Thursday, February 28, 2013

Applications Thursday: Multiple applications, one reader

A recent article published by Ndjomou et al in the journal Antiviral Research highlights the ability of the FLUOstar Omega to perform a variety of applications. The paper entitled: 'Fluorescent Primuline Derivatives Inhibit Hepatitis C Virus NS3-Catalyzed RNA Unwinding, Peptide Hydrolysis and Viral Replicase Formation' used the microplate reader from BMG LABTECH in four separate applications.

WHO-Guide: Hepatitis C, 2002
The effect of the compounds being studied on HCV replication was assessed using an HCV Renilla luciferase reporter gene assay that employed the optional on board injectors available on the FLUOstar Omega and the luminescent detection capabilities. Luminescent detection was also used in a cell viability assay to assess compound cytotoxicity.

The fluorescence detection capabilities of the FLUOstar Omega were used in an NS3 protease activity assay that employed a 5-Carboxyfluorescein-labeled substrate. Fluorescence detection was also employed in a  molecular beacon-based helicase assay (MBHA) in which DNA or RNA test strands were labeled with fluorophores and/or quenchers which are initially annealed to a complimentay strand. The released strands were designed to form hairpins to prevent reannealing. Therefore helicase activity could be determined based on a change in fluorescence intensity.

In the end 4 distinct tests, using 2 detection modes on 1 multi-functional microplate reader from BMG LABTECH.

Wednesday, February 27, 2013

Fun fact: Saccharin was discovered on this date in 1879

Ball-and-stick model of the saccharin molecule, C7H5NO3S,
as found in the crystal structure
by Ben Mills
In what would now be considered poor laboratory practice Constantin Fahlberg began to consume a meal with hands unwashed after leaving his work. He accidentally discovered saccharin when the bread and water that he was eating at the start of his meal were uncharacteristically sweet due to the residue that was on his hands. Then, in his excitement, he rushed back to the lab and proceeded to taste the contents of the beakers and evaporating dishes one of which contained impure saccharin derived from the coal tar which he was studying.

As with many fortuitous discoveries that is when the real work started. Constantin worked for months determining the modes of production, characteristic reactions and chemical composition of saccharin. Though initially disregarded by the scientific community
his discovery was embraced by the public
 and brought the chemist wealth when he
established a production facility.

For more information on Constantin Fahlberg and his discovery you can visit this site:

http://todayinsci.com/F/Fahlberg_Constantin/FahlbergConstantin-Saccharin.htm



Tuesday, February 26, 2013

FAQ: What is 'junk' DNA?

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.
Christoph Bock (Max Planck Institute for Informatics)
When the human genome was sequenced it became clear that the vast majority of the DNA contained within the human genome does not code for proteins or functional RNA's. With no explanation for the function of this non-coding DNA scientist coined the term 'junk' DNA.

However, the recently published findings of the collaborative efforts of researchers from the Karolinska Institutet in Sweden and the Scripps Research Institute in California, USA indicates that much of this sequence indeed has a function. It had been previously shown that pseudogenes, regions that do not code for proteins despite their similar sequence, are able to regulate the expression of the tumor suppressor gene PTEN. However, this report found a regulatory network of pseudogenes that alter PTEN transcription by epigenetic modification and mRNA stability by RNA-RNA pairing.

Clearly this 'junk' DNA can no longer be considered trash!

Article information:
  1. Per Johnsson, Amanda Ackley, Linda Vidarsdottir, Weng-Onn Lui, Martin Corcoran, Dan Grandér, Kevin V Morris. A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. Nature Structural & Molecular Biology, 2013; DOI: 10.1038/nsmb.2516

Monday, February 25, 2013

Did you know: HIF-1 α plays a role in replication as well as transcription?

Hypoxia inducible factor 1 alpha has been well characterized for its role in the response to low oxygen. Its expression is regulated at the transcriptional level, as well as by modulators of its translation and degradation. HIF-1 α then regulates the expression of a variety of genes that allows cells to survive in changing oxygen conditions.

