The possible protective effect of L-arginine against 5-fluorouracil-induced nephrotoxicity in male albino rats

5-fluorouracil (5-FU) is a potent antineoplastic agent used for the treatment of various malignancies. The L-arginine nitric oxide (NO) pathway involved in the pathogenesis of chemotherapy induced kidney damage. This work investigated the beneficial mechanism of L-arginine supplementation in 5-FU induced nephropathy. Eighty male Wistar rats were divided into four equal groups: control group; L-arginine group (378 mg/rat/day for 4 weeks); 5-FU group (189 mg/rat/week for 4 weeks) and L-arginine for 1 week before and 4 weeks concomitant with 5-FU group. At the end of experiment, the kidney functions were assessed and kidneys specimens were processed for paraffin sections and stained with haematoxylin and eosin (H&E), Masson’s trichome (MT) and periodic acid-Schiff (PAS) stains. Other sections were processed for immunohistochemical demonstration of caspase-3 and inducible NO synthase (iNOS). Image analyser was used to analyse the results morphometrically and statistically. L-arginine administration to 5-FU treated animals elicited significant reduction in serum urea and creatinine levels, urine volume, urinary protein excretion and kidney/body weight ratio in comparison to fluorouracil treated group. L-arginine improved glomeruloscelerosis, degeneration of convoluted tubules and interstitial fibrosis in 5-FU treated animals. L-arginine attenuated effectively some biochemical and histological changes in 5-FU nephrotoxicity

Design, synthesis, and biological investigation of oxadiazolyl, thiadiazolyl, and pyrimidinyl linked antipyrine derivatives as potential non-acidic anti-inflammatory agents

A novel series of 12 antipyrine derivatives containing 1,3,4-oxadiazoles (4a-d), 1,3,4-thiadiazoles (6a-d), and pyrimidines (8a-d), was preparedand assessed for its potential in vitro COX-2 inhibitors. Compared to Celecoxib, compounds 4b-d and 8d were the most potent derivatives c with a half-maximal inhibitory concentration range of 53-69 nM. Considering COX-2 selectivity index, compounds 4 b and 4c were chosen among these most potent derivatives for further investigation. The in vivo ability of compounds 4 b and 4c to counteract carrageenan-induced paw edoema has been assessed and their potential underlying mechanisms have been elucidated and the results have been further validated using molecular docking simulations

Biomarkers of apoptosis

Within the era of molecularly targeted anticancer agents, it has become increasingly important to provide proof of mechanism as early on as possible in the drug development cycle, especially in the clinic. Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. Thus, the present minireview focuses on a discussion of the pros and cons of a variety of methodological approaches to detect different components of the apoptotic cascade as potential biomarkers of programmed cell death. The bulk of the discussion centres on serological assays utilising the technique of ELISA, since here there is an obvious advantage of sampling multiple time points. Potential biomarkers of apoptosis including circulating tumour cells, cytokeratins and DNA nucleosomes are discussed at length. However, accepting that a single biomarker may not have the power to predict proof of concept and patient outcome, it is clear that in the future more emphasis will be placed on technologies that can analyse panels of biomarkers in small volumes of samples. To this end the increased throughput afforded by multiplex ELISA technologies is discussed

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

BACKGROUND: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success. METHODOLOGY/PRINCIPAL FINDINGS: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally. CONCLUSIONS/SIGNIFICANCE: The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network

In Vitro and In Vivo Anti-Inflammatory Activity of 17-O-Acetylacuminolide through the Inhibition of Cytokines, NF-κB Translocation and IKKβ Activity

BACKGROUND AND PURPOSE: 17-O-acetylacuminolide (AA), a diterpenoid labdane, was isolated for the first time from the plant species Neouvaria foetida. The anti-inflammatory effects of this compound were studied both in vitro and in vivo. EXPERIMENTAL APPROACH: Plant extracts were initially tested against LPS-stimulated release of tumor necrosis factor alpha (TNF-α) from murine macrophages (RAW264.7 cells). Based on bioassay-guided fractionation, the active compound was identified as AA. AA was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. The inhibition of a panel of inflammatory cytokines (TNF, IL-1β, IL-6, KC, and GM-CSF) by AA was assessed at the expression and the mRNA levels. Moreover, the effect of AA on the translocation of the transcription factor nuclear factor kappa B (NF-κB) was evaluated in LPS-stimulated RAW264.7 cells and in TNF-stimulated L929 cells. Subsequently, AA was tested in the inhibitor of NF-κB kinase beta (IKKβ) activity assay. Lastly, the anti-inflammatory activity of AA in vivo was evaluated by testing TNF production in LPS-stimulated Balb/c mice. KEY RESULTS: AA effectively inhibited TNF-α release with an IC(50) of 2.7 µg/mL. Moreover, AA significantly inhibited both NO production and iNOS expression. It significantly and dose-dependently inhibited TNF and IL-1β proteins and mRNA expression; as well as IL-6 and KC proteins. Additionally, AA prevented the translocation of NF-κB in both cell lines; suggesting that it is acting at a post receptor level. This was confirmed by AA's ability to inhibit IKKβ activity, a kinase responsible for activating NF-κB, hence providing an insight on AA's mechanism of action. Finally, AA significantly reduced TNF production in vivo. CONCLUSIONS AND IMPLICATIONS: This study presents the potential utilization of this compound, as a lead for the development of an anti-inflammatory drug

Systematic evaluation of immune regulation and modulation

Cancer immunotherapies are showing promising clinical results in a variety of malignancies. Monitoring the immune as well as the tumor response following these therapies has led to significant advancements in the field. Moreover, the identification and assessment of both predictive and prognostic biomarkers has become a key component to advancing these therapies. Thus, it is critical to develop systematic approaches to monitor the immune response and to interpret the data obtained from these assays. In order to address these issues and make recommendations to the field, the Society for Immunotherapy of Cancer reconvened the Immune Biomarkers Task Force. As a part of this Task Force, Working Group 3 (WG3) consisting of multidisciplinary experts from industry, academia, and government focused on the systematic assessment of immune regulation and modulation. In this review, the tumor microenvironment, microbiome, bone marrow, and adoptively transferred T cells will be used as examples to discuss the type and timing of sample collection. In addition, potential types of measurements, assays, and analyses will be discussed for each sample. Specifically, these recommendations will focus on the unique collection and assay requirements for the analysis of various samples as well as the high-throughput assays to evaluate potential biomarkers

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms

An expanded evaluation of protein function prediction methods shows an improvement in accuracy

Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging.Results: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2.Conclusions: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent