New Glycan Array – Sweet 17? Sweet 100!

Glycocalyx, literally meaning ‘sugar coat’, is an extracellular polymeric coating surrounding many prokaryotic and eukaryotic cells that consists of glycoproteins, glycolipids, proteoglycans and glycosaminoglycans. The constituents of the glycocalyx play an important role in the process of cell signaling, virus transfection, and immunity and glycosylation status is important in processes such as inflammation and tumour microenvironment. However, the range of detection tools available for glycobiology research is quite limited, and in most cases, studies aimed at characterising glycosylation patterns have to be done with HPLC using home-based methods. [Read more…]

Blood as a cells highway

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IF on CTCs. HepG2 cells were stained with CD44-FITC labeled monoclonal antibody (Green). The cell nucleus were counterstained with DAPI (Blue) (Cat. No. 157MAB3838).

For years, we already knew that a lot of biomarkers are circulating in the blood (i.e. the secretome), as if it were a massive highway. Cells (apart from haematological cells, that is) also circulate.

There is much research now being focused on the role of several types of circulating cells in different diseases. Rapid advances in CRC research are pushing their utility to the in vitro diagnostic (IVD) arena, specifically in non-invasive diagnoses, companion diagnostics, and therapeutic monitoring. Up to date, several circulating rare cell (CRC) types have been described: Circulating Stem Cell (CSC), Circulating Endothelial Cell (CEC), Circulating Hematopoiectic Stem & Progenitor Cell (CHSPC), Circulating Mesenchymal Cell (CMC) and Circulating Tumour Cell (CTC).

Role of these CRCs still has to be elucidated, but there are some things we already know. For example, CSCs have a role in epithelial tumours & metastasis, as well as leukemia & lymphoma, together with CHPSCs and CTCs. CECs play a role in some tumour types. For cardiovascular, neurodegenerative and autoimmune diseases, CECs, together with CMSCs seem to be also involved.

Major challenges for the research on these CRCs is the optimisation of isolation methods, to make sure that we are indeed isolating CRCs and not other cell types. Current isolation methods depend on surface markers specific for these cell types, including EPCAM (1), CD44 (2), CD34 (3),  CD38 (4), CTNNB1 (5), etc.

Working on CRCs and their role in your disease? Leave your comments below!

 

 

References

1.- Zhao, M. et alAnal Chem. Oct 15, 2013; 85(20). doi: 10.1021/ac401985r.

2.- Qin, J. et al. Int J Clin Exp Pathol. 2014; 7(6): 3235–3244. PMCID: PMC4097256.

3.- Sho, E. et al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2004; 24: 1916-1921.

4.- Ferrero, E. & Malavasi, F. J Leukoc Biol. 1999 Feb;65(2):151-61.

5.- Nowak, M. et al. Folia Histochem Cytobiol. 2007;45(3):233-8.

 

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…]

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.

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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.

Identification of a MicroRNA signature for Fibromyalgia diagnosis

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Heatmap of PBMCs miRNome from FM patients and controls. The microarray analysis was performed with Toray’s 3D-Gene Human miRNA Oligo chips (v.16.0; current version is v21.0). Patient samples are labeled (FM 1–11) and controls (C 1–10). Color palette is included to indicate signal intensity.

A recent paper by Cerdá-Olmedo, G. et al. unravels the miRNA signature in fibromyalgia (FM). Diagnosis of FM, a chronic musculoskeletal pain syndrome characterised by generalized body pain, hyperalgesia and other functional and emotional comorbidities, is a challenging process hindered by symptom heterogeneity and clinical overlap with other disorders. No objective diagnostic method exists at present.

This study aimed at identifying changes in miRNA expression profiles (miRNome) of FM patients for the development of a quantitative diagnostic method of FM. In addition, knowledge of FM patient miRNomes would lead to a deeper understanding of the etiology and/or symptom severity of this complex disease.

A broad profiling was first performed using Toray’s technology. miRNAs found were validated by qPCR in a later step. The profiling of FM patients PBMCs showed a marked downregulation of hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p, hsa-miR145-5p and hsa-miR-21-5p (4-fold or more).

