Friday, June 28, 2013

Focus on: Telomeres and Cancer

Telomeres are the protective extensions on the end of chromosomes which shorten during the life of a cell each time it divides. Without these protective extensions the chromosome would lose genetic material and be susceptible to fusion with other chromosomes. Usually the shortening of the telomere is a signal to end the lifespan of a cell through senescence or cell death. However, in cancer cells; telomeres are shorter than in normal cells but are maintained at a constant short length by telomerase. Telomerase is an enzyme that adds length to the telomeres which allows the cells to continue to grow.

An image showing how telomerase elongates
telomeric DNA progressively

Fatma Uzbas
A research group from Japan was curious why cancer cells had shorter telomeres than normal cells. They published the results of there studies in the recent Molecular and Cellular Biology paper entitled 'Telomere length influences cancer cell differentiation in vivo'. In the paper they elongated the telomeres of human prostate cancer cells by enhancing telomerase activity. The result was cancer cells that had long rather than short telomeres. These cells retained the ability to form tumors, however, they had hallmarks of a more differentiated cell and lacked the expression of genes associated with poor prognosis cancers. The authors conclude short telomeres contribute to the malignancy of the tumor by affecting differentiation of the cancer cells and propose a potential role for telomeres in normal cell differentiation.




Thursday, June 27, 2013

Identification and characterization of novel PAM's of the Galanin 2 receptor

Nociceptive pathways
The galanin system is implicated in nociception or the perception of painful stimuli. As a result the galanin system, and specifically the galanin 2 receptor (GalR2) has been identified as a potential target for novel therapeutics. In BMG LABTECH application note 236 the authors describe the screening procedure used to identify positive allosteric modulators of GalR2 using the PHERAstar microplate reader.


In the application note the authors exploit the fact that GalR2 is a G-protein coupled receptor and used the HTRF IP1 assay from Cisbio as a read out of the activity of this receptor. Using this assay a high throughput screen was performed which was simplified by the availability of an HTRF optic module for the PHERAstar from BMG LABTECH. The screen exhibited strong statistical results which indicate that the approach employed is indeed suitable for high through put and can be used for additional screening as necessary.

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

Wednesday, June 26, 2013

Fun facts: On this date in science history

William Thomson, 1st Baron Kelvin (1824–1907)
On June 26th in 1824 William Thomson was born, although you may know him better as Lord Kelvin.

He was a child prodigy in the field of mathematics who began his studies at Glasgow University at the age of 10 and published the first of his more 600 scientific papers when he was 16.

Lord Kelvin is, of course, best known for his studies on heat and specifically for his 1848 paper: 'On an Absolute Thermometric Scale.' In this paper he wrote of the need for a scale where absolute zero was the null point and which used the degree Celsius for the unit increment. In this paper he also calculated that absolute zero is equivalent to -273 Celsius. This absolute scale is known today as the Kelvin thermodynamic temperature scale.

Tuesday, June 25, 2013

FAQ: Why does transcription occur primarily in the direction of genes?

Surface model of RNA polymerase II from yeast, chains colored uniquely
DNA transcription occurs when RNA polymerase binds to unwound DNA at a promoter. RNA polymerase can bind to either strand of DNA and can therefore proceed in the sense direction (toward the gene) or anti-sense direction (away from the gene). However, productive elongation occurs primarily in the gene coding direction in mammalian species. An article published in a recent issue of Nature provides some insight into why gene coding elongation is preferred.


In an article entitled: 'Promoter directionality is controlled by U1 snRNP and polyadenylation signals' the authors from MIT describe two processes that confer the bias toward gene coding elongation. The authors propose that the asymmetric presence of polyadenylation sites and U1 small nuclear ribonucleoprotein binding sites is at the heart of this phenomenon.

The role of polyadenylation sites in the cessation of transcription and aid in nuclear export of mRNA transcribed from genes is well characterized. These polyadenylation sites also appear to be abundant in regions in the anti-sense direction proximal to a promoter. This leads to the early cessation of transcription in this direction.

In contrast U1 small nuclear ribonuculeoprotein binding sites are more abundant in the sense direction proximal to the promoter. This protects the elongating mRNA from early termination and cleavage. The corresponding paucity of binding sites in the antisense direction combined with the early cessation associated with increased polyadenylation sites leads to short RNA's produced in this direction which are rapidly cleaved.

This situation is clearly preferential from a standpoint of energy consumption; production of long non-coding RNA's would be an unnecessary burden. This is especially relevant when you consider the fact that only about 15 percent of the genome encodes for protein-coding genes.

