Pictures from Switzerland: Bern

Pictures from Switzerland: mountains & valleys

Pictures from Switzerland: Zurich

Deconvolution of the cellular oxidative stress response with organelle-specific peptide conjugates

Chemistry & Biology, one of the journals of the famous Cell Press publishing group (2007, 14:923-930), published an article which uses CoLocalizer Pro software to analyze images and thus help to understand the mechanism of oxidative stress response. One of the images presented in the paper was selected to be the issue cover.

Review on quantitative colocalization analysis

Acta Histochemica et Cytochemica, an Official Journal of the Japan Society of Histochemistry and Cytochemistry, published our review on quantitative colocalization analysis. The review is entitled "Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena" (Acta Histochem Cytochem 2007 40:101-111). This is the most comprehensive review on this subject available. It should be very helpful to the beginners and to those familiar with the technique alike, as it not only gives the basics of the colocalization theory, but provides examples of the practical use of quantitative colocalization analysis as well.

Interesting paper appeared in Journal of Clinical Investigation

Journal of Clinical Investigation (Impact Factor 16 in 2006), published a very interesting paper about requirement of Foxo3 (forkhead box O3) for the regulation of oxidative stress in erythropoiesis (J. Clin. Invest. 117:2133-2144 (2007). The study is a collaborative effort performed by Dr D Marinkovic and colleagues from Departments of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, USA, Laboratory Medicine, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA, Black Family Stem Cell Institute, and Departments of Molecular, Cell, and Developmental Biology, and Medicine, Division of Hematology and Medical Oncology, Mount Sinai School of Medicine, New York, New York, USA.

The study found that Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21CIP1/WAF1/Sdi1 (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role. The study used CoLocalizer Pro software to perform quantification of immunofluorescence images and employed Manders' coefficient to obtain important conclusions.

Interesting article published by PNAS

Proceedings of the National Academy of Sciences the United States of America (PNAS), one of the most respected and highly acclaimed scientific journals, published in its June issue (104: 10205-10210, 2007) a very interesting article about involvement of host cellular multivesicular body functions in hepatitis B virus budding. The paper was written by Dr. Watanabe and colleagues from Institute for Molecular Virology, McArdle Laboratory for Cancer Research and Howard Hughes Medical Institute, University of Wisconsin, Madison. The authors found that host multivesicular bodies (MVBs) functions are required for efficient budding and release of enveloped Hepatitis B virus (HBV) virions and may be a valuable target for HBV control. Moreover, HBV enveloped virions, enveloped subviral particles, and unenveloped nucleocapsids are all released by distinct pathways with separate host factor requirements. The study elegantly used CoLocalizer Pro software to estimate colocalization of HBV envelope protein with class E proteins in Huh-7 cells.

Zymogen activation and other early events in secretagogue-induced acute pancreatitis

Dr. Van Ecker and collaborators from Tufts-New England Medical Center and Tufts University School of Medicine in Boston published a very interesting article about cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis. The article appered in American Journal of Physiology Gastrointestinal and Liver Physiology (292: G1738-G1746, 2007). It was concluded: 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity. The authors made a very good use of CoLocalizer Pro software to obtain these important results.

Tyrosine phosphorylation and lipid raft association of pseudorabies virus

Virology, the leading source of new information in its field (2007 362:60-66), published an article of Dr. Desplanques and colleagues from Ghen University, Belgium, on tyrosine phosphorylation and association of lipid raft of pseudorabies virus (PRV) glycoprotein E during antibody-mediated capping. It was found that PRV gE-mediated viral capping process results in increased Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of gE and that a fraction of gE associates with lipid rafts, all very reminiscent of immunoreceptor capping. These results provide evidence that gE-mediated capping is a viral mimicry of immunoreceptor capping. The authors made a good use of CoLocalizer Pro software not only performing calculations of various colocalization coefficients, but analyzing scatter grams of images as well. Interesting that images used for quantification of colocalization in this study were selected for the journal issue cover. Good job!

Ethanol consumption alters expression and colocalization of bile salt export pump and multidrug resistance protein 2 in the rat

Histochemistry & Cell Biology (2007, 127:503-512), an Official Journal of the Society for Histochemistry, published our article on the effect of ethanol toward bile salt export pump and multidrug resistance protein 2. In this paper, we calculated several colocalization coefficients to understand the changes occurring in hepatocytes in greater details. We believe that this article should be of significant interest not only to basic science researchers, but to clinicians dealing with alcohol abuse as well.

Colocalization of zymogens with lysosomal hydrolases

Observation of colocalziation played central role in understanding of the cause-effect relatioships between zymogen activation and other early events in secretagogue-induced acute pancreatitis. CoLocalizer Pro software was used by Dr. Van Acker and colleagues from Tufts-New England Medical Center in Boston to show that inhibition of cathepsin B by pre-treatment with CA-074me prevents intrapancreatic zymogen activation and reduces organellar fragility but it does not alter the caerulein-induced co-localization phenomenon or sub-cellular F-actin redistribution or prevent caerulein-induced activation of NF-B, ERK 1/2, and JNK, or up-regulated expression of cyto-chemokines. The authors concluded (a) that the co-localization phenomenon, F-actin redistribution, activation of pro-inflammatory transcription factors, and up-regulated expression of cyto-chemokines are not the result of zymogen activation, and (b) that these early events in pancreatitis are not dependent upon cathepsin B activity. The paper appeared in in American Journal of Physiology (Gastrointestinal and Liver Physiology), an official journal of The American Physiological Society ((2007) 292 G1738-G1746).

American Journal of Pathology paper cites CoLocalizer Express software

Dr. Rocker and colleagues from departments of biophysics and cardiology at the University of Ulm and physics at the University of Illinois at Urbana–Champaign published interesting article about enhancement of the affinity of C-reactive protein toward FcgammaRI by the gamma-chain. The publication appeared in American Journal of Pathology (2007) 170:755-763, an official journal of the American Society for Investigative Pathology. This study investigates C-reactive protein (CRP), the prototype human acute phase protein, which is widely regarded as a key player in cardiovascular disease. Taking into consideration the structure of CRP, the study concludes that multivalent binding and receptor clustering are crucially involved in the interaction of CRP with nucleated cells. The article cite CoLocalizer Express software, wich has been used to understand the details of the studies processes.

Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes

International Immunology (Int Immunol (2007) 19:19-30), new but already highly respected journal in the field of immunology, published interesting article about role of activation-induced endocytosis of the raft-associated transmembrane adaptor protein Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) in B lymphocytes in internalization of the B cell receptor (BCR). The authors lead by Dr. Deans from Department of Biochemistry and Molecular Biology at the University of Calgary used a complex methodological approach, which included quantitative colocalization analysis using CoLocalizer Pro software. The paper sheds light onto the role of LAB/NTAL in the internalization of BCR and describes intrinsic details of this process. Congratulations to the authors on this elegant study.

Regulation of adenylyl cyclase in cultured cardiac myocytes

Journal of Biological Chemistry (JBC) editors seem to like quantitative colocalization analysis, because the journal published three articles which employed CoLocalizer Pro software in 2006 alone. This is very encouraging, because JBC is one of the best journals in the medico-biological field. The paper which I did not mention here yet is written by Dr. Head and colleagues from the University of California in San Diego (J Biol Chem (2006) 281:38730-38737). It reports about regulation of localization of adenylyl cyclase in cultured cardiac myocytes. In an elegant way, Dr. Head and coworkers showed that microtubules and actin filaments restrict formation of cyclic AMP by regulating the localization and interaction of G protein-coupled receptors with adenylyl cyclase in lipid rafts/caveolae. The paper is an interesting read.

CoLocalizer Express 1.2 released!

We have updated CoLocalizer Express to version 1.2. This update is a universal binary version. It includes updates to the Help and User Guides and to the License Agreement. It can be downloaded here: http://homepage.mac.com/colocalizerpro/purchasesupport.html. Enjoy!

CoLocalizer Pro papers keep coming...

Dr. Osvaldo Rey and colleagues from the University of California at Los Angeles published a very interesting paper about the role of calcium-sensing receptor (CaR) in the generation of transient Ca2+ oscillations. Dr. Rey used CoLocalizer Pro software to elegantly quantify the colocalization of CaR and transient receptor potential gene, TRPC1, according to Manders' overlap coefficient. They employed CoLocalizer Pro to quantify colocalization of CaR and TRPC1 and presented corresponding scatter grams. The paper is a good read and leaves a very positive impression. The paper appeared in the Journal of Biological Chemistry (281:38730-38737 (2006), one of the best journals in the medico-biological field.

To do or not to do

A number of researchers contacted CoLocalization Research Software Support lately and said they tried either CoLocalizer Pro or Express versions of colocalziation analysis software and the results of colocalization coefficients were not what they expected. They wondered why is this so and what should they do to obtain more suitable calculations. After asking them few questions about the way they prepared samples and how they used confocal microscopes, it became clear that their images were simply unusable for quantitation purposes. For these images, even calculations which seem correct will no doubts be erroneous. We put together some simple guidelines for researchers using confocal microscopy and doing quantitative colocalziation analysis so that they can avoid mistakes in future. Here they are:

Sample preparation:
- Make sure that the used antibodies are specific and do not cross-react.
- Choose fluorophores with well-separated excitation and emission spectra.
- Use the same mounting medium for the samples you will be comparing.
- Try to avoid using anti-fading reagents, as they can increase background fluorescence.

Microscope set-up:
- Use plan apochromatic lenses to reduce chromatic shift.
- Use optimized emission filters to maximize emission collection while avoiding bleed-through effect.
- Use the same objective lens for the samples you will be comparing.
- Make sure that the size of microscope pinhole is properly set.

Image acquisition and handling:
- Acquire images sequentially to minimize bleed-through effect.
- Do not acquire too bright and too contrast images, as it will result in their saturation.
- Do not manipulate images in graphics-editing programs, as it may ruin usability of your images for quantitation.
- Do not resave files in any other format than TIFF. It may cause loss of original image data needed to perform calculations.

If you have not followed one of these guidelines, just don`t use your images for calculating colocalization. You will not get reliable results.

CoLocalizer Pro 2 released!

We upgraded CoLocalizer Pro to version 2. New version of CoLocalizer Pro is a significant improvement over the previous one. CoLocalizer Pro 2 adds one of the most requested features: the ability to reveal colocalized and selected pixels. The upgrade is free to all registered users! It can be downloaded here: http://homepage.mac.com/colocalizerpro/purchasesupport.html

Colocalization studies performed using multidimensional microscopic robot technology

October issue of Nature Biotechnology published an article about the latest trend in studying the high-throughput protein colocalization studies: multidimensional microscopic robot technology. The technology consists of cycles of fluorescence tagging, imaging and bleaching in situ. It is said that the technology combines three advances: a) a fluorescence technique capable of mapping hundreads of different proteins in one tissue section or sample, b) a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors, c) and a system for imaging the distribution of these protein clusters in a so called toponome map. It is claimed that this approach is able to reveal rules of hierarchical protein network organization, in which state-specific lead proteins control protein network topology and function.

The method is relied on a so called multi-epitope-ligand cartography (MELC), that can map the location of several proteins in the one samlel of cells or tissue using sequential rounds of fluorescent detection in situ. Importantly, it appears that MELC can be used to show when and where proteins colocalize in cellular or tissue compartments, which proteins are excluded, how these associations are altered by pathology and how proteins are functionally interrelated.

MELC appears to be a significant step forward in colocalization analysis, because it enables obtaining considerably more reliable images for latter use with colocalization analysis software. It would be interesting to see whether these images will still require background correction, a critical step performed by the current software.

Study colocalized antigens? Make sure you`ve got CoLocalizer...!

A whole lot of new researchers doing confocal microscopy will now learn about amazing capabilities of CoLocalizer Pro and CoLocalizer Express! Microscopy and Analysis, the premier international journal for users of microscopical, analytical and imaging equipment with 46,000 subscribers and 120,000 readers worldwide, in its special issue dedicated to "Light Microscopy in the Life Sciences" published our review article about quantitative colocalzition analysis. The article describes the applicability of our colocalization analysis software for studying dynamics of cellular responses: Microscopy and Analysis 2006, September. For the convenience of users, we included table with characteristics of coefficients used to estimate colocalization. We describe the range of their values and give tips on how they should be applied.