Wednesday, December 3, 2014

Selective Inhibitors Identified in Labware Plastics

It is undeniable that the extensive use of plastic materials in labware has contributed to increased productivity and decreased costs. These plastics are generally considered to be chemically inert. However, a recent paper in the Journal of Biomolecular Screening is just the most recent example of the mounting evidence that plastics can contain trace chemical additives, also known as extractables and leachables, that can exhibit bioassay specific interference.

In the current paper, scientists from Bristol-Myers were using their PHERAstar microplate reader to perform a screen to identify compounds that inhibit monoamine oxidase using a fluoresence based assay. During the screen they observed an abrupt and drastic decrease in the signal-to-background ratio. After much investigation they finally discovered that 3 compounds, which were present in a new microplate used for compound storage and delivery, were the culprit. Further investigation revealed that these compounds were selective monoamine oxidase B inhibitors!

Article citation: Labware additives identified to be selective monoamine oxidase-B inhibitors.Stewart, J. et al J Biomol Screen (2014) 19:1409-14.

Thursday, September 25, 2014

Using the CLARIOstar and LVF-Monochromator to Identify Wnt Modulators

BMG LABTECH recently posted a new application note (#255) that describes the use of the Leading LightTM Sclerostin-LRP Screening System from EnzoLife Sciences which enables the detection of Wnt-pathway modulators.

This application is important in the study of Wnt effects on bone formation which have been characterized to involve the binding to receptors on bone cells leading to regulation of transcription that promotes bone formation. Specifically, Wnt binds to the LRP 5/6 receptor and the effect of Wnt can be atagonized by Sclerostin when it interacts with the LRP 5/6 receptor leading to inhibition of bone formation. Enzo has exploited the LRP-Sclerostin interaction to produce a system that can identify compounds which block this interaction and could serve as treatments for osteoporosis.

Sclerostin-LRP System Assay Principle


In the Leading LightTM Sclerostin-LRP Interaction System, 96 well plates are coated with Sclerostin and binding of LRP can be seen since LRP5 is linked to the enzyme alkaline phosphatase (AP). After washing steps an AP substrate is added so that LRP5 which remains bound to the Sclerostin on the plates will be revealed by a chemiluminescent signal. Treatments which alter the interaction between Sclerostin and LRP5 can thus be identified based on changes in the luminescent signal which can be detected.


In this application note we found that detection of this chemiluminescent signal can be accomplished in a highly sensitive manner using the LVF-monochromator available on the CLARIOstar®! Using the CLARIOstar® the emission profile for the chemiluminescent signal can be observed. Furthermore, the unique ability of the CLARIOstar® to employ bandpasses up to 100 nm is displayed!


If you would like more information on this and other applications using BMGLABTECH microplate readers please visit the Applications Center on our website!

Wednesday, September 17, 2014

Measuring GPCR signaling with a fast kinetic BRET assay.

G-protein coupled receptors or GPCR's remain a very important subject of study due to their integral role in a variety of physiological processes. For example, the dopamine receptor is a GPCR that is a major target of drugs designed to assist in conditions such as Parkinsons disease. Therefore, continued efforts are being made to monitor the activity of GPCR's in order to identify new treatment options.

In a recent paper in the journal PLOS ONE the authors describe an approach based on bioluminescence resonance energy transfer or BRET to monitor dopamine receptor activation. As with other BRET approaches this assay monitors a protein-protein interaction which in this case is indicative of dopamine receptor activation. A binding peptide was linked to the recently engineered NanoLuc luciferase and a protein that binds to this peptide was linked to the Venus fluorophore. Treatment with agonist leads to association between the peptide and protein which brings NanoLuc into proximity of the Venus leading to a BRET signal.

