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.

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.

[Read more…]

8 criteria for selecting your ELISA kits

Biomarkers specialists are often asked to select an ELISA kit for researchers: with thousands of ELISA references available on the market, the choice can be tricky regarding proteins for which several kits available.

When researchers have to choose a new ELISA kit, the price is regularly the first parameter of selection. But my experience with long term projects shows that it should in fact be the very last one…

[Read more…]

Discovery of new biomarkers… 3 tips regarding controls

One of the recurrent questions that we get at the Biomarkers team at tebu-bio is on what controls should be included in a given experiment. Either if the experiment is done by researchers in their lab, or if we collect their samples and perform the analysis in our lab, a good design starts by using the most convenient controls.

One of the controls is related to the study itself. In this sense, definition of what a control population is, and how we want to study it vs. a cohort of patients has been discussed elsewhere in a proteomics post. Today, we will put our spotlight on the “technical” controls, i.e. those related to the technique itself.

Control # 1 – positive control

Obvious. We need to check that the technology we are using is able to detect what we want to detect. And before starting with unknown samples, we need to check that it works in samples we know well.Multicoloured wells - Blog Thumbnail

Ideally, a positive control should be as similar as the samples we want to analyse. In this sense, samples for a given health state (be it with a disease or not), are commercially available, or they can be found if not yet available. tebu-bio has a network of collaborations with private companies that can provide validated samples, fulfilling all ethical and clinical criteria.

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Background can be an issue…only if you are not able to detect it. If you can see it, then you can either modify your protocol, or discard that sample. Picture shows an example of what can be seen with slightly-hemolysed plasma.

Alternatively, we can use recombinant or chemically synthesised controls. If we take the example of recombinant proteins (either in pure form or spiked in biological samples), there are many which are commercially available, or they can be made upon demand. Here it is important that the recombinant protein is very similar to the one found in an organism, including glycosylations and other post-translational changes. In this sense, for many control proteins, HEK293 is preferred over E.coli as an expression system.

Control #2 – negative control

Water. Or PBS. Or not?

Ideally, a negative sample should be as similar as to our case samples as possible. Meaning that it has the same clinical and biological parameters than our samples of interest…

Commercially available samples mentioned previously can be a good approach. For immunological studies involving cell culture supernatants, it is important to include a control with the culture medium only, as FBS can affect the specificity of the assay and can render false-negatives due to background.

Background due to FBS is not detected by technologies such as ELISAs or bead-based, whereas it is detected in optical-based technologies such as arrays and Q-plex.

Control #3 – technical replicates

Every technology has an inherent coefficient of variation (CV).

Genomic technologies usually are under 5 % CV. Immunoassays are around 10 to 25 % (or even more). This means that, for some biomarkers where the difference between healthy vs. disease is small, CV may hide the relevance of these biomarkers. This is especially dramatic in studies related to signal transduction, where differences are usually very small.

6-plicates in an antibody array.

6-plicates in an antibody array.

A way to make sure about whether a result comes from real biology or artificial CV is the performance of replicates.

Triplicates (or even 4-plicates) have been popular with ELISA users. Nowadays, however, most researchers perform duplicates, and repeat the analysis of the sample if the results are very discordant. This approach is quite practical, and still allows to have accurate results in a sensible way (i.e. not doing 4-plicates for every sample!).

Antibody arrays in the market usually have replicates spotted onto the same slide (from duplicates to 8-plicates), which can be considered as semi-independent technical replicates. Therefore, there is no need, in most cases, to perform additional technical replicates.

In any case, every project is different, so we are continously advising our customers on what is the best approach for one given study. From its design to the technology best suited to get the best results, we are glad to contribute to the advance of the understanding of biomarkers in several diseases.

Wondering what controls to include in your experiment? Don’t hesitate to contact us!

Vive la différence! To pool or not to pool…

In a previous post on whether samples should be pooled or not for proteomic profiling, we discussed this approach, which can be quite cost-wise, while still allowing to see the main biomarkers differentiating one physiological condition from another (e.g. disease vs healthy control).

