Wednesday, December 18, 2013

Advanced Spectroscopy Technique Allows Scientists to Analyze Protein Structure with Infrared Light

Proteins come in many forms and are essential for cellular function. Each protein has a defined structure that is necessary for the protein to perform its function. The most common structures found in proteins are alpha helices and beta pleated sheets. Disruption of the normal common structures in a protein is associated with diseases such as Alzheimer or Parkinsons disease.

Interferometer for FTIR
Until now spectroscopic techniques lacked the sensitivity to analyze protein structure. However, a recent paper published in Nature Communications describes the work of European scientist using a technique called Fourier transform infrared nanospectroscopy (nano FTIR) that allows mapping of protein structure with a 30 nm resolution. This allowed them to identify the structure of proteins in a complex or to identify the protein structures characteristic of a virus (alpha helices) mixed in with insulin fibrils (beta pleated sheets).

The authors believe that this approach will be very useful, with the potential to map the structure of cellular receptors and proteins within complexes.



Original article: Structural analysis and mapping of individual protein complexes by infrared nanospectroscopy

Some information for this blog was obtained from: Infrared Sheds Light On Single Protein Complexes

Tuesday, December 17, 2013

Newly Discovered Regulation of KRas may Result in Novel Cancer Therapies

KRas has been well characterized for its role in several cancers. Mutations of KRas are seen in more than 90% of pancreatic cancer patients, and are prevalent in colon and lung cancer. However, targeting KRas for cancer therapy has been difficult.

Dictyostelium
cytokinesis
Now, an international collaboration of scientists has revealed a new mechanism for regulating KRas function that they hope will provide a new approach for identification of therapeutic targets in cancers with a KRas mutation. Their findings are published in a recent issue of JBC in the article entitled: 'Degradation of Activated K-Ras Orthologue via K-Ras Specific Lysine Residues is Required for Cytokinesis'

This article describes the use of a social ameboid, Dictyostelium, as a powerful model system to study Ras signaling and KRas regulation. They introduced the cancer causing (oncogenic) form of KRas into Dictyostelium, and compared its regulation with normal KRas. They found that these cells recognize the oncogenic KRas and mark it by ubiquitination. Ubiquitination is a process that cells normally use to mark a protein that needs to be recycled, so the ubiquitination of oncogenic KRas leads to it being chopped up and cleared from cells.

The next step is to identify the protein that ubiquitinates oncogenic KRas so that its activity can be increased in cancer cells with mutated KRas.

Some information for this blog was obtained from: Regulation of Cancer-Causing Protein Could Lead to New Therapeutic Targets

Monday, December 16, 2013

Scientists study essential enzyme to better understand molecular evolution

Scientists at the University of Iowa analyzed various forms of the enzyme dihydrofolate reductase (DHFR) using bioinformatics, computer-based calculations, artificial mutagenesis and kinetic measurements. The result of their work is published in the recent JBC paper entitled: 'Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution'.

DHFR with dihydrofolate (left)
and NADPH (right) bound

The scientists chose DHFR due to its well characterized role and the fact that it is present in nearly all organisms. DHFR is involved in DNA biosynthesis and cell replication, therefore, it is essential to survival. Indeed, DHFR has been targeted in several therapeutic applications where antagonists have been used as anti-cancer treatments and anti-bacterial agents.

The results of the current paper showed that although bacteria and human DHFR are different in terms of genetic and protein sequence, the chemical conversion performed by this enzyme is quite well conserved. Indeed the bacterial enzyme already has reactants perfectly aligned in its active site! This was quite surprising as the human enzyme is not only genetically different but much faster.

The results of this work are important in the design of drugs against DHFR and the design of catalysts that are inspired by nature.

Some information for this blog was obtained from: 'Evolution On Molecular Level'


Wednesday, November 27, 2013

Scientists now able to observe molecular motor movement during cell division

The molecular motor Xkid is well characterized for its critical role in aligning chromosomes during cell division. This has been determined based on results obtained from in vitro experiments using purified Xkid, which showed directed movement of this protein on microtubules. However, until now scientists had not been able to observe Xkid behavior on intact spindles.