"Dopamine Electron Map" by Jaelkoury - Rendered on Spartan.
Licensed under Creative Commons Attribution-Share Alike 3.0
via Wikimedia Commons http://commons.wikimedia.org
/wiki/File:Dopamine_Electron_Map.png
#mediaviewer/File:Dopamine_Electron_Map.png
To achieve detection of the BRET signal the authors used a POLARstar Omega plate reader from BMG LABTECH to detect light emitted by Venus and NanoLuc. Furthermore, they were able to perform detection with a 50 millisecond resolution which enabled detailed analysis of activation kinetics in response to dopamine as well as deactivation kinetics.

For more information on the POLARstar Omega and other microplate readers from BMG LABTECH please visit: http://www.bmglabtech.com/

Article citation: J.C. Octeau, et al. "G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization" PLoS One20149(8)

Thursday, September 11, 2014

Applications: BRET beta-Arrestin Interaction Assay

Bioluminescence resonance energy transfer, or BRET for short, has been used for many years now by scientists seeking to monitor molecular interactions such as protein-protein interactions.

General bioluminescence reaction of coelenterazine
by Yikrazuul
The original method used Renilla luciferase (Rluc) and yellow flourescent protein (YFP). In the example of protein-protein interactions one protein is labeled with Rluc and another with YFP. When the proteins associate the Rluc and YFP are brought into proximity, such that when the Rluc substrate coelenterazine is present Rluc produces light whose energy can be transfered to the YFP acceptor.

The BRET method has seen several modifications over the years including NanoBRET which was discussed in a BMG LABTECH webinar last year. However, a recent JBC paper employs the classic BRET approach to analyze the interaction of the long-chain fatty acid receptor, FFA4, with beta-arrestin in order to study which ligands bind to FFA4.

This analysis employed the PHERAstar FS from BMG LABTECH. When the FS is equipped with BRET 1 optic module it can perform simultaneous dual emission detection and measure the luminescent emission at 530 and 490 nm produced by YFP and Rluc respectively.

For more information on how BMG LABTECH equipment can help you measure BRET and many other applications please visit our website: http://www.bmglabtech.com/

Original article citation: B.D. Hudson, et al. "The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)" J Biol Chem. 2014 289(29): 20345–20358

Thursday, September 4, 2014

Applications: Screening for Parkinson's disease treatments

Parkinson’s disease is the second most common neurodegenerative disease that affects 7 million people worldwide. It is an incurable, progressive disease with treatments currently restricted to symptom reduction, with debatable efficacy.

TEM image of mammalian lung showing mitochondria
by Louisa Howard
One of the potential causes of the disease is poorly functioning mitochondria in the neurons of affected
individuals. A recent study has also shown that mitochondria are also abnormal in the skin cells of patients. This observation was used to test skin cells derived from Parkinson’s sufferers for potential drugs that can restore mitochondrial function.  Previous compound screens have typically used toxin induced models of Parkinson’s in unaffected cell lines. The authors adopted the approach that even though their model uses skin cells, as they are patient derived they will be more physiologically relevant.

As a proof of principle, patient fibroblasts were treated with 2000 small molecules, and the effect on their mitochondrial membrane potential was measured using a BMG LABTECH FLUOstar Omega plate reader. After performing confirmation experiments, including checking the efficacy on cells from different patients, 15 compounds were found to have a significant effect on  mitochondria. Of these, 2 were taken for further study and were shown to be effective in models of inherited Parkinson’s disease as well as with neuronal models of the disease.


This paper proposes a very interesting method of screening for compounds with efficacy in cells of individuals affected by Parkinson’s in order to relatively cheaply identify compounds worthy of more extensive, neuron focused investigation.

Original article: 
  • H. Mortiboys
  • J. Aasly
  • and O. Bandmann 
  • Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson’s disease Brain (2013) 136 (10): 3038-3050 

    Tuesday, September 2, 2014

    "Stuck fermentation": an example of prion-based transformation

    Prions are best known for their deleterious effect on higher mammals, for example, in the diseases CJD in humans and BSE in cows. Indeed, BMG LABTECH has provided some instrumenation that has helped with the analysis of these diseases (see application note #232). However, a recent article in Cell, provides an example of a prion-based change that is advantageous, at least to the bacteria and yeast involved.

