Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of Gram negative and some Gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.     

Is low dose inhaled corticosteroid therapy as effective for inflammation and remodeling in asthma? A randomized,parallel group study

Background

While most of the clinical benefits of inhaled corticosteroid (ICS) therapy may occur at low doses, results of dose-ranging studies are inconsistent. Although symptom/lung function response to low and high dose ICS medication is comparable, it is uncertain whether low dose ICSs are as effective as high dose in the treatment of inflammation and remodeling.

Methods

22 mild or moderate asthmatic adult subjects (corticosteroid free for > 2 months) participated in a randomized, parallel group study to compare effects of fluticasone propionate (FP) 200 mcg/day and 1000 mcg/day. Alveolar macrophage (AM)-derived cytokines and basement membrane thickness (BMT) were measured at baseline and after 7 weeks treatment while symptoms, spirometry, exhaled nitric oxide (eNO) and airway hyperresponsiveness (AHR) to mannitol at baseline and 6 weeks.

Results

FP improved spirometry, eNO, symptoms and AHR with no difference between low and high dose FP. Both high and low dose FP reduced GM-CSF, TNF-alpha and IL-1ra, with no change in BMT and with no differences between low and high dose FP.

Conclusions

200 μg/day of FP was as effective as 1000 μg/day in improving asthma control, airway inflammation, lung function and AHR in adults in the short term. Future studies should examine potential differential effects between low and high dose combination therapy (ICS/long acting beta agonist) on inflammation and airway remodeling over longer treatment periods.     

Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA-protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.     

Substrate Inhibition Competes with Halide Inhibition in Polyphenol Oxidase

Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis?CMenten kinetics at catechol concentrations of up to 1?mM, it slowly decreased at catechol concentrations above 2?mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.     

Sterically Stabilized Phospholipid Micelles Reduce Activity of a Candidate Antimicrobial Wound Healing Adjunct

KSLW is an antimicrobial decapeptide, presumed to associate with micelles. Linear polymeric chains of hydrophobic phospholipids tend to form micelles, spontaneously, and function as efficient drug-stabilizing delivery systems. Our goal was to examine whether association of a cationic decapeptide with sterically stabilized nanomicelles (SSMs), improves stability and antimicrobial effect in vivo, using an impaired healing model. KSLW solutions were prepared in either saline or 12?mM SSM. Bilateral circular excisional wounds were created on the backs of SKH-1 mice followed by intradermal delivery of peptide solutions. Bacterial assays were conducted to assess bioactivity of KSLW in different formulations. Fluorescence analyses demonstrated an optimum lipid:peptide ratio for loading KSLW in PEGylated phospholipid micelles to be 15:1. Stressed animals treated with KSLW?CSSM preparations demonstrated no differences in microbial load at post-operative time points. In vitro assays against Staphylococcus epidermidis confirmed diminished activity of KSLW in SSM solution. The loss of KSLW antimicrobial activity may be based on electrostatic interactions with the anionic surface of SSM, which interfere with the peptide??s interaction with bacterial membranes. This study emphasizes the importance of antimicrobial peptide charge, size, and bioactivity, when designing delivery systems for wound healing agents.     

Studies on the characteristics of drug-loaded gelatin nanoparticles prepared by nanoprecipitation

The morphology of gelatin nanoparticles loaded with three different drugs (Tizanidine hydrochloride, Gatifloxacin and Fluconazole) and their characteristics of entrapment and release from gelatin nanoparticles were investigated by the analysis on nanoparticle size distribution, SEM and FT-IR in this study. The particles were prepared by nanoprecipitation using water and ethanol as a solvent and a nonsolvent, respectively. The exclusion of a crosslinking agent from the procedure led the system to have an irregularly-shaped morphology. Nonetheless, the uncrosslinked case of Gatifloxacin loading generally led to a more homogeneous population of nanoparticles than the uncrosslinked case of Tizanidine hydrochloride loading. No loading was achieved in the case of Fluconazole, whereas both Tizanidine hydrochloride and Gatifloxacin are observed of being capable of being loaded by nanoprecipitation. Tizanidine hydrochloride-loaded, blank and Gatifloxacin-loaded nanoparticles yielded, under crosslinked condition, 59.3, 23.1 and 10.6% of the used dried mass. The crosslinked Tizanidine hydrochloride-loaded particles showed the loading efficiency of 13.8%, which was decreased to 1.1% without crosslinking. A crosslinker such as glutaraldehyde is indispensable to enhance the Tizanidine hydrochloride-loading efficiency. To the contrary, the Gatifloxacin-loading efficiency for crosslinked ones was lower by a factor of 2-3 times than that for uncrosslinked ones. This is due to the carboxylic groups of Gatifloxacin and the aldehyde groups of glutaraldehyde competing with each other during the crosslinking process, to react with the amino groups of gelatin molecules. The loading efficiency of gelatin nanoparticles reported by other investigators greatly varies. Nevertheless, the loading efficiency reported by us is in good agreement with the drug-loading data of gelatin nanoparticles reported by other investigators. The 80% of loaded Tizanidine hydrochloride was released around 15 h after start-up of the release experiment, while the 20% of loaded Gatifloxacin was released more rapidly, as free Gatifloxacin, than the loaded Tizanidine hydrochloride and it showed the trend of sustained slow release during the remaining period of its release experiment. Furthermore, the result of comparative FT-IR analysis is consistent to that of the corresponding drug release study.