Globally, 20% of the miRNAs analyzed (233/1212) showed downregulation of at least 2-fold in patients. This might indicate a general de-regulation of the miRNA synthetic pathway in FM. No significant correlations between miRNA inhibition and FM cardinal symptoms could be identified. However, the patient with the lowest score for mental fatigue coincided with the mildest inhibition in four of the five miRNAs associated with the FM-group.

Therefore, the authors propose a signature of five strikingly downregulated miRNAs (hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p and hsa-miR145-5p) to be used as biomarkers of FM. Validation in larger study groups would be required before the results can be transferred to the clinic, as the authors indicate.

Looking for miRNA signatures? Don’t hesitate to leave your comments below!

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…]

Good news for Illumina mRNA array users

Recently, Illumina informed their customers that their whole-genome arrays for mouse and FFPE-human samples were to be discontinued. This came as a shock to some researchers that had already started studies using them, or had grants approved and would use them in the future.

Considering other technologies when you have one that is working is really difficult. In Science as well as in life in general, if something works, don’t touch it. That said, if the products you are using are discontinued, you really have to start looking for alternatives.dna5_img_01

Today, let’s take a look at an alternative to Illumina microarrays… that in fact has some advantages, including lower background and higher robustness, among other benefits.

This alternative is based on the 3D-Gene technology developed by Toray. We have discussed this technology, and its use for miRNA studies, in previous posts, but today we will focus on its use for mRNA studies.

The mRNA Oligo chip from Toray was selected from the well-established oligo DNA set for microarray from Operon Biotechnologies Inc. – AROSTM v 3.0 and v 4.0. The selection was made with a focus on genes with substantial annotation information. Therefore, a researcher can perform an analysis without being misled by ambiguous information.
The main public databases used in the design of the probes include Ensembl human database, NCBI human RefSeq and H-Invitational Database.

tebu-bio, Toray Reach Agreement for miRNA, mRNA Profiling TechnologyArrays are available for human (25k), mouse (24k) and rat (20k). Access to this technology is easy, as it is also provided as a service by tebu-bio’s laboratorios located near Paris. You only have to tell them when to collect your samples, and they’ll take care of the whole process!

If you’re interested in learning more about these arrays, as well as a comparison with other microarrays in the market, leave your questions below!

D-dimer and Cardiovascular Disease

Fibrinogen is the main protein of the blood coagulation system. It consists of two identical subunits that contain three polypeptide chains: alpha, beta and gamma. The process of blood coagulation results in the activation of fibrinogen into fibrin by thrombin and fibrin polymerization. Fibrin clot is then digested by plasmin, and fibrin degradation products of different molecular weights are released into the bloodstream.

D-dimer is one of these fibrinogen degradation products, and is a biomarker for Cardiovascular damage. Therefore, it is widely used in many detection systems in the market, including ELISAs and lateral flow tests.

Clone 8D3, which has been used in many of these detection systems, is nolonger available. So unless you have the hybridoma in your facility, if you were using 8D3, you will have to consider switching to another monoclonal. [Read more…]

Wnt4 and Rspo1 to promote mammary stem cell self-renewal

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WNT4 antibody detects WNT4 protein by western blot analysis. Non-transfected (-) and Wnt4-transfected (+, including V5-tag) 293T whole cell extracts (100 μg) were separated by 10% SDS-PAGE, and the membrane was blotted with WNT4 antibody (Cat. No. 210GTX101085) at a dilution of 1:2500. The V5 was used as internal control (Cat. No. 210GTX117997, 1:2500) shown at the bottom panel.

The behavior of adult mammary stem cells (MaSCs) is precisely controlled by the activities of hormones and local factors, though the underlying mechanisms remain obscure. A recent report by Cai et al in Genes & Development illustrates the dynamic interaction between systemic ovarian hormones, Wnt signaling, and the Wnt agonist R-spondin1 (Rspo1) to promote MaSC self-renewal.

In response to estradiol and progesterone, R-spondin1 and Wnt4, but not Wnt7B, act as niche factors to drive MaSC regeneration, with Wnt4 acting through the canonical Wnt/β-catenin signaling pathway. This work establishes a clear mechanistic link between locally acting Wnt signals and the systemic hormone growth response of MaSCs, unveiling the intriguing concept that hormones induce a collaborative local niche environment for stem cells.

Working on unveiling the mechanisms of stem cell fate and differentiation? We’d like to hear from you!