    S. cerevisiae plasma membrane marked with some membrane
    proteins fused with RFP and GFP markers
    The article entitled: 'Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism' describes how bacteria can communicate with yeast to get them to use carbon-sources other than glucose when it is present.

    Typically, there is a very strong preference for glucose use by yeast, when it is present. This is especially true in strains such as Saccharomyces cerevisiae which is why it has been exploited for the production of ethanol. Biologist have known for years about this 'glucose repression' circuit based in the membranes of yeast cells that blocks yeast from using other carbon sources when glucose is present.

    In the current article the researchers describe how diverse bacteria can induce a stable trait, without affecting the DNA of yeast, through the production of protein-based element. This protein fits the characteristics of a prion in that it: 1) is dominant, 2) is inherited in a non-Mendelian fashion, 3) is transferred without the exchange of nuclei 4) does not involve the mitochondrial genome, and 5) employs a molecular chaperone in propagation. This provides an example of a beneficial trait induced by prions that has likely been established to increase the likelyhood of yeast and bacteria survival in a variety of challenging environments.

    Although the use of this prion is beneficial to the yeast and bacteria involved it is a problem for the wine-makers of the world. However, it is believed that now that the means of communication is understood they will be able to combat 'stuck fermentation' by knocking out the bacteria that can trigger the process, avoiding the introduction of bacteria that can induce the process or using yeast strains that are capable of overpowering the bacteria.

    Some information for this blog post was obtained from the Science Daily article: "Prions can trigger 'stuck' wine fermentations, researchers find"

    Journal Reference: D. F. Jarosz, et al. Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of MetabolismCell, 2014; 158 (5): 1083

    Thursday, August 28, 2014

    Focus on: Novel tuberculosis treatments

    Tuberculosis (TB) is a widely prevalent disease, causing millions of deaths across the world. Although it is treatable, this is difficult and requires administration of multiple antibiotics. There has recently been a growing problem associated with the appearance of resistant strains. There is also a growing problem of tuberculosis amongst cattle in the UK, with the recent badger cull an attempt to address the transmission of TB to cattle.

    Scanning electron micrograph of Mycobacterium
    tuberculosis 
    bacteria, which cause TB


    One of the traditional classes of antibiotics known as caprazamycins target the enzyme MraY, which is involved in the production of peptidoglycans, an integral part of the bacterial cell wall. Some caprazamycin resistant strains are known to overexpress MraY, making MraY expression and activity an important area of research. A recent research paper published in the Journal of Biological Chemistry 1 describes an approach where a caprazamycin derivative was created that is effective against strains overexpressing MraY as it targets WecA, an enzyme involved in the synthesis of an integral part of the cell wall structure. WecA could therefore be a promising novel target for a new class of antibiotics in the fight against antibiotic resistant strains of Tuberculosis.



    In addition, BMG LABTECH recently released an application note for a FRET screening approach to identify modulators of MraY activity entitled: A high-throughput, homogeneous, FRET-based assay to detect bacterial membrane-bound enzyme (MraY) activity

    1. Y. Ishizaki, et al 'Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core, Catalyzed by the GlcN Ac-1-phosphate Transferase WecA, by the Novel Caprazymycin Derivative CPZEN-45' J. Biol. Chem 2013, 288:30309-30319


    1         

    Tuesday, August 26, 2014

    Application: High throughput screening assay to identify anti-malarial drugs

    Malaria is a parasitic disease spread through mosquitos. It is widespread in many areas with poor access to good quality medical care, and is one of the most devastating diseases on the planet, often leading to coma or death. In fact, the World Health Organisation estimate that in 2010, the disease killed between 660,000 and 1.2 million people (1). Although anti-malarial drugs are available, resistance to these is increasing, with potential impact on the health of millions of people, as well as having a major negative effect on the economies of those countries affected.

    A family of ATP dependent plasma membrane pumps have been implicated by Genome Wide Association Studies to have roles in a variety of different conditions. One particular member of this family (PMCA4) has been shown to be associated with resistance to malarial infection. Due to the wide ranging effects of the PMCA family, a group in Manchester devised an assay to screen for potential drugs targeting this group of proteins, using PMCA4 as a model (2).