A recent article in Science Signaling suggests additional functions for this regulator of hypoxic survival. In 'A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication' the authors describe how, during hypoxia, HIF-1α bound to a complex essential for firing of replication origins and decreased the phosphorylation and activation of this complex despite an increase in association of the complex with chromatin. The results is cell cycle arrest that results from HIF-1α stabilization which is independent of transcription.

BMG's ACU
If you are interested in performing cell-based analysis in different oxygen conditions, the atmospheric control unit available with the Omega series of microplate readers from BMG LABTECH could be a useful tool.

Thursday, February 21, 2013

Applications Thursday - Faster, More Sensitive Prion Seeding Assay




A newly developed prion seeding assay called Real-Time Quaking Induced Conversion assay (RT-QuIC) now permits researchers to measure prion seeding at lower levels and in less than a few days in microplates. This is in contrast to standard animal bioassays that are sensitive enough, but are more expensive and much slower (they can years versus only days). RT-QuIC is an improvement on the previously employed methods of quaking-induced conversion (QuIC) (Atarashi 2008) and amyloid seeding assay (ASA) (Colby 2007).

In order to better understand the spread of various infectious prion diseases (Transmissible Spongiform Encephalopathies ), including those that affect humans (Creutzfeldt-Jakob disease (CJD)) or animals (i.e. Scrapie in sheep or Chronic Wasting Disease in deer), researchers need a better and faster way to quantitate the infectivity or number of prions in the infected host. RT-QuIC, when used on BMG LABTECH’s Omega Series of readers, helps solve this problem.

For RT-QuIC to be successfully performed in a microplate reader, three requirements need to be met:  the assay must be incubated at 42°C for the entire measurement period from 20-60 hours, it must have alternating cycles of one minute of shaking at high speed (700 RPM) and one minute of resting, and it must be measured every 15 minutes. BMG LABTECH microplate readers fulfill all three of these requirements with the FLUOstar Omega and POLARstar Omega microplate readers having the hardware and software to run the RT-QuIC prion assay.

To learn more about this exciting new assay please see BMG LABTECH’s Application Note or the publication: Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays in PLoS Pathog 6(12): e1001217. 

Temperature effects on trp fluorescencec
RT-QuIC end-point dilution analysis of three 263K-inoculated preclinical 10 days post injection hamster BHs. In this case, the approximate SD50 was achieved with a 2 μl aliquot (the seed volume) of a 10E−5 dilution of the scrapie 10dpi 263K BH stock (green line). This gave an SD50/2μL of 10E5.5 and an SD50/g of 10E8.2. This figure was adapted from reference 2.

Friday, February 15, 2013

Focus on Events

S. aureus bacteria escaping destruction by human white blood cells
http://www.niaid.nih.gov/topics/antimicrobialResistance/Pages/aureusBacteria.asp


Next week, on February 20-22 BMG LABTECH will be present at the 3rd Lorne Infection and Immunity Conference in Austrailia. Please come by and visit BMG representatives at booth no. 18 and see how we can assist you to make your research into areas such as bacterial infections higher throughput.





Also, on February 20th BMG will attend LAB-SUPPLY in Frankfurt, Germany. This one day trade fair will help you keep up to date with the newest developments in laboratory equipment. There, you will have the opportunity to speak to our specialists about your biochemistry, cell and molecular biology applications and how our microplate readers fit into your experimental approach.


Thursday, February 14, 2013

Applications Thursday: Assessing antibody stability

Structure of an IgG2 antibody. Created from PDB 1IGT
As medical and research based use of antibodies proliferates so to does the need to accurately determine antibody stability to assure that the product does not exhibit a loss of bioactivity.

