Monoclonal validated for Post-Translational Modification studies

Knowing the Human genome better has allowed major advancements in Personalised Medicine. Nowadays, we can know (if we want) the likelihood to develop a given disease and/or how we will react to different pharmacological treatments. Examples of this include diseases like breast cancer (for diagnosis or estimation of likelihood) and lung cancer (for response to treatment), to name just a few.

That said, our genotype does not have the last word. Research in the last couple of decades has shown the power of other regulatory mechanisms, that may enhance or diminish the effect that our genotype will have on our health. Starting from basic healthy life styles, to other more subtle mechanisms, our genotype defines us, but not completely. Above genetics, we have epigenetics… and everything at the protein level. This post will focus on Post-Translational Modifications (PTM), because, after all, it’s the proteins that are the final effectors of a given response to a treatment or to an environmental stimulus.

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How to get clear best-quality data in IP and WB protocols?

Immuno-precipitation & Western Blots often suffer from heavy  /light chain blotting, contamination, and ongoing interferences. This can prevent from obtaining biologically-relevant data in a given experimental model, especially when the target of interest has a size similar to the IgG molecules that may remain. Getting rid of these “artificial” bands can be tricky. In this post, you will discover the robustness of the TrueBlot® products when facing this issues.

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Oxygen and miRNAs in Cosmetology and Dermatology

A recent review by Nadim et al. casts some light on a Cosmetology and Dermatology, where circulating biomarkers, though studied to a certain extent so far, are yet unknown for many skin models. (1) A first element to have in mind when considering the skin-related experimental model is the “Oxygen level”. Oxygen levels may contribute to different findings, and the in vitro models used so far may not be so physiologically relevant as initially thought.

Hypoxia and miRNAs

Tissue oxygenation (which is a major part of the cell microenvironment) regulates the expression of the microRNAs called oxymiRs. OxymiRs may be categorized into three groups:

  1. microRNAs whose expression is directly modied by the Oxygen partial pressure,
  2. microRNAs whose expression is indirectly (pH, metabolites, etc.) modified by the Oxygen partial pressure, andpO2 percentage levels in various tissues
  3. microRNAs that target mediators of Oxygen sensing pathways to regulate biological networks for cell survival.

Examples of miRNAs important in skin physiology include miR-17, miR-21, miR-24, miR-27, miR-29b, miR-99 family, miR-125, miR-146a, miR-155, miR-203 and miR-205, among many other.

For example, in injured tissue, disruption of the vascular supply is associated with a low oxygen partial pressure, or hypoxia, which induces the expression of specic microRNAs referred to as HypoxamiRs (included in group (1) of the oxymiRs classification).

miR-210, known as the master HypoxamiR, is robustly induced under hypoxic conditions in nearly all kinds of cells. Under hypoxia, miR-21 also induces angiogenesis by targeting PTEN, leading to activation of AKT and ERK1/2 signaling pathways.

Transfection Factors and microRNAs

Other transcription factors such as p53 and NF-kB have been shown to affect the expression of microRNAs under hypoxia/anoxia conditions.

Numerous studies have demonstrated the modulation of microRNA expression and particularly that of the miR-200 family with oxidative stress due to excessive ROS levels.

Up to now, very few studies have shown the role of the intermediate Oxygen level (physioxia) in regulating microRNA expression in skin cells. Taken together, these results strongly support the idea that physioxia should be an important criterion in determining the microRNA expression level and consequently protein expression and skin functions.

Therefore, when trying to understand the role of microRNAs in skin models, it is important to choose a cell culture system that is as physiological as possible, and have the tools to analyse the different biomarkers (no only microRNA, but also signaling pathways, secretome, etc).

Should you like to have more information on this review, or know what we can do for you to support you with cell culture technologies with controlled oxygen levels or to support you for your biomarker discovery studies, do not hesitate to contact me by leaving a message below.

Source:

(1) Nadim M. et al. “Physioxia and MicroRNAs As Key Factors in the Skin Microenvironment” (2015) FSCC Magazine, Vol. 18 – #1, pp: 35-43

miRNAs: potent biomarkers in cancer research?

journal.pone.0118220.g002

Expression signals of validated miRNAs that differentiated pancreato-biliary cancer from non-malignant abnormalities (A), or from cancers of other types (B).

A recent paper by Kojima, M. et al. has found a signature of miRNAs to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.

Namely, a combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively.

In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort.