Diagram of a mitotic spindle



During cell division chromosomes must be precisely segregated so that they can be divided between the two daughter cells. If this segregation is not performed correctly this can result in severe illness or malignant tumor transformation. The spindle apparatus is composed of numerous microtubules and is required for cell division. Xkid molecules are located in the spindle apparatus so a collaboration of scientists from Asia sought to characterize the movement of Xkid in its native environment. The results of their collaboration are published in an article entitled: 'Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array'.


In this article, the authors describe the binding of up to four Xkid molecules to a quantum dot (Qdot) and monitored the movement of the Qdot on the microtubules of the meiotic spindle of Xenopus egg extracts. The Xkid-Qdots were able to travel on average 5 micrometers and up to 17 micrometers, which is quite far by cell size standards. They were able to move these relatively long distances by changing which microtubule track they used, always moving along the microtubule with a defined polarity, until they accumulated around the metaphase plate.


These results will contribute to our understanding of how chromosome segregation occurs and thus what goes wrong when chromosomes are incorrectly segregated leading to medical disorders.


Some information for this blog was obtained from the Science Daily article: Molecular Motors' Involved in Chromosome Transport Observed

Tuesday, November 26, 2013

Two separate studies identify new potential AML treatments

AML, or acute-myeloid leukemia is a cancer characterized by rapid growth of abnormal blood cells. AML is a disease that is actually characterized by a wide range of diversity in terms of genetics. A genetic hallmark of about 10% of AML is mutation of epigenetic repression or C/EBPα. Two separate studies have investigated AML with C/EBPα dysfunction and found two separate potential targets for treatment. Both studies represent the work of international collaborations.

BONE MARROW: ACUTE MONOCYTIC LEUKEMIA
by 
The Armed Forces Institute of Pathology (AFIP)
The first study was published in a recent issue of Cancer Cell in an article entitled: 'Sox4 Is a Key Oncogenic Target in C/EBPα Mutant Acute Myeloid Leukemia'. This article describes how Sox4 expression is normally repressed by C/EBPα but when this regulation is silenced Sox4 is overexpressed leading to leukemic growth. Overexpression of Sox4 independent of C/EBPα silencing led to similar gene expression profiles as those seen when Sox4 overexpression is the result of C/EBPα silencing. Thus Sox4 is a viable target for drug intervention and treatment of AML. The search now begins to find suitable compounds that will inhibit Sox4 function and can therefore be used to treat AML in which C/EBPα dysfunction is seen.

The second study produced an article entitled: 'The gene signature in CCAAT-enhancer-binding protein α dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors' was published in a recent issue of the journal Haematologica. This study sought a way to reactivate C/EBPα through the use of small molecule inhibitors where they found that histone deacetylase inhibitors had a positive connection. Histone deacetylases (HDAC's) are involved in epigenetic regulation so it is expected that they will be useful in treatments of AML which involves epigenetic repression of C/EBPα.

Structure of the CEBPA protein
by Emw
Both of these studies provide an exciting opportunity to discover new compounds that will be useful in treating AML with C/EBPα dysfunction. The PHERAstar FS is an excellent tool for drug discovery and high throughput screening that will be necessary to discover new compounds that either inhibit Sox4 or inhibit the relevant HDAC's.

Please visit BMG LABTECH's website at: http://www.bmglabtech.com/ to find out more about the PHERAstar FS and other microplate readers that will assist your research.

Monday, November 25, 2013

X-ray laser protein modeling may be useful for solving membrane protein structures

Membrane proteins, such as G-protein coupled receptors and receptor tyrosine kinases, are well characterized for their role in important cellular functions and represent the major targets for new drug development. However, only a small fraction of membrane proteins have had their three dimensional structure completely mapped by traditional techniques. This lack of structural information is due, in large part, to the inability to obtain large crystals of pure membrane proteins.

Now, a recent article in Science explains an approach that does not require macroscopic crystals and can generate a 3-D structure without prior structural knowledge. The results of a collaboration between German and U.S. scientist are reported in the article entitled: 'De novo protein crystal structure determination from X-ray free-electron laser data'. In this article, the authors describe an approach which uses nanometer to micrometer sized crystals which are in a complex with a lanthanide compound, in this case gadolinium.

Single Protein crystal of Lysozyme
Photographed by Mathias Klode

New technique does not require a macroscopic crystal such as this
As proof of principle, the scientists obtained the essential information to complete the structure of a well characterized protein, lysozyme. They found that the de novo structure obtained using this technique was equivalent to that previously determined. It is hoped that this new approach will enable the characterization of proteins that had previously been elusive, such as membrane proteins. With the new structural information, scientists will be able to better understand interactions of potential drug compounds, which should lead to more effective treatment options.

Friday, November 22, 2013

Scientists discover how seasonal flu typically escapes immunity

The three-dimensional structure of influenza virus
from electron tomography
It is that time of year again, flu season. Every year seasonal flu is responsible for severe illness, hospitalization and even death worldwide. In order to combat this worldwide health issue, flu vaccines are prepared that consist of inactivated flu virus from three major different types of flu virus that infect humans. This vaccine initiates an immune response which produces antibodies against these viruses which will have a protective effect when someone is exposed to the actual virus.

However, the flu virus is constantly evolving. Specifically, the outer coat changes and the antibodies that once were protective eventually become ineffective. Because of this constant change, the World Health Organization meets twice a year to decide whether or not to change the strains of flu virus which are included in the vaccine.

In the interest of better understanding how the seasonal flu virus escapes immunity a collaboration of scientists from around the world worked together on a project to understand the molecular basis for the changes that result in the loss of virus-specific antibody response. The results of this work are published in a recent issue of Science in a paper entitled: 'Substitutions Near the Receptor Binding Site Determine Major Antigenic Change During Influenza Virus Evolution'. In this study they found that changing even a single amino acid allows the virus to evade detection by antibodies. However, these changes occur at only 7 places on the virus coat, as opposed to the 130 places previously believed. All 7 of the sites where single amino acid changes occur are near the area where the flu virus binds to and infects host cells also known as the receptor binding site. This is somewhat surprising as the virus need to conserve the receptor binding site so that it can recognize and infect cells.

These scientists hope that this new information will help to improve flu vaccines!

Some information for this blog post was obtained from the Science Daily article: How Flu Evolves to Escape Immunity


Thursday, November 21, 2013

Recently discovered enzyme found to play a role in DNA repair

DNA ligase I repairing chromosomal damage
Courtesy of Tom Ellenberger
Every time a new cell is formed the parent cell must first make of copy of its DNA that it will pass along to the daughter cell in a process called replication. When DNA damage is encountered the progress of DNA replication is halted which can lead to DNA double strand breaks and threaten genomic stability. The final result of genomic instability is cell death.

In order restart DNA replication and repair DNA damage the cell has two options if it proceeds with replication: either perform an error prone synthesis of the damaged DNA or skip the damage and reinitiate DNA replication beyond the damage. The unreplicated gap can then be repaired after replication.

A recent paper published in Nature Structural & Molecular Biology entitled: 'Repriming of DNA synthesis at stalled replication forks by human PrimPol' describes how the recently discovered enzyme PrimPol uses its primase activity to continue replication progression and reinitiate DNA synthesis. Thus, PrimPol allows cells to survive but it will introduce mutations into DNA at a higher rate than normal.

This paper is the work of a group Spanish scientist who previously reported their discovery of PrimPol and characterized its biochemistry. They have also found that this enzyme is related to proteins found in archaebacteria which means it is a very old enzyme in evolutionary terms since archaebacteria are among the first life forms to inhabit earth. Archaebacteria are characterized by their ability to survive in harsh environmental conditions. These harsh conditions result in more extensive DNA damage so having an enzyme like PrimPol would be beneficial even though it can't duplicate DNA as precisely as other enzymes. The authors postulate that this inexact replication may be important for the evolution of genomes.