    To do this, they used the CisbioTranscreener assay which uses FRET technology in combination with long lived fluorophores to assay for ATPase activity with a Z’ of greater than 0.5.  Using this assay, over 20,000 compounds were screened in a single day using the FLUOstar OMEGA, leading to nearly 1500 compounds that inhibited PMCA4 activity by more than 50%.
    The Cisbio Transcreener assay used to identify drugs that target PMCA4
    was detected with the FLUOstar OMEGA from BMG LABTECH

    Although it is very early days in the drug discovery process, someday a descendant of one of these compounds may have an application as an anti-malarial treatment, bringing potential relief to millions of people.

    1.  NayyarGML, Breman JG, Newton PN, Herrington J (2012). "Poor-quality antimalarialdrugs in southeast Asia and sub-Saharan Africa". Lancet InfectiousDiseases 12 (6): 488–96.
    2. Mohamed TM, Zakeri SA, Baudoin F, Wolf M, Oceandy D, Cartwright EJ, Gul S, Neyses L (2013). “Optimisation and validation of a high throughput screening compatibleassay to identify inhibitors of the plasma membrane calcium ATPase pump--anovel therapeutic target for contraception and malaria”. J Pharm Pharm Sci 16 (2): 217-30

    Thursday, August 21, 2014

    Applications: Monitoring Calcium Flux in 3D Heart Tissues in Real Time

    In order for a drug to attain approval for use it must not only be shown that it has the desired effect but that it does not have undesirable side-effects. Traditionally, laboratory animals are used to predict possible side-effects but the animals may respond differently than humans so an effective in vitro system is highly desirable.

    In order for an in vitro system to be most useful it should best mimic the in vivo tissue. For this reason 3D cell culture approaches have become more prevalent and have proven to exhibit fewer misleading results than their 2D counterparts.

    The scientists at InvivoSciences have recognized the importance of using 3D cultures for drug assessment and have met the challenge of creating a suitable in vitro system with their Mini-Construct ChambersTM .

    The CLARIOstar® captures periodic Ca2+-transients 
    that induce cardiac contractions

    In an application note now available on the BMG LABTECH website InvivoSciences scientists describe the use of the CLARIOstar® to monitor the function of engineered heart tissues. A calcium indicator was used to monitor the calcium transients that induce cardiac contractions. The ability of the CLARIOstar® to read 100 data points per second allow it to measure the rapid changes in fluorescence intensity that are associated with the calcium flux from a beating heart.

    Together, BMG and InvivoSciences can provide a platform for the analysis of drug effects on heart function and do so at the early of stages drug discovery!

    BMG LABTECH Application Note 253: Real time calcium flux measurements in iPSC derived 3D heart tissues.

    Tuesday, August 19, 2014

    New software enables detection of disease associated insertion deletion mutations

    Insertion deletion mutations (indel mutations or indels) can have a profound effect on biological function. As their name suggests this is when genetic sequence is either added or removed.  The effect of these mutations is especially evident when these mutations occur in a protein coding region as they can lead to the production of proteins that are truncated or otherwise altered so that function is severely impacted. However, these indel mutations can vary in length, from 1 base pair to thousands. Furthermore the sequence that is inserted or deleted can be highly variable.The variability of indels has made them difficult to detect within the genome, until now!

    Photograph of CSHL in the fall of 2008
    by AdmOxalate
    A group of scientists at Cold Spring Harbor Labs have developed and used a new software program called Scalpel to precisely see where indels are causing cuts in the genome. The results of their work are published in a recent issue of the journal Nature Methods in an article entitled: 'Accurate de novo and transmitted indel detection in exome-capture data using microassembly'.



    The group used Scalpel on a genome database produced from families in which one child suffered from autism and the rest of the family is unaffected. This approach allowed them to identify indels that likely disupt genes in autistic children.