In real life, however, this discrimination between physiological conditions may be difficult to define. Let’s take, for example, a study aimed at studying the differential immune response to an infection, and how this can be used to design more efficient therapies in different population subgroups. [Read more…]

Invasion of influenza A

The influenza season in Europe is over… until next year. Influenza remains one of the most important public healthcare problems, especially in risk groups.

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Figure 1. Influenza A life cycle, indicating target molecules for which specific antibodies have been developed.Influenza virus can cause annual epidemics of approximately 500,000 deaths per year worlwide, which is facilitated by the high mutation rate of the viral genome and polymerase. The function interplay between hemagglutinin and neuraminidase glycoprotein characterises the pathogenic profile of each viral subtype (1).

The Influenza A virus (IAV), mimicked as misfolded protein aggregates, carries unanchored ubiquitin chains to exploit host cell’s aggresome processing machinery for efficient IAV entry and capsid dissociation (2).

For studies involving IAV, it is important that antibodies recognise the specific viral subtype, with high specificity, to avoid any cross-reactions with other subtypes that may not be relevant that season. [Read more…]

Notch and renal failure revisited

In our series of posts on different signaling pathways, let’s take a look today on Notch and its relevance in Acute Renal Failure (ARF).

A recent paper by Gupta et al. elucidated the role of the Notch pathway in kidney regeneration. This paper means an advance towards understanding potential therapeutic targeting of Notch signaling to enhance renal repair. Activation of the Notch pathway occurs following ARF. Pretreatment with the Notch ligand DLL4 enhanced recovery from ARF and represents a potential novel therapeutic option for regenerating the injured kidney.

100-401-407-Anti-Notch1-Antibody-1-IHC-4x3

Anti-Notch 1 (Cleaved N terminal) (Human specific) (RABBIT) Antibody (Cat. No. 039100-401-407).

However, compared to previous publications, as the authors mention in the paper, the use of different antibodies can affect the overall result of the experiment (as we all know!). In this specific case, Gupta et al. demonstrated increased expression of cleaved Notch1 and cleaved Notch2 as early as 1 h following reperfusion after 45 min of ischemia, and their findings are consistent with studies by Kobayashi et al. in a similar model of ARF with a few exceptions.

The paper by Kobayashi showed increased mRNA and protein expression of Delta-1, cleaved Notch2 only, while cleaved Notch1 was minimally detected under basal conditions or following injury. However, Gupta used the cleaved Notch-1 antibody from Rockland (see figure), and detected a robust signal for cleaved Notch1 with increased expression seen as early as 1 h following injury. These results were confirmed by immunohistochemistry using the Val1744 antibody. Therefore, both Notch1 and Notch2 are activated in the kidney following ARF.

Notch signaling has many roles, from neuronal function and development to the expansion of the hematopoietic stem cell compartment during bone development. Notch signaling pathways are a booming area of pharmacological research, due largely to the direct connection to human disease intervention.

IL-1 family quantified simultaneously…at last!

The IL-1 family has long been known as a key player in the effector role of macrophages in innate defence against tumours and infections. It plays an important role in the field of natural / innate immunity effector mechanisms, especially as far as macrophage and macrophage-derived cytokines are concerned, in defence response. It also seems to be involved in pregnancy and pre-eclampsia (1), as well as cardiovascular diseases and type 2 diabetes (2). It is also involved in the response to Carbon Nanotubes (CNTs) exposure, where IL-1 family members activate the JNK pathway, but not ERK (3). [Read more…]

Focus on apolipoproteins

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Role of apolipoproteins in different diseases.

Apolipoproteins, with amphipathic properties, form lipoprotein particles with phospholipids and transport hydrophobic lipids through lymphatic and blood circulations to designated peripheral tissues or organs for energy supply, biomolecule synthesis, or degradation. Off-balance of apolipoproteins has been implicated in numerous diseases such as SLE, myocardial infarction, Alzheimer’s, and diabetes. Apolipoproteins emerge as risk markers to pinpoint different diseases.

For Cardiovascular diseases, apoF and apoB seem to have a predominant role. [Read more…]