Thursday, June 20, 2013

Dual Luciferase Reporter (DLR) Assay Certification on the CLARIOstar® using LVF monochromators™

The Dual Luciferase Reporter Assay is an essential tool used in the studies of transcriptional regulation. As such, it important that the capability of existing microplate readers is confirmed for the performance of this assay. This is the basis for the DLReady certification program. BMG LABTECH is proud to say that the new CLARIOstar has passed this certification process and the results that led to this certification are presented in BMG application note #239.

Dual Luciferase Reaction: Luciferase Assay Reagent II
(LAR II) is injected in step 1 to initiate the Firefly
luciferase reaction. In the second step the injection of Stop
and Glo® step quenches the Firefly reaction while starting
the reaction for Renilla Luciferase.
In order to attain DLReady certification 3 criteria must be attained based on three tests. 1) A quenching experiment which indicates that the Stop and Glo injection sufficiently suppresses the reaction produced by Luciferase Reagent II. 2) A consistency experiment that shows that replicates at different concentrations of Firefly and Renilla luciferase deviate by less than 5% CV. 3) A tubing adsorption experiment that shows that reproducible results are obtained after waiting for 10 minutes. The CLARIOstar exceeded all criteria and has attained official DLReady certification!

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

Wednesday, June 19, 2013

Fun fact: Popular sushi item contains the first vertebrate fluorescent protein

The Japanese freshwater eel has long been a staple at sushi bars. However, based on a report from Cell this eel may hold the key to advancing biological imaging. The report entitled: 'A Bilirubin-Inducible Fluorescent Protein from Eel Muscle' describes the cloning and characterization of UnaG; the first vertebrate fluorescent protein.

Anguilla japonica the Japanese freshwater eel

The authors show that UnaG belongs to the fatty-acid binding protein family whose fluorescence is induced by binding bilirubin rather than using a chromophore; the portion of a common fluorescent protein molecule that absorbs excitation light energy at a specific wavelength and produces colored light at a distinct emission wavelength. Bilirubin is a heme metabolite which has been used in hospital tests for liver function. As a result the authors also designed a human bilirubin detection assay that has potential clinical application as it is simpler, more sensitive and uses smaller sample volume.

Another interesting characteristic of UnaG is that it fluoresces in anaerobic environments. This may allow it to be used to analyze cells where oxygen levels are low such as the interior of tumors. The possible implications of this new ligand-induced fluorescent protein are numerous and we at BMG LABTECH are interested to see where this leads the field of fluorescent detection!

Tuesday, June 18, 2013

FAQ: Is the CLARIOstar certified DLReady?

image of the thermostable Firefly Luciferase
(Photinus pyralis), taken from pdb 2d1s.
In yellow are the bound Adenosine and luciferin analoge.
 Drawn by PyMOL
Author: Yikrazuul
Based on the results presented in BMG application note 239 we can proudly answer: yes!

In order to attain DLReady status a microplate reader must show that they meet several performance criteria. The CLARIOstar exceeds all these criteria. Although this is not unexpected as other microplate readers from BMG LABTECH, such as the Omega and Optima, have previously received certification it is useful to have the performance of the CLARIOstar confirmed.

The DLR (Dual Luciferase Reporter) Assay remains a frequently used approach to perform transcriptional regulation analysis. The DLR certification of the CLARIOstar is one more indication that with the CLARIOstar anything is possible!


Monday, June 17, 2013

Did You Know: The New CLARIOstar is Certified DLReady?

Data from MARS data evaluation software shows
a certification experiment graphically


The Dual Luciferase Reporter (DLR) assay remains a common approach to perform gene expression analysis. Therefore, it is important to verify that a microplate reader is capable of performing this commonly used assay. Application note 239 from BMG LABTECH shows some of the results that allow the CLARIOstar to be certified DLReady.

This result comes as no surprise when you consider that all BMG microplate readers that have been tested for certification have exceeded the standards necessary to attain certification. You can be confident that a BMG microplate reader will be an excellent tool for your gene expression analyses using DLR.

Thursday, June 13, 2013

Applications Thursday: High Throughput Screening for FEN 1 Inhibitors Using a Fluorescence Polarization Assay.

Cartoon representation of the molecular structure of FEN 1
Jawahar Swaminathan and MSD staff at the
European Bioinformatics Institute

Flap endonuclease 1 (FEN 1) is a highly conserved endonuclease that is involved in a number of DNA replication and repair pathways. Overexpression of FEN 1 has been observed in multiple cancers and down regulation of FEN 1 sensitizes cells to chemotherapeutic agents. As of now, no inhibitors of FEN 1 that are sufficient to be pursued as drugs have been identified. Therefore, a research group from AstraZeneca sought to design a high throughput screening approach using the PHERAstar microplate reader from BMG LABTECH to identify novel inhibitors of FEN 1. The results of their efforts are published in the current issue of the Journal of Biomolecular Screening.