These scientists are now investigating the possible role of PrimPol in diseases such as cancer where DNA damage leads to mutations which lead to abnormal growth. It is not unreasonable to speculate that PrimPol, which is involved DNA damage repair but also leads to mutations, could have a role in cancer.

Some information for this blog post was obtained from the Science Daily article: Newly Discovered Ancestral Enzyme Facilitates DNA Repair

Wednesday, November 20, 2013

BMG LABTECH and InvivoSciences, Inc. Collaborate on 3-D Heart Function Application

Representative data collected on the CLARIOstar,
showing the ability to detect physiological changes in heart
function resulting from drug treatment
BMG LABTECH was proud to participate in the announcement of a collaboration with InvivoSciences, Inc. this week. InvivoSciences, Inc. provide novel drug discovery solutions with their engineered stem cell tissues. The collaboration between BMG LABTECH and InvivoSciences, Inc. will focus on a microplate assay which can monitor 3-D heart tissue function in a time-dependent manner by detecting changes in fluorescence that result from calcium flux. This calcium flux, which is a normal part of heart function, is detected by fluorescent calcium-sensing dyes and the heart tissue can then be assessed for the alterations in function that are observed with drug treatment.

As a part of this collaboration BMG LABTECH will have a short presentation during an InvivoSciences sponsored workshop at the Cell Based Assay Summit in Munich, Germany.

In addition, BMG LABTECH and InvivoSciences will co-present a workshop at the Society for Laboratory Automation and Screening (SLAS) conference on January 20th, 2014 in San Diego, CA.

For more information on this collaboration please visit the BMG LABTECH News page on our website.

Tuesday, November 19, 2013

Applications: DNA Detection Using PicoGreen® and the CLARIOstar®


The advantage of using PicoGreen® for detecting nucleic acids such as DNA is that this fluorescence based assay is highly sensitive and also allows users to discriminate between double stranded DNA, single stranded DNA and RNA.

This application note shows the utility of the CLARIOstar®’s  LVF MonochromatorTM which is used to perform spectral scanning that is important in determining the optimal settings for the monochromator. Using the optimized excitation and emission wavelengths and bandpasses this application note shows that performance of this assay is comparable to that seen with filters.

 PicoGreen® assay comparison
using either filters or LVF Monochromators™ in the LVis Plate.
The final volume in the well was 2 µl.
Another interesting aspect shown in this note is the ability to perform this fluorescence assay using the LVis plate. This will allow users to perform fluorescent detection AND use low volumes thus saving your valuable sample for its intended use.


For more information on this and other applications for the CLARIOstar® and other BMG LABTECH microplate readers please visit our website: http://www.bmglabtech.com/

Monday, November 18, 2013

New Technique Identifies Novel Human Gene Regions

Human genome
by
Webridge
A collaboration of Swedish scientists has used a new protein analysis method which allowed them to identify 98 previously undiscovered protein coding loci in the human genome. The approach is a liquid chromatography- mass spectrometry based method that uses high-resolution isoelectric focusing. Essentially this approach allows scientists to isolate and obtain peptide sequence information for more of the proteins present in a sample. The results of this research are published in the recent Nature Methods article entitled: 'HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics'

The results of the human genome project found that only about 1.5% of the genome is actually involved in coding for protein producing genes. Some of the remainder is certainly involved in the regulating the expression of these genes, however, the majority of the DNA in the genome has no currently understood function. The lack of known function for this DNA has earned it the name 'junk DNA' and within this 'junk DNA' are sequences known as pseudogenes. Pseudogenes are believed to be remnants of genes which lost their function.

The technique used in the current paper makes it possible to discover protein coding genes in 'junk DNA', which was previously impossible. The authors found that some pseudogenes actually produce protein, indicating that they have some function. They also found that many of the proteins encoded by pseudogenes were also expressed in cancer cell lines, raising the possibility that expression of the pseudogene encoded proteins may play a role in disease.

Some information for this blog post was obtained from the Science Daily article: Protein Coding 'Junk Genes' May Be Linked to Cancer

Friday, November 15, 2013

Another Potential Treatment for Cancers with p53 Mutations Identified

About half of all cancers are associated with a mutation in a gene called p53 which, in normal cells, is essential for discovering DNA damage and eliminating cells whose DNA damage is too extensive for repair. Just last week we discussed the role of Type 2 PIP kinases in the survival of p53 mutant cancers here. Now another potential target that may enhance treatment of cancers with p53 mutations has been reported!

The image shows cisplatin crystals,
which is a platinum compound,
and used as a chemotherapy drug

by
Larry Ostby


A recent paper in the journal Cell Reports describes the work of biologists at MIT that found that cancers with mutated p53 could be made more susceptible to chemotherapy if they also lack another gene called MK2. The paper entitled: 'A Reversible Gene-Targeting Strategy Identifies Synthetic Lethal Interactions between MK2 and p53 in the DNA Damage Response In Vivo' describes a study performed in mice where treatment with cisplatin caused dramatic tumor shrinkage when both p53 and MK2 were deleted. Similar treatment of mice with functional MK2 exhibited continued tumor growth. This animal study focused on non-small-cell lung tumors, however, similar results have been observed in cancer cells derived from other tumor types. It is hoped that these results will extend to multiple cancer types and studies are ongoing to investigate mouse models of colon and ovarian cancer.

Since drugs that inhibit MK2 are already available and approved for use to treat inflammatory diseases such as arthritis it is hoped that combining these drugs with current chemotherapy treatments could greatly improve the efficacy of these treatments. The potential combination is not an obvious choice as the usual combinations chosen for cancer treatment each have anti-tumor effects individually, while in this case the MK2 inhibitor does not directly affect cancer growth alone.

Some information for this blog was obtained from the Science Daily article: Biologists ID New Cancer Weakness

Thursday, November 14, 2013

Scientist Uncover Novel Bioluminescent Properties of the Parchment Tube Worm

It has been known for decades that the Chaetopterus marine worm, commonly known as the 'parchment tube worm' is capable of producing light in the form of a mucus that is secreted from any part of the worms body. A recent paper in the journal Physiological and Biochemical Zoology entitled: 'Optical and Physicochemical Characterization of the Luminous Mucous Secreted by the Marine Worm Chaetopterus sp.' describes the work that is the result of collaborative efforts by scientists at the Scripps Institute and Georgetown University.

Polychaete worm Chaetopterus sp
PD-US-not renewed
In this paper the authors described in detail the light produced in the worms mucus. The light is a long glow in the blue range (455 nm) which is an unusual color among this class of invertebrates. The scientists also found that the light is produced by a photoprotein and that light production is independent of oxygen and not strongly increased by iron.

Numerous biotechnological applications already employ bioluminescence as their means of detecting gene expression, enzyme activity, protein-protein interactions and other biological/biochemical phenomena. We at BMG LABTECH will be watching the progress of this research with interest to see if the long-lived blue light can be harnessed for a biotechnological application. You can be sure that if this becomes an important detection method that BMG LABTECH will have the microplate readers capable of performing this detection.

Some information for this blog post was obtained from the Science Daily article: Nature's Glowing Slime: Scientists Peek Into Hidden Sea Worm's Light

Wednesday, November 13, 2013

Study Identifies RUNX3 as 'First Line of Defense' in Tumor Suppression

RUNX3 has been intensively studied for its role as a tumor suppressor. However, a recent paper in the journal Cancer Cell indicates that RUNX3 may be even more important than previously thought and scientists hope that this understanding will lead to improved cancer treatments.

The report is a collaboration of Chinese scientists entitled: 'Runx3 Inactivation Is a Crucial Early Event in the Development of Lung Adenocarcinoma'. These scientists describe how they were able to perform targeted inactivation of Runx3 and show that this leads to adenoma formation and more rapid formation of adenocarcinoma in the lungs of mice. Furthermore Runx3 was observed to be frequently inactivated in human lung adenocarcinomas that had mutated K-Ras. Although this study focused on lung cancer it opens the possibility that RUNX3 could be similarly pivotal for other cancers.

Structure of the RUNX3 protein
by Emw
The means of Runx3 inactivation may play a key role in future therapies in which this inactivation takes place. Runx3 was epigenetically inactivated, meaning that DNA methylation silences the gene without any alteration to Runx3 coding information. Since it is known that epigenetically inactivated genes can chemically reactivated the search now begins for a means to reverse the epigenetic inactivation of Runx3.

Some information for this post was obtained from the Science Daily article: Scientists Find 'Missing Link' in Important Tumor Suppression Mechanism

Tuesday, November 12, 2013

Support for use of Crispr Technique as Treatment for Hereditary Disease Growing

As previously reported here, earlier this year scientists employed a new technique called Crispr in order to perform genetic alterations to the human genome. What sets the Crispr technique apart is its ability to make accurate and detailed alterations to specific positions in the genome of humans, or in fact, any organism. This development is now being hailed as a revolution that will allow for better treatment of cancer, incurable viruses and inherited genetic disorders.

Secondary structure image for CRISPR-DR57
by Rfam database


The Crispr technique is truly a triumph of basic science. The process was first identified as a natural defense system used by bacteria to protect themselves from viruses. The applicability of the Crispr technique to other organisms was quickly realized and finally applied to the human genome earlier this year.



It is hoped that having a tool that so accurately alters the genome will reduce concerns about making alterations to the genome of IVF embryos. If this technique is successfully employed some doctors believe that it could be used to eliminate genetic diseases from affected families.




Information for this blog was obtained from the following:
http://www.independent.co.uk/news/science/exclusive-jawdropping-breakthrough-hailed-as-landmark-in-fight-against-hereditary-diseases-as-crispr-technique-heralds-genetic-revolution-8925295.html

Friday, November 8, 2013

New Study Provides Hope for Treatment of Cancers with p53 Mutations

P53 is the most frequently mutated gene across all types of cancers. However, directly targeting this vital protein with drugs has been difficult. A report in the current issue of Cell entitled: 'Depletion of a Putatively Druggable Class of Phosphatidylinositol Kinases Inhibits Growth of p53-Null Tumors' describes the work of a multi-institutional collaboration that has identified potentially important enzymes that are essential for growth of cancers where p53 is mutated but not critical for the growth of normal cells. By targeting these enzymes it is hoped that a broad group of cancer patients will see benefits including those with breast, lung and brain tumors.

Cartoon representation of a complex between
DNA and the protein p53

by Thomas Splettstoesser

The enzymes in question are called Type 2 phosphatidylinositol-5-phosphate 4-kinases alpha and beta (Type 2 PIP kinases). The scientists knew that one of the critical roles of p53 is to 'rescue' cells which are producing too much reactive oxygen species (ROS) which is a by-product of rapid growth. In the absence of p53 excessive ROS can cause further DNA damage and cancer growth can become even more aggressive. However, too much ROS will cause excessive damage to cellular components and eventually death of the cell. Type 2 PIP kinases appear to be a backup to p53 and reduce ROS enough to keep the cells from dying. The research described in the current paper employed breast cancer cells which were known to have higher expression of Type 2 PIP kinases. They found that genetically targeting these enzymes effectively shuts down the growth of these cancer cells and these scientists believe that the results will extend to other cancers with mutated p53.

The search now begins for drugs that target Type 2 PIP kinases using appropriate inhibitor screening technology like the HTS capable PHERAstar FS microplate reader from BMG LABTECH.

