Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

Antioxidant and anti-inflammatory activities of lycopene against 5-fluorouracil-induced cytotoxicity in Caco2 cells

5-fluorouracil (5FU) is widely used to treat colorectal cancer (CC) and its main mechanisms of anticancer action are through generation of ROS which often result in inflammation. Here, we test the effect of Lycopene against 5FU in Caco2 cell line. Caco2 cells were exposed to 3 µg/ml of 5FU alone or with 60, 90, 120 µg/ml of lycopene. This was followed by assessment of cytotoxicity, oxidative stress, and gene expression of inflammatory genes. Our findings showed that Lycopene and 5FU co-exposure induced dose-dependent cytotoxic effect without compromising the membrane integrity based on the LDH assay. Lycopene also significantly enhanced 5FU-induced SOD activity and GSH level compared to control for all mixture concentrations (p < 0.01). Lycopene alone and combination with 5FU-induced expression of IL-1β, TNF-α, and IL-6. Furthermore, IFN-γ expression was significantly enhanced by only mixture of lycopene (90 µg/ml) and 5FU (p < 0.05). In conclusion, Lycopene supplementation with 5FU therapy resulted in improvement in antioxidant parameters such as catalase and GSH levels giving the cell capacity to cope with 5FU-mediated oxidative stress. Lycopene also enhanced IFN-γ expression in the presence of 5FU, which may activate antitumor effects further enhancing the cancer killing effect of 5FU.     

The relationships among monocyte subsets,miRNAs and inflammatory cytokines in patients with acute myocardial infarction

Background

Acute myocardial infarction (AMI) causes irreversible myocardial damage and release of inflammatory mediators, including cytokines, chemokines and miRNAs. We aimed to investigate changes in the levels of cytokines (IL-6, TNF-α and IL-10), miRNAs profiles (miR-146 and miR-155) and distribution of different monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) in the acute and post-healing phases of AMI.

NADPH oxidase inhibition rescues keratinocytes from elevated oxidative stress in a 2D atopic dermatitis and psoriasis model

Emerging evidence suggests oxidative stress plays a role in the pathophysiology of both atopic dermatitis (AD) and psoriasis (PSO). We established in vitro models of AD and PSO skin, and characterized these models in regard to their oxidative stress state. Both AD and PSO model keratinocytes exhibited elevated reactive oxygen species (ROS) levels and accumulated more DNA damage than control cells after oxidative stress induced by 250 µmol/L H₂O₂. Elevated ROS levels and DNA damage accumulation could be inhibited by the NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI). Further, immunofluorescence analysis revealed the presence of both NOX1 and NOX4 in keratinocytes. By inhibiting NOX1, stress-related signalling cascades and elevated ROS levels could be abrogated, and survival of AD and PSO cells improved. Taken together, this study reveals that inhibition of NOX inhibition could abrogate elevated oxidative stress in a 2D model of AD and PSO.     

Enhanced generation of reactive oxygen species and photocatalytic activity by Pt-based metallic nanostructures: the composition matters

The modification of semiconductor nanostructures with metallic nanocomponents can promote the separation of electron/hole from photoexited semiconductors by forming heterojunctions, thus exhibit enhanced photocatalytic activities and potential applications. In this study, Pt-based NPs, including Pt, PtCu, and PtCuCo are employed as model co-catalysts to comparatively study their capability to enhance the photocatalytic activity of TiO₂ nanosheets. It was found that each of Pt, PtCu, and PtCuCo can greatly enhance the photocatalytic activity of TiO₂ toward degradation of organic dyes. Using electron spin resonance spectroscopy, we demonstrated that deposition of Pt-based NPs resulted in more production of reactive oxygen species including hydroxyl radicals, superoxide, and singlet oxygen. The enhancing effects of Pt-based NPs on generation of ROS and photocatalytic activity showed same trend: PtCuCo?>?PtCu?>?Pt. The mechanism underlying the enhancement differences in Pt-based NPs may be mainly related to electronic structure change of Pt in alloying with Cu and Co. These results are valuable for designing hybrid nanomaterials with high photocatalytic efficiency for applications in water purification and antibacterial products.     

Rescue of the Cold Preserved Rat Liver by Hypothermic Oxygenated Machine Perfusion

The aim of the study was to investigate whether hypothermic oxygenated liver perfusion after cold liver preservation resuscitated metabolic parameters and whether this treatment had a benefit for liver viability upon reperfusion.
We preserved rat livers either by cold storage (UW) for 10 h, or by perfusion for 3 h (oxygenated modified UW) after 10 h cold storage. We assessed viability of livers after preservation and after ischemic rewarming + normothermic reperfusion ex vivo . Ten hour cold storage reduced mitochondrial cytochrome oxidase and metabolically depleted the livers. Oxygenated perfusion after cold storage resulted in uploaded cellular energy charge and oxidized mitochondrial cytochrome oxidase. Reperfusion after 10 h cold storage increased formation of superoxid anions, release of cytosolic LDH, lipid peroxidation, caspase activities and led to disruption of sinusoidal endothelial cells. In contrast, reperfusion after 10 h cold storage + 3 h hypothermic oxygenated perfusion resulted in no changes of lipid peroxidation, bile flow, energy charge, total glutathione, LDH release and of caspase activation, as compared to fresh resected livers.
This study demonstrates, that a metabolically depleted liver due to cold storage can be energy recharged by short-termed cold machine perfusion. The machine perfused graft exhibited improved viability and functional integrity.     

人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。     

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.