    The scientist are now seeking to apply Scalpel more broadly and are collaborating with any scientists, such as cancer biologists, that are looking for indels.

    Some material for this post was obtained from the Science Daily article: A shift in the code: New method reveals hidden genetic landscape

    Original Article citation: Giuseppe Narzisi, Jason A O'Rawe, Ivan Iossifov, Han Fang, Yoon-ha Lee, Zihua Wang, Yiyang Wu, Gholson J Lyon, Michael Wigler, Michael C Schatz. Accurate de novo and transmitted indel detection in exome-capture data using microassemblyNature Methods, 2014

    Thursday, August 14, 2014

    Applications: A novel ultrahigh-throughput fluorescence anisotropy-based assay for ATP-competitive inhibitors of TilS

    TilS (tRNAIle lysidine synthetase) is an essential, ATP-dependent enzyme which is conserved in bacterial pathogens. The high degree of conservation of TilS among pathogenic bacteria combined with its absence in human nuclear and mitochondrial genomes make it an attractive potential target for novel antibacterial drugs.

    E. coli
    With this in mind scientists at AstraZeneca created a screening platform which is described in the recent Journal of Biomolecular Screening article entitled: ‘Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase by High-Throughput Screening’. To enable this screening these scientists used ATP linked with either the fluorophore BODIPY or TAMARA. Binding of the labeled ATP to TilS enzymes from E. coli and S. aureus could be measured by an increase in fluorescence anisotropy and treatment with an ATP-competitive inhibitor would displace the labeled ATP leading to a decrease in fluorescence anisotropy.

    We at BMG are happy that our PHERAstar HTS microplate reader could be used in this novel screening approach!


    For more information on the PHERAstar and other BMG LABTECH microplate readers please visit our website: www.bmglabtech.com

    Thursday, July 31, 2014

    Applications: Measuring Tryptophan UV Fluorescence with the CLARIOstar

    Tryptophan 3-D Model

    Tryptophan is an essential amino acid that is also a precursor for other compounds important for normal function such as vitamins. Furthermore, tryptophan has been characterized for its fluorescent characteristics which have been employed in research endeavors such as protein folding analysis. BMG LABTECH has now characterized our newest instrument the CLARIOstar for its ability to perform tryptophan detection. The results of this characterization are presented in Application Note 254:' Tryptophan quantificaiton using UV fluorescence measurements on the CLARIOstar mult-mode microplate reader'.


    The CLARIOstar has a unique system for wavelength selection with its LVF monochromator and  can use filters when necessary. In this application note another unique capability of the CLARIOstar is on display, it can combine the use of filters and monochromator! In this case a filter was used for UV excitation at 280 nm and the monochromator used to detect emission at approximately 360 nm. Furthermore, the CLARIOstar can automatically select the appropriate dichroic setting with its adjustable dichroic. The results for tryptophan sensitivity obtained with a filter in combination with the monochromator were compared to those obtained with filters alone and showed again the filter-like performance of the LVF monochromator!

    For more information on the capabilities of the CLARIOstar please visit: www.bmglabtech.com

    For a copy of this application note please the visit the Applications Center on BMG's website.

    Monday, July 28, 2014

    New study provides evidence for direct role of epigenetic changes in cancer development

    Epigenetics, the ability to change a genes expression without changing the DNA sequence, has long been proposed as a mechanism to turn off the expression of certain genes and thus lead to cancer in a fashion similar to that seen as the result of a genetic mutation. However, until now direct evidence that this was indeed the case was lacking. In a recent article in the Journal of Clinical Investigation, scientists at Baylor College of Medicine describe the creation of a mouse model that provides evidence that epigenetic changes, specifically DNA methylation, can cause cancer.

    This work appears in the article entitled: 'Targeted p16Ink4a epimutation causes tumorigenesis and reduces survival in mice'. The article describes the modification of the promoter for p16, an important tumor suppressor gene, such that DNA methyltransferases were attracted to this site and hypermethylation occured, with the anticipated result of p16 gene silencing. The authors provide a variety of controls to indicate that the loss of p16 is due to hypermethylation of its promoter and show that the result of this loss was a higher incidence of cancer and reduced rate of survival.