In the BMG LABTECH application note 212: 'Promotion of Aggregation as a Means of Assessing the Stability of Antibody Molecules', the utility of the FLUOstar Omega microplate reader to assess antibody stability is described. The application takes advantage of not only the ability to read fluorescence but the temperature control and shaking capacity of this instrument as well.

The results show that in the presence of a teflon bead aggregates form and Thioflavin T is incorporated into the aggregates thus enhancing the fluorescence of this dye. Dynamic Light scattering confirmed that aggregation required the teflon bead and further tests showed that promotion of aggregation was similar to that seen by the method of agitation by vortexing.

In short the FLUOstar Omega from BMG LABTECH provides an excellent platform to perform high throughput assessment of antibody aggregation.

For more information about this and other BMG LABTECH applications please visit the Applications Center on our website: http://www.bmglabtech.com/application-notes/application-notes-list.cfm

Wednesday, February 13, 2013

Changes on horizon for mice models?

"Genomic responses in mouse models poorly mimic human inflammatory diseases" is now available in the online version of PNAS. It calls into question the suitability of mouse models for diseases such as sepsis.

Two NOD/SCID mice expressing enhanced green fluorescent
 protein (eGFP) under UV-illumination flanking one plain
 NOD/SCID mouse from the non-transgenic parental line
doi:10.1186/1471-2407-12-21 PDF
For years mice have been the go to model for numerous diseases when testing the effects of potential drugs. The current report compared the genes that were being activated in white blood cells of human that had experienced sepsis, trauma or severe burns. They found that the genetic response to these injuries was similar. However, when they sought to compare their results to the accepted mice models there were no similarities between human and mouse and each mouse model for the conditions used different genes!

The failure to find effective drug treatments for these conditions is believed to stem from the lack of similarity between mouse and human responses. Nearly 150 drugs for sepsis have been tested in humans based on results from mice and all have failed. However, there is hope that since we now know that the human response to burns, trauma or sepsis are similar that a drug that aids with one will aid with the others.

By no means is this the end of mice as an animal model. There are too many cases to innumerate where they have been effective. In the end we must remember that the goal is to cure humans and not mice.

For more information see this article from the NY Times: Mice Fall Short as Test Subjects for Humans’ Deadly Ills


Tuesday, February 12, 2013

FAQ: Will BMG LABTECH attend the 25th anniversary Lorne Cancer Conference?

Metastasis Illustration (National Cancer Institute)
BMG LABTECH is excited to attend the Lorne Cancer Meeting where speakers will enlighten participants about how mutations and genomic instability result in a situation where cells sustain the proliferative signal, resist cell death and evade growth suppression. The altered metabolism of these cells allow them to evade immunity and actively invade other tissues.

On February 14-16 you can meet members of the BMG team at booth 18 in Mantra Lorne, Australia. We believe that as The Microplate Reader Company we have the tools to help you with your cancer research. Our instruments provide outstanding sensitivity, speed and multi-functional capabilities that will allow you to efficiently employ your resources.

Monday, February 11, 2013

Did you know: BMG LABTECH will be attending The 38th Lorne Conference on Protein Structure and Function

CCR5 receptor (based on PDB 1ND8) in cell membrane
Thomas Splettstoesser
Held in Mantra Lorne, Australia February 10-14, BMG The Microplate Reader Company, is glad to be able to attend this years conference whose goal is to highlight leading edge protein science.

The program will include sessions on: Developing drugs to membrane receptors, Immunity, Enzymes and Protein dynamics.

Please stop by booth 18 and say hello to the representatives from BMG LABTECH and find out how we can help you with your protein science studies.

To find about this event and other events that BMG LABTECH is attending visit the BMG LABTECH Events page on our website.

Friday, February 8, 2013

Focus on: Paternal epigenetics

A recent study from Duke Medicine entitled: 'Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort' has been published in BMC Medicine. While the effects of nutrition and environment on the mothers during pregnancy have been primarily studied this is the first study to show that obesity in the father can affect a child's health.