Finding of this eight miRNAs was possible by using Toray’s 3D profiling technology. This finding is especially important, as it is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology.

microRNAs are stably present in peripheral blood, and are therefore a good candidate for finding prognostic or diagnostic biomarkers.

Studying only the miRNAs available in the literature may limit the novelty of the biomarkers found, so for a wide variety of diseases, a profiling is mandatory in order to find really specific circulating biomarkers.

Toray’s technology is available in Europe as fee-for-services via tebu-bio Laboratories (see Press Release). By exploring the full miRnome (composed by 2,000 miRNAs) with Toray’s 3D-Gene® technology, one might identify slight miRNA expression level changes (including low abundance miRNAs) in blood samples, biopsies FFPE specimens…

Interested in miRNA profiling in cancer research? Leave a message below or browse tebu-bio’s 3D-Gene® miRNA profiling platform web page!

Array profiling to study PPAR-alpha in progenitor cell

Peroxisome proliferator-activated receptor alpha (PPAR-alpha also known as NR1C1)  regulates a myriad of biological processes. It is a key modulator of lipid metabolism.

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services)

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services).

Vergori, L. et al. have shown in murine models how PPAR-alpha regulates endothelial progenitor cell maturation and myeloid lineage differentiation via a NADPH oxidase-dependent mechanism. (1) All the data described in this publication suggest that PPAR-alpha, in murine models, is a critical regulator of recruitment, homing and maturation of Bone Marrow-derived progenitor cells.

This conclusion was made (in part) possible with the analysis of secretome markers by using C-Series profiling arrays for the analysis of bone marrow-derived cells in PPAR-alpha wild-type vs. KO mice.

RayBio® Membrane-Based Antibody Arrays (C-Series) are tools for screening and comparing expression levels of many cytokines between samples. C-series Arrays are available as ready-to-use kits or lab services. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012.

tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Interested in studying the secretome biomarkers relevant in your model for Cardiovascular diseases?

Leave your comment or contact us!tebu-bio: European RaybioTech's Certified Laboratory service provider

Source:
(1) Vergori L. et al. “PPARa regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice” (2015) Stem Cells – 33 (4) :1292-303. DOI: 10.1002/stem.1924.

Phosphorylation studies made with Antibody Arrays

Oropharyngeal squamous cell carcinomas (OSCCs) can be either Human papillomavirus (HPV)-positive or HPV-negative. Profiles of druggable Receptor Tyrosine Kinases (RTKs) are different in both groups, as shown in a paper by Cortelazzi, B. et al. The authors chose a cohort of 17 HPV-positive and 59 HVP-negative Formalin-Fixed OSCCs, in order to study E5 expression and RTK alterations. RTK activation was explored in further 12 Frozen OSCCs.

HPV-positive and HPV-negative OSCCs showed different RTK profiles, including differences in E5 and HER2 levels, as well as in HER3 activation and heterodimerisation (HER3/EGFR, also seen for HER2/EGFR). PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases.

The authors stated that, for HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. They also indicate that in HPV-negative cases, HER3 may be a promising druggable biomarker that would deserve further investigation. Finally, PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCCs, particularly in HPV-positive/PI3KCA-mutated OSCCs.

This study was possible in part thanks to an approach based on arrays to detect multiple biomarkers in biological samples at the same time, followed by validation using simplex technologies. These experimental approaches are known for cytokine profiling but also exist for phosphorylation studies (RTKs, EGFR, mTOR phospho-pathways…).

To ensure top quality in the data obtained, you might outsource your biomarker profiling and validation to certified service providers fully trained. tebu-bio: European RaybioTech's Certified Laboratory service provider

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012 but also by Quansys BioSciences and FullMoon BioSystems technologies. The Biomarkers team will be proud to support you to publish novel discoveries by providing innovative discovery & validation tools.

Genetic fingerprint of aggressive colon tumours

1.organoides_tumorales_de_pacientes

Patient-derived tumour organoids (mini colon tumours). In blue: cellular nuclei; in red: cellular membranes (Image: Alexandre Calon, IRB Barcelona).

Researchers at the IRB in Barcelona have found a signature of 4-6 genes able to predict the aggressivity of colon tumours, by analysing the tissue surrounding the tumour cells.

The scientists are currently developing a test that enables the identification of patients at risk of relapse after surgical removal of the tumour by measuring these found genes. They also propose to test in patients a particular drug that blocks the metastatic capacity of colorectal cancers in mice. This drug has been already tested using organoids derived from patients’ samples.