    This image shows a DNA molecule that is methylated
     on both strands on the center cytosine.
    DNA methylation plays an important role for epigenetic
    gene regulation in development and cancer.
    by Christopher Bock
    This work represents just the latest evidence of the importance of epigenetic study. We at BMG recognize this importance and the resulting need for tools to identify possible epigenetic treatments. This is why we have recently published application notes which employ the PHERAstar FS to identify possible modifiers of epigentic enzymes.

    For more information on how microplate readers from BMG LABTECH can help with epigenetic or other screening assays please visit our website: http://www.bmglabtech.com/

    Some information for this post was obtained from the Science Daily article: 'Epigenetic changes can drive cancer, study shows'


    Original article citation: Da-Hai Yu, Robert A. Waterland, Pumin Zhang, Deborah Schady, Miao-Hsueh Chen, Yongtao Guan, Manasi Gadkari, Lanlan Shen. Targeted p16Ink4a epimutation causes tumorigenesis and reduces survival in miceJournal of Clinical Investigation

    BMG LABTECH application notes:

    247 Screening for Histone Deacetylase (HDAC) Active Compounds

    249 Assessing Epigenetic Enzyme Activity using HTRF® Epigenetic Assays from Cisbio with the PHERAstar FS from BMG LABTECH

    Thursday, July 3, 2014

    Applications: Assessing Snake Venom for Hemoglobin-modifying Activity

    BMG LABTECH in cooperation with Venomtech recently released an application note which describes the use of the PHERAstar FS to create a simple 384 well test for the ability of snake venom to modify hemoglobin. Although snake venom is well characterized to effect hemostasis, there is little literature covering the direct effect of venom on hemoglobin.
    A time-dependent change in hemoglobin absorbance is
    seen when mixed with the venom for the black-necked
    spitting cobra


    In this application note the spectrometer available on the PHERAstar FS was employed to obtain the absorbance spectrum from 400-700 nm of samples blood samples treated with venom from a variety of snake species. Using BMG LABTECH's ultra-fast spectrometer these readings can be obtained in less than one second per well.



    The results showed that characteristic absorbance peaks for blood at 540 and 570 nm were lost in the presence of the venom from some snake species. Furthermore, the effect was time dependent. Based on this and other evidence the observed effect is believed to be enzyme-mediated.

    For more information please go to the BMG LABTECH website and visit the Applications Center.

    Application note # 251: Identification of novel haemoglobin-modifying activity in snake venom libraries using the PHERAstar FS

    Wednesday, July 2, 2014

    Brighter fluorescent proteins could improve biotechnology applications

    Green fluorescent proteins (GFP's) and their various relatives with other colors have become important tools for tracking gene expression and monitoring protein-protein interactions, as well as other applications. A recent study in Scientific Reports could improve our understanding of how GFP's can be brighter and therefore improve the signal-to-noise in these applications.

    GFP structure
    by Richard Wheeler
    The results of this study are found in the article entitled: 'Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus'. This paper is a detailed analysis of the GFP's in Amphioxus, a small marine invertebrate also know as lancelets. This animal has two forms of GFP, one which is very bright, in part due to 100% quantum efficiency and one that has low (0.1%) quantum efficiency. The comparison of these two GFP's showed that in the bright GFP the interaction of 3 amino acids yielded a change in protein conformation and increased stability which are believed to improve GFP brightness. The authors believe that by understanding the relationship between structural environment and the level of brightness current FP's can be made brighter thus improving their performance in a wide variety of biotechnology applications.


    Many of the GFP biotechnology applications can be performed on microplate readers like those from BMG LABTECH!

