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.

Anti cellulite compound evaluation with in vitro adipocyte-assays

Mechanisms leading to cellulite formation is complex. It involves lipid regulatory pathways and proinflammatory cross-talk that represent promising molecular targets in cosmetology. This post introduces a clever in vitro adipocyte-based assay targeting adipocytokines to better determine the anti cellulite effects of cosmetics compounds.

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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.

mTOR revisited

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Figure taken from Ref. 1.

The PI3K/Akt/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle. It is directly related to cellular quiescence, proliferation, cancer, and longevity. This pathway can be regulated by genes involved in response to hypoxia. Discoveries that have been made over the last decade show that the mTOR pathway is activated during various cellular processes (e.g. tumour formation and angiogenesis, insulin resistance, adipogenesis and T-lymphocyte activation) and is deregulated in human diseases such as cancer and type 2 diabetes (1). [Read more…]

Exosomes as a trigger for signaling

Extracellular vesicles in general, or exosomes in particular, are becoming a hot topic for research, especially in cancer. They seem to have different roles in tumour progression or can be used as targets to develop new therapies. In our series of posts on exosomes, we will focus today on their role in signal transduction.

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Release of microvesicles and exosomes (taken from Ref. 2).

Knowledge of the content of exosomes, and their role to in cell-to-cell communication by mediation of signal transduction, is of interest on two sides. [Read more…]

The TRPV1 Pain Receptor activates T cells

The Transient Receptor Potential (TRP) ion channel family participates in numerous functions of the Nervous System. A recent publication tends to indicate that TRP might also be of interest for therapeutical strategies for controlling pro-inflammatory  CD4+ T cell reactions.

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Tumour microenvironment and kinome studies

We all react to external factors, even the most cold-blooded person. We might hide our emotions, but there they are.

The same happens in cancer. Cells (both the tumour cells and the normal ones) react to the environment in one direction or another. In this post, we will see how “kinome” analysis might help Researchers in better understanding cellular interactions in tumour microenvironment (TME).

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IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake by Deak et al.

Deak AT et al. decipher the mechanisms by which mitochondria contribute to Ca2+ intracellular signaling. In their recent paper (1), they show that mitochondrial “Ca2+ buffering” close to the Endoplasmic Reticulum predominately shapes cytosolic Ca2+ micro-domains. To perform their studies, the authors used stable knock-down (KD) HeLa cellular models optimized for loss-of-function analysis.

Two proteins known to be essential for mitochondrial Ca2+ uptake (Mitochondrial Calcium Uniporter (MCU) or Uncoupling Protein 2 (UCP2)) have thus been stably silenced with sh-RNA engineered HeLa cell lines (MCU-KD and UCP2-KD HeLa cell lines). This shRNA technology has already been used in Drug Discovery approaches (ex. PARP inhibitors efficiency by Synthetic Lethality in Hypoxic conditions). (2)

Finally, the authors present two diagrams illustrating the role of mitochondrial Ca2+ uptake for Stromal Interacting Molecule 1 (STIM1) oligomerization and Store-Operated Ca2+ Entry (SOCE) maintainance in their HeLa cellular models.

Want to know more?

(1) Deak AT et al. “Inositol-1,4,5-trisphosphate (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake” (2014) J Cell Sci. 2014 May 7. DOI: 10.1242/?jcs.149807

(2) Mennesson E. et al. “SilenciX®, novel stable knock-down cellular models to screen new molecular targets through the synthetic lethality approach” (“Experimental and Molecular Therapeutics” poster session – AACR 2014, San Diego) Abstract n° 3733

Congratulations to the authors!

Focus on the JAK-STAT pathway…

The Janus kinase (JAK) / Signal Transducer and Activator of Transcription (STAT) pathway a key regulator of cellular proliferation, differentiation, migration, apoptosis and survival. Numerous cytokines, growth factors and Interferons interact with the JAK/STAT signaling pathway which in turn   regulates many biological processes, especially in hematopoietic development and immune responses.

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Focus on the NFkB Signaling Pathway…

 NF-κB (Nuclear Factor κ-light-chain-enhancer of activated B cells) is a protein complex that controls the transcription of DNA. Find the updated version of the NF-κB cell signaling pathway.

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