A FEN 1 oligonucleotide substrate was designed which incorporated a fluorescent label and included key features which have been shown to be important for FEN 1 specificity. The assay then uses fluorescence polarization performed on the PHERAstar to monitor the cleavage of this substrate. The PHERAstar features easy to use assay specific optic modules and a module designed to detect to fluorescence polarization was used in this assay. After assay validation, a screen of 850,000 compounds was performed. A Z' of greater than 0.6 was observed throughout the screening indicating that the PHERAstar is important in delivering a simple and robust assay even in low volumes; like the 1536 well plates used in this assay. In the end 6261 compounds were confirmed as FEN 1 inhibitors.

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

Article citation: Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

Wednesday, June 12, 2013

Fun fact: On this date in science history

Gossamer Albatross in flight.
Author: NASA
On June 12, 1979 the Gossamer Albatross completed a successful crossing of the English Channel.

The Gossamer Albatross was a human-powered aircraft that was designed and built by a team led by Paul B MacCready. It used pedals to drive two large bladed propellers and was piloted by Bryan Allen on the June 12 flight. The 22.2 mile crossing was completed in 2 hours 49 minutes at an average altitude of 5 feet. It achieved a top speed of 18 miles per hour.

The back-up plane the Albatross II is currently on display at the Museum of Flight in Seattle, WA.

Tuesday, June 11, 2013

FAQ: How do bacteria become resistant to antibiotics?

Scientist continue to struggle to answer the question of how bacteria obtain antibiotic resistance. However, a recent paper published in Nature entitled: 'Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome' suggests that bacteriophages may be involved.

Transmission electron micrograph of the bacteriophage Qβ
attached to sex pilus of the bacterium Escherichia coli

 by Dr Graham Beards
Bacteriophages, or simply phages, are viruses that infect bacteria. Phages are known for their capacity to move genes from one bacteria to another. With this in mind, the scientists from Boston University and Harvard, decided to focus on the effect of drug treatment on phages rather than bacteria. They gave mice one of two commonly prescribed antibiotics and subsequently harvested viruses from the fecal matter after 8 weeks. They then identified the viral genes present by comparing them to a database of known genes.

The results showed that phages from antibiotic treated animals did indeed carry a higher number of genes for drug-resistance. Furthermore, they were able to transfer the resistance genes to bacteria. When phages isolated from antibiotic treated mice were combined with bacteria from untreated mice the researchers saw an increase in antibiotic resistance that was 2 to 3 times what is normally seen.

These results open the door for new avenues of treatment that target the phage rather than the bacteria. Assessing viral susceptibility to drug treatment is a potential application that could employ the NEPHELOstar Plus from BMG LABTECH. Since a nephelometer detects insoluble particles with high sensitivity using a laser, the NEPHELOstar Plus has the potential to be able to monitor changes in the amount of virus in a sample and to do so in a microplate format.

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

Monday, June 10, 2013

Did you know: A potential drug target for treatment-resistant anemias has been identified?

Erythropoietin Created with data set "1buy"
from Protein Data Bank and the free program PyMOL

Anemia occurs when the multistep process that creates red blood cells breaks down. This process is called erythropoiesis. Common anemias can be treated with a recombinant hormone erythropoietin (EPO). However, some anemias fail to respond to EPO treatment. When EPO fails to help anemic patients doctors commonly turn to glucocorticoids, which have been shown to increase the number of times that red blood cell progenitors divide before they are induced to differentiate by EPO. Unfortunately, glucocorticoids have a number of negative side effects including: loss of bone density and immune suppression.

Scientists at Whitehead Institute have identified a protein whose expression is upregulated by glucocorticoids. They reported their findings in the Nature article entitled: 'ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.' They found that ZFP36L2 binds to mRNA's that would normally lead to differentiation. Therefore, when ZFP36L2 is present continued proliferation of red blood cell progenitors is favored over differentiation.

With this new information in hand it will now be possible to screen for other compounds that similarly increase expression of ZFP36L2. Hopefully this will identify a compound that will have the desired effect on erythropoiesis without the negative side effects of glucocorticoids.

The microplate readers available from BMG LABTECH are ideally suited to the high throughput protein expression analysis that will be required to screen compounds that increase ZFP36L2. For more information on how BMG LABTECH readers like the PHERAstar or CLARIOstar can assist your protein expression analyses please visit the Applications Center on the BMG LABTECH website.