    Original article: E.K. Bomati, et al, Spectral and structural comparison between bright and dim green fluorescent proteins in AmphioxusScientific Reports, 2014; 4

    Some information for this post was taken from the Science Daily article: Behind a marine creature's bright green fluorescent glow

    Friday, June 27, 2014

    Fun fact: LEGO bricks used to study plants

    LEGO bricks
    by Priwo

    A group of scientists at the Iowa State University were seeking to find a frugal alternative that would allow
    them to hold soil substitutes for germinating and growing plants. They wanted something that was modular, scalable, and capable of housing simultaneous experiments.


    They found what they were looking for with LEGO blocks, which despite being a toy, are made of high quality plastic, held to very precise standards. And, of course, you can build things with them! Which they did! Their results are reported in the PLoS ONE paper entitled : LEGO Bricks as Building Blocks for Centimeter-Scale Biological Environments: The Case of Plants.




    Some information for this post obtained from the Science Direct article: LEGO bricks turned into scientific tool to study plant growth

    Thursday, June 26, 2014

    Application: Monitoring hydrogen peroxide production in cell monolayers

    Hydrogen peroxide (H2O2) has been characterized for its role in a wide variety of biological process. Some beneficial, such as regulation of immune system function, and others viewed detrimental, such as involvement in aging. Therefore, there are many instances where understanding the cellular level of H2O2 would be useful. Application note 252, recently added to BMG LABTECH's website describes the use of the PHERAstar FS to detect a genetically encoded redox probe specifically designed for H2O2 detection.

    Lung adenocarcinoma H1975 cells expressing the cytosolic
    roGFP2-Orp1 probe. Cells were exposed to hydrogen 
    peroxide as indicated, and the probe response followed using 
    the PHERAstar FS
    This note entitled: 'Real-time monitoring of genetically encoded redox probes in mammalian cell monolayers' describes a very useful tool that can be used to monitor cellular redox changes. This note employs biosensors based on redox sensitive green fluorescent protein (roGFP). RoGFPs are GFPs that have had 2 surface exposed residues changed to cysteines. These cysteines are in appropriate positions to form a disulfide bond. In the oxidized state this bond is strained and the protein contains localized structural changes which are believed to be responsible for the spectral changes of roGFP that change the excitation maxima from 485 nm to 400 nm. Furthermore, roGFP can be engineered to respond to specific redox species. In this case,  fusion to the thiol peroxidase Orp1 generates a H2O2 sensitive roGFP.

    The PHERAstar FS proved to be an excellent tool for detecting changes in H2O2 using this biosensor. To find out more please visit the Applications Page on BMG LABTECH's website.

    Application note: #252 Real-time monitoring of genetically encoded redox probes in mammalian cell monolayers

    Wednesday, June 25, 2014

    Did you know your microbiome can be detected on your phone?

    Structure and shape of E. coli 70S
    ribosome. For the 30S subunit, the
    16S rRNA (dark blue) is shown.
    by Vossman


    According to a study published recently in Peer J. the bacteria that are present on a cell phone closely resembles that which was sampled from the owners fingers. The analysis, performed by University of Oregon scientists, was a proof of concept experiment that used short-read 16S sequencing to categorize the whole microbial community.

    This type of sequencing refers to sequencing of the gene for the 16S ribosomal RNA component of a subunit of the prokaryotic ribosome. Previous work has indicated that this gene is highly conserved in bacteria species and thus can be used in phylogenetic studies such as this.

    While it may not be surprising that the bacteria that reside on our hands can also be found on our phones, the authors propose that the phones could be used as a non-invasive way to monitor our health. The uses of this screening could include real-time assessment of possible exposure to pathogens that could be carried into and out of a medical facility.

    Some information was obtained from the Science Direct article:  Cell phones reflect our personal microbiome

    Original article: J.F. Meadows et al Mobile phones carry the personal microbiome of their owners. Peer J, 2014

    Thursday, June 19, 2014

    How well do we understand water?

    Water is all around us and inside us, it is essential for life as we know it.

    It's chemical formula is one that nearly everyone knows regardless of their background. Water has a very simplistic structure as well so what else can we learn about water?

    It turns out there is still a lot we can learn about water as was exhibited by two papers that were published in a recent issue of the journal Nature.