Source:

On a more funny note, have a look at the video done this last summer by researchers at the IRB Barcelona!

Any exciting research (or videos!) done over at your laboratory or institute?

We would like to know!

3 validated tools for ubiquitination studies

Elongation of ubiquitin chains, regardless of the linkage, can form polyubiquitin fibrils to initiate the Autophagy pathway. A deficiency of the autophagy causes cytotoxic accumulation of ubiquitin-positive aggregates leading thus  to neurodegenerative diseases (1). A new deubiquitinating enzyme, USP36, highly expressed in human breast and lung cancers, was identified to regulate c-Myc oncoprotein stability in nucleolus. (2) Also, Itch, an E3 HECT Ubiquitin ligase, inhibits MAPK p38α activation through ubiquitylation can be exploited therapeutically to prevent chronic skin inflammation (3). A recent publication by Yumimoto K. et al. determined that expression of FBXW7, the F-box Protein of SCF Ubiquitin Ligase, can suppress cancer metastasis in either non-cell-autonomous or cell-autonomous manner. (4)

Ub proteolysis pathway

Ub and F-box proteins proteolysis pathways.

 

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

In this work, researchers used the anti FBXW7 monoclonal antibody  to perform immuno-cyto-chemical staining on breast tissues.

Other tools to study ubiquitination include the use of Tandem Ubiquitin Binding Entities (TUBES) for the isolation and detection of polyubiquitylated proteins. TUBES can be used, in combination with Anti Linear Polyubiquitin antibody to investifgate Uquitin linkages on substrates.

References

  1. Morimoto, D., et.al. (2015). Nature communications. DOI: 10.1038/ncomms7116.
  2. Sun, X.-X., et.al. (2015). PNAS. DOI: 10.1073/pnas.1411713112.
  3. Theivanthiran, B., et.al. (2015). Science. DOI: 10.1126/scisignal.2005903.
  4. Yumimoto, K., et.al. (2015) JCI. DOI: 10.172/JCI78782.

If you are studying the role of ubiquitin and ubiquitination in your model, please leave your comment!

Cytokine signature in airway inflammation with arrays

Chronic Mucosal Inflammation is the hallmark of common airway diseases (ex.  Allergic Rhinitis and asthma). Lipoxin B4(LXB4) is an endogenous mucosal protective mediator decreasing such diseases. LXB4 mechanisms of action remain poorly understood.

Cytokine Quantibody Arrays from Raybiotech and tebu-bio laboratories

Cytokine Quantibody Arrays from Raybiotech and tebu-bio laboratories.

In a recent paper in Mucosal Immunology (Karra, L. et al. (2015) 8; DOI:10.1038/mi.2014.116), Allergic Rhinitis  and asthma murine models have been described to better investigate the role of LXB4 in Mucosal Inflammation. The authors conclude that, in the upper airway, LXB4 significantly decreases nasal mucosal leukocytes and degranulation of Mast Cells (MCs) and Eosinophils. They also show that, in the lower airway, LXB4 significantly decreased airway inflammation, mucus metaplasia, and hyper-responsiveness.

Inhibition of MC degranulation in vivo by LXB4 is more potent than Dexamethasone (a well-known apoptosis inducing glucocorticoid) with unique profiles for cytokine regulation. This latter  is proved by  quantitative analysis of 20 murine cytokines in a single array (Quantibody Cytokine Array).

These findings indicate that LXB4 carries cell type selective and mucosal protective actions and need to be translated to human models. This publication opens the way to consider lipoxins as new therapeutical candidates.

tebu-bio: European RaybioTech's Certified Laboratory service provider

tebu-bio laboratories are the European RaybioTech’s Certified Laboratory service provider.

The Cytokine Arrays are available as ready-to-use assays. They can also be outsourced to Certified Service Laboratory Providers as fee-for-services. In that case, researchers just send their samples and in return receive the experimental data set. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012. tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Normalisation assays in biomarker studies – all’s well that starts well

The normalisation of quantitative assay data is critical when interpreting effective biological system status. With cells grown in culture and lysed, a simple total protein determination such as the Bradford assay (developed by Marion Bradford at the University of Georgia in 1976) can be enough by giving an estimate of the total cellular proteins. However, this type of measurement, along with Lowry and other dye binding assays, can be prone to errirs due to various factors such as detergent, chelators… Standardization also involves the use of a protein of interest. Here again, the protein used is crucial for the accuracy of the overall assay.

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