Epigenetic_mechanisms.jpg
It is becoming increasingly clear that these effects are the result of epigenetics or modifications that change gene expression without affecting the underlying DNA sequence. In this study the level methylation of the insulin-like growth factor 2 (IGF2) gene was monitored and showed that children with obese fathers had lower IGF2 methylation. Decreased IGF2 methylation has been associated with increased risk for developing cancers such as colorectal and ovarian cancer.



In order to better understand the epigenetic process it is necessary to understand the enzymes, such as methyltransferases that are involved. A procedure to characterize methyltranferases using a PHERAstar microplate reader from BMG LABTECH is described in application note 225, available at the applications center on BMG's website.

Thursday, February 7, 2013

Applications Thursday: Miniaturized Platelet Aggregation Assay

Blood clot diagram (Thrombus)
 
http://en.wikipedia.org/wiki/Image:Blood_clot_diagram.png
Platelet aggregation is an assay that studies platelet function. Understanding platelet function is essential to our understanding of the regulatory mechanisms involved in thrombus formation that prevent blood loss as well as pathological conditions where platelets are inappropriately activated triggering heart attacks or strokes.

The conventional assay requires the use of single purpose equipment and a relatively large amount of blood. This hampers efforts to analyze multiple conditions simultaneously or to employ animal models where blood supply is limited. As an alternative the BMG LABTECH application note: 'Miniaturised Platelet Aggregation Assays using the NOVOstar Microplate Reader' allows users to analyze a large number of small volume samples simultaneously.

The assay employs the temperature control and shaking capabilities of the NOVOstar with absorbance measurement at a wavelength of 405nm. A comparison of the three shaking modes available using the NOVOstar showed that double orbital shaking was superior to either linear or orbital shaking. Results show that aggregation seen by addition of collagen or collagen related peptide were highly reproducible within the same experiment or when platelets from different donors were employed. Furthermore, results were similar to conventional aggregation assays.

Although not employed in the initial analysis; the presence of two injectors and a robotic pipette in the NOVOstar plate reader could provide additional utility to perform dose concentration analysis or compound screening.

To the view the complete application note visit the BMG LABTECH website where you can see this and other applications using our plate readers.


Wednesday, February 6, 2013

Fun fact: Mary Leakey was born 100 years ago today

The renowned archaeologist and anthropologist, who passed away in 1996, was born on February 6, 1913. Best known for discovering an extinct ape thought to be ancestral to humans, she also discovered tools, fossils and footprints of ancient hominids and is credited with the development of a system of classification for stone tools.

Louis Seymour Bazett Leakey (1903-1972) and his wife, archeologist and anthropologist Mary Douglas Nicol Leakey (1913-1996), digging at Oduvai Gorge, Tanzania, Africa.
Anthropology was truly a family affair for the Leakeys as Mary spent much of her career working with her husband Louis and she eventually trained her son Richard in the field as well.

Tuesday, February 5, 2013

FAQ:Do I need to update the filter list when I install software for PolarSTAR Omega on a new computer?

Yes!

Upon each installation of BMG LABTECH software for an Omega reader you will need to fill the filter table with the appropriate information. If you have access to the list from another computer that controls the microplate reader the information is easily transferred.

If not follow the instructions in the users manual to see how to access the filter wheels and determine the location of the different filters within the filter wheel.

Monday, February 4, 2013

Did you know: February is American Heart Month?

Heikenwaelder Hugo, Austria, heikenwaelder@aon.at, www.heikenwaelder.at
The American Heart Association celebrated 10 years of going red on Friday, a campaign to raise awareness that the number one killer of women is heart disease. This served to kick-off a month of awareness regarding heart health.

Studying heart function and health is an area of intense research and BMG LABTECH instruments have been cited for their use in at least 39 articles that investigate heart health.

For more information visit the BMG website and investigate the many applications and citations that The Microplate Reader Company can help you perform.