Friday, June 7, 2013

Focus on: Events

Once again, BMG LABTECH will be attending meetings throughout Europe next week!

BMG LABTECH will be at Oxygen 2013 on June 8 -12 in Oulu, Finland. The focus of this meeting is dealing with hypoxia and BMG LABTECH is proud to serve as one of the meetings sponsors.

On June 11-13 BMG LABTECH will be hosting a workshop in Hamburg, Germany in association with the EPR. This practical workshop will focus on cell based assays for screening.

Finally, if you are at the Dundee University Supplier Trade Fair on June 13 please introduce yourself to the BMG LABTECH representatives who will also be in attendance.

For more information on these and other upcoming events that BMG LABTECH will attend please visit the BMG LABTECH Events Page on our website.





Thursday, June 6, 2013

Applications Thursday: HTS Assay for SUMO proteases based on AlphaScreen

The current issue of JBS has a technical note from scientists at Duke University entitled 'Development of a High-Throughput Screening Assay for Inhibitors of Small Ubiquitin-Like Modifier Proteases'. The assay is based on developing a substrate whose cleavage can be monitored using AlphaScreen technology and employs the PHERAstar microplate reader from BMG LABTECH.

structure schematic of human SUMO1 protein made with
iMol and PDB file 1A5R, an NMR structure; ribbon view
Jakob Suckale at en.wikipedia
Small ubiquitin-like modifiers (SUMO 1-3) are a group of proteins which can be ligated to target proteins at lysine residues. SUMO conjugation/deconjugation is a highly dynamic process which plays a pivotal role in a number of pathological conditions such as diabetes and cancer. Therefore, it is of clinical significance to find compounds that interfere with the conjugation/deconjugation pathway. The current paper describes a specific HTS assay to discover SUMO protease inhibitors.

The technical note describes how the authors used a bacterial SUMOylation system to create a substrate that had SUMO3 modified with streptavidin conjugated to SUMO 2 which was His-tagged. In order to employ AlphaScreen this intact substrate is mixed with Strep-Tactin donor beads and Nickel-Chelate acceptor beads. Therefore in the absence of protease (or protease activity) the substrate will remain intact and excitation at 680 nm will lead to an emission signal at 520-620 nm; while if protease activity is present a decrease in emission signal will be detected. When run in a 384 well format and detected using the PHERAstar from BMG LABTECH they observed an average Z' = 0.83 indicating that this assay is indeed suitable for HTS applications.

The PHERAstar and CLARIOstar from BMG are both ideally suited to the performance of AlphaScreen as described in this application as either can be equipped with a red diode AlphaScreen laser.

For more information please visit the BMG LABTECH website: www.bmglabtech.com

Wednesday, June 5, 2013

Fun fact: Genomics and particle physics are the two hottest areas of scientific research

An example of simulated data modeled for the CMS particle
detector
on the Large Hadron Collider (LHC) at CERN.
http://cdsweb.cern.ch/record/628469
Lucas Taylor
For the third straight year genomics research produced the most highly cited scientific papers. This indicates the central importance of genetics in biology and medicine to help us understand how DNA sequences lead to disease. The highly cited genetic research papers include the studies that sequenced the genome of a cancer patient in order to find genetic indicators of disease development.

Most of the citations in particle physics were related to the search for the Higgs boson particle. This research is viewed as vital in describing the formation of the universe. The top two most cited papers both dealt with the Higgs boson particle.

The citation data was tabulated by Science Watch which is a division of Thomson Reuters.


Monday, June 3, 2013

Did you know: There is a new way to produce DNA oligonucleotides?

Schematic showing how antisense DNA prevents protein translation.
RNAi Therapeutics: How Likely, How Soon? Robinson R PLoS Biology
 Vol. 2, No. 1, e28 doi:10.1371/journal.pbio.0020028
by Robinson R

DNA oligonucleotides have a number uses including as antisense inhibitors of protein synthesis and as oligonucleotide based drugs. In order for them to be used as drugs the product must be very pure and should be inexpensive. A recent report describes a technique that could address both issues.

A paper published in the online version of Nature Methods on June 2nd describes the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. The work in the paper entitled: 'Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides' is a collaboration between U.S. and Swedish scientists.

Single-stranded DNA oligonucleotides are typically produced using polymer chemistries in which the number of errors increases as oligonucleotide length increases which can lead to problematic sequence diversity. MOSIC uses bacteria to amplify the oligonucleotides and allowed for the preparation of oligonucleotides up to 378 nucleotides in length. By using bacteria they are able to improve quality while making it possible to scale-up production and produce larger quantities of DNA cheaply.