    Image of the electron localization of water
    by JVertrees
    One aspect of water which we still can't fully explain is why it floats when it freezes. This characteristic is unlike other molecular substances. Without this characteristic marine life could not survive as lakes, rivers and oceans could freeze solid. To investigate the properties of water as it freezes, scientists at Princeton University used computer modeling. Their results indicate that at very cold temperatures and above a certain pressure water actually splits into two different liquid phases. This finding supports the 'liquid-liquid transition' proposed by Eugene Stanley in 1992. The authors propose that these two liquid phases may be responsible for the areas of low and high density that are known to exist in forming ice. As ice solidifies the low density regions predominate leading to a solid form that is less dense than the liquid form. If this dual nature can be observed in experiments it could improve the predictive ability of our weather and climate models.

    The second paper could go a long way toward providing the experimental evidence necessary to study water under different conditions. In this paper scientists at the Stanford Linear Accelerator Center used the Linac Coherent Light Source X-ray laser to collect rapid-fire snapshots of waters molecular structure in the instant before it freezes.

    For their experiments the researchers produced a steady stream of very pure water droplets in a vacuum that were directed toward a laser that was pulsing with a femtosecond shutter speed. As the water traveled towards the laser beam some of the liquid evaporated supercooling the remaining water to temperatures below minus 42 F ( -41 C). Their results showed the molecular structure of water is continuously transforming just before freezing and further cooling dramatically accelerates the structural changes observed. It is hoped that by pushing these experiments into colder regions closer to the transition to a solid that they will be able to identity the theories that best explain waters behavior.

    Some information for this post was obtained from the Science Daily articles:

    Familiar yet strange: Water's split personality revealed by computer model

    Scientist take first dip into water's mysterious 'no man's land'

    Original articles are:

    J.C. Palmer et al Metastable liquid–liquid transition in a molecular model of waterNature, 2014; 510 (7505): 385 

    J.A. Sellberg et al  Ultrafast X-ray probing of water structure below the homogeneous ice nucleation temperatureNature, 2014; 510 (7505): 381 

    Thursday, June 5, 2014

    Applications: HTRF Epigenetic Assays using the PHERAstar FS


    Epigenetics remains a hot topic in the biological scientific community due to its role in cancer and autoimmune diseases. This field describes how the regulation of gene activity and expression are not solely dependent on genetic sequence but can be influenced by DNA methylation and histone modification.

    Enzyme inhibition curves for three G9a compounds

    In this application note we verify the performance of a Cisbio HTRF® assay that allows you to detect the activity of the histone methyltransferase G9a. G9a is a member of a class of enzymes that mediate the methylation of histone H3 and is an excellent epigenetic target due to its reported role in embryogenesis and cancer cell proliferation.

    The use of laser excitation, HTRF® optimized optic module and matched PMT’s dedicated for TRF detection make the PHERAstar FS an excellent choice to perform this assay. Combined with BMG LABTECH’s MARS data analysis software you will be able to perform enzyme inhibition curve analysis quickly and easily with high quality data.


    For more information please visit the Applications Page on BMG’s website or access the application note at the following web address:

    Monday, June 2, 2014

    Experimental drug could be useful MERS treatment

    MERS virus 3-D image
    by Scinceside
    MERS (Middle Eastern Respiratory Syndrome) has been featured in the news of late due to the spread of the disease globally.

     The virus that causes MERS belongs to a group called coronaviruses. Currently, no treatment for coronaviruses exists. However, a chemical called K22 was recently shown to inhibit the growth of various strains of coronavirus, including those that cause MERS.



    The results are reported in the PLOS pathogens article entitled:'Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus.' The work is a collaborative effort of scientist at a number of European Universities and describes how K22 inhibits viral RNA synthesis and the formation of double membrane vesicles. Therefore, K22 halts viral replication at a very early stage. The authors propose that the recruitment of cellular membranes for viral replication represents a druggable target for antiviral treatment and expect to be able to exploit this to develop new drugs to combat viral infections!

     The original article can be found at:

    http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004166