, we have conducted electrochemical impedance spectroscopy experiments on bare titanium substrates. The results showed that electrochemical transformations occurred within the titanium oxide layer; its impedance and polarization resistance were found to decrease steadily upon both cathodic and anodic polarization resulting in the polyelectrolyte desorption from the titanium surface.     

Nanoparticles facilitate gene delivery to microorganisms via an electrospray process

In this study, we developed a technique for delivering genes to microorganisms via electrospray of gold nanoparticles. During the electrospray process, charged monodisperse nano-droplets (a mixture of pET30a-GFP plasmid and nano-sized gold particles) were accelerated and deposited on a thin layer of non-competent Escherichia coli cells. Via antibiotic selection, transformed cells containing green fluorescent protein appeared on the agar plates. PCR amplification and restriction enzyme analysis further confirmed that pET30a-GFP plasmid had successfully been delivered into the non-competent E. coli cells. The transformation efficiencies were optimized under different electrospray conditions. Among several electrospray buffer solutions, CaCl₂ (0.01 M) was found to be the best for gene delivery. Furthermore, gold nanoparticles (NPs, 50 nm diameter) significantly improved plasmid transformation efficiency by 5- 7 fold (up to 2 × 10⁶ CFU/μg plasmid) compared with that obtained using naked plasmid. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmids remained unchanged during the electrospray process, but cellular membrane integrity was reduced after being electrosprayed with gold NPs and CaCl₂ buffer solutions. This gene delivery method has the potential to work for many other microorganisms.     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

Proteomic screening of anaerobically regulated promoters from Salmonella and its antitumor applications

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.     

TAK1 regulates SCF expression to modulate PKBα activity that protects keratinocytes from ROS-induced apoptosis

Dysregulated reactive oxygen species (ROS) generation contributes to many human pathologies, including cancer and diabetes. During normal wound repair, inflammation-induced ROS production must be tightly controlled, but the mechanisms reining their generation remain unclear. Herein, we show that transforming growth factor β-activated kinase 1 (TAK1) directly regulates stem cell factor (SCF) expression, which activates the protein kinase B (PKB)α pro-survival pathway in a cell-autonomous manner to protect keratinocytes from ROS-mediated cell death. TAK1 is a pivotal inflammatory mediator whose expression was transiently elevated during wound healing, paralleling the ROS production profile. TAK1 deficiency in keratinocytes led to increased apoptosis in response to anoikis and TNF-α treatment and was associated with elevated ROS level as analyzed by FACS. Using organotypic skin co-culture and comparative growth factor array analysis, we revealed a cell-autonomous mechanism that involved the SCF/c-Kit/PKBα signaling cascade. Ectopic expression of TAK1 or treatment with exogenous recombinant SCF restored the increased ROS production and apoptotic cell death in TAK1-deficient keratinocytes. Conversely, normal keratinocytes treated with various inhibitors targeting the SCF/c-Kit/PKBα pathway exhibited increased ROS production and TNF-α- or anoikis-induced apoptosis. Our study reveals a novel anti-apoptotic role for SCF in keratinocytes and identifies TAK1 as a novel player uniting inflammation and ROS regulation in skin redox biology.