Thursday, November 7, 2013

Applications: Screening for Small Molecule Inhibitors of BRD 4

BRD4 is a member of the bromodomain family of proteins which serve as epigenetic readers. Essentially there are 3 groups of important proteins that are involved in epigenetic regulation. Writers and erasers, enzymes which add and remove marks to DNA and associated proteins, and the epigenetic readers that determine how those marks are interpreted with resulting increased or decreased expression of genes.

Structure of the BRD4 protein.
Based on PyMOL rendering of PDB 2oss
by Emw 
The importance of epigenetic readers is indicated by their implication in a number of diseases and the search for inhibitors of these proteins is ongoing. BRD4 has been identified as a therapeutic target in advanced myeloid leukemia and other cancers as well as inflammatory diseases. In the recent article entitled: 'Discovery of Novel Small-Molecule Inhibitors of BRD4 Using Structure-Based Virtual Screening' the authors describe an approach to mine new potential inhibitors based on knowledge of BRD4 structure. In order to validate these inhibitors they used an AlphaScreen based approach and chose the PHERAstar FS from BMG LABTECH to detect these biochemical assays.


The assay employed was previously described in a paper entitled: 'Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery.' It identifies the ability of bromodomains to bind to acetylated histone peptides via protein-protein interaction that can be detected using AlphaScreen technology.

We at BMG LABTECH are proud that our instruments could contribute in some way to this very interesting research!

Wednesday, November 6, 2013

Masked cytotoxic agents could provide selective killing of cancer cells.

The goal of all cancer treatments is to eradicate the diseased cells while leaving the normal cells unharmed. The reality is that all current cancer treatment options come with a price, damage to normal cells that results in the undesirable side-effects associated with the treatment.

A current article in the journal Nature Communications provides some real hope that there may be a way to selectively target cancer cells. This article, entitled "Selective cancer targeting with prodrugs activated by histone deacetylases and a tumour-associated protease" describes work done by scientists at Stony Brook University in New York. These scientists found that all the cancer cells that they tested had high levels of two enzymes, histone deacetylase (HDAC) and a protease called cathepsin L (CTSL).

Space-filling model of puromycin, an antibiotic
by Jynto
The team wondered if they could take advantage of the high levels of HDAC and CTSL. Their approach was to modify a known cytotoxic agent in such a way that it would be masked in the absence of HDAC and CTSL activity. They modified puromycin, a protein synthesis inhibitor, with a tag that consisted of an acetylated lysine. This modified puromycin will not have cytotoxic effects until the acetylated lysine is removed. Unmasking of puromycin is accomplished by the sequential activity of HDAC removing the acetyl group and the CTSL removing the lysine. Proof of concept experiments in cancer cells and mice afflicted with human tumors point to the powerful inhibition of growth using this approach.

A similar approach could revive other compounds that were deemed too cytotoxic for clinical use.

Monday, November 4, 2013

Archaea species exhibits accelerated growth and ability to sidestep normal replication process

A recent report in Nature entitled: 'Accelerated growth in the absence of DNA replication origins' describes the work of The University of Nottingham scientists studying the archaea species Haloferax volcanii. Archaea are single celled organisms that are best known for their ability to survive and thrive in some of the worlds harshest environments, such as extreme temperatures and pH's. Haloferax volcanii are able to live in high-salt conditions and the ones used in this study originate from the Dead Sea.

Salt deposit at Dead Sea
Archaea are single-celled, and therefore similar to bacteria, but also have other characteristics that are more similar to eukaryotes, such as humans. Therefore, archaea fall into their own category of classification. The arahaea characteristic that was the focus of this study was the fact that when they copy their DNA prior to cell division they exhibit multiple origins of replication, similar to eukaryotes, where bacteria have a single origin of replication. In humans, if these replication origins are eliminated DNA replication cannot proceed and cells eventually die. When all replication origins were eliminated in a strain of  Haloferax volcanii not only were no growth defects apparent but growth faster than wild type was observed!

The origin-less replication occurred at dispersed sites rather than discrete origins and in order to perform this origin-less replication Haloferax volcanii adapted another cellular function, homologous recombination, to initiate DNA replication. In fact, they found that the recombinase RadA was absolutely essential for replication in the absence of origins.

The unregulated and accelerated growth of the origin-less Haloferax volcanii strain can be compared to cancer cells. It is the hope of the authors of this study that by better understanding the phenomena they observed they may shed light on how cancer cells avoid normal controls and this understanding could lead to new targets to selectively effect cancer cells.


Thursday, October 31, 2013

Review details potential role of epigenetic targets in cancer therapy

Scientist from Boston University School of Medicine recently published a review article in the International Journal of Molecular Sciences entitled: Cancer Development, Progression and Therapy: An Epigenetic Overview. In this article they propose that epigenetic and other changes mediate the development of cancer progenitor cells. Based on the acknowledgement of the key role of epigenetic changes in cancer progression they hope that better epigenetic drugs will become available.

Epigenetics refers to external modifications made to DNA and proteins associated with DNA. These modifications turn genes on or off but do not affect the DNA sequence. Epigenetic regulation is an important part of normal cell development and cell fate determination but when dysregulation of epigenetic mechanisms occur the results can be detrimental. The dysregulation of these epigenetic mechanisms may be in response to internal and external, or environmental, factors.

DNA molecule that is methylated on both strands on cytosine
by Christoph Bock
The importance of epigenetics is clearly on the rise and there are a number of different assays available to study modulators of epigenetic enzymes. At BMG LABTECH we have multiple microplate reader platforms that are capable of detecting these epigenetic enzyme assays. Please visit our website to see the capabilities of all of our instruments: www.bmglabtech.com

Wednesday, October 30, 2013

Three new vertebrate species found in Australia

Cape Melville, Cape York, Queensland, Australia
Cape Melville in Australia contains a rugged mountain range that consists of large black granite boulders stacked high. The unique nature of this mountain range has allowed surveys of the base of the mountain but restricted access to the rainforest that sits atop a plateau. Now, a National Geographic expedition finally made it there, with the help of a helicopter to fly them in. Within several days of their arrival the scientists and film crew were surprised to find 3 new vertebrate species! The new species were named and described by Dr. Conrad Hoskins from James Cook University.

One of the species is especially unique, the Cape Melville Leaf-tailed Gecko, which, as its name suggests, has a broad flat tail at the end of its 20 cm body. The other species are the Cape Melville Shade Skink which is golden in color and the Blotched Boulder-frog whose range is completely restricted to the boulder fields at Cape Melville.

It is very surprising to find three new species anywhere, let alone in Australia which was thought to have been thoroughly explored.

Information for this blog was obtained from Science Daily

Friday, October 25, 2013

Three-dimesional structure of RNA polymerase I solved

Diagram of the essential subunit of
human RNA polymerase I, II, III
by Nevit Dilmen

Three different RNA polymerases are employed in order to make all of the different types of RNA required for normal cell function. The structure of RNA polymerase II, which makes mRNA, has been known for over 10 years but the structure of RNA polymerase I, which is responsible for making RNA which will form ribosomes has been elusive, until now. Details of the structure of RNA polymerase I may be found in a recent publication in the journal Nature. The report is a collaboration between German and Spanish scientists entitled:  'Crystal structure of the 14-subunit RNA polymerase I.'



One aspect that had previously confounded the study of RNA polymerase I is its size, which is much larger than RNA polymerase II. The results of the 3-D structure indicate that some of the larger size is the result of additional modules that are very similar in structure to proteins that assist RNA polymerase II function. Having these modules constantly attached may, in part, explain why RNA polymerase I produces RNA faster than RNA polymerase II. However, the permanent association provides the cell with fewer means of regulating RNA polymerase II function.

To find out more about this discovery you can read the ScienceDaily entry: Bigger, Better, Faster: 3D Structure Reveals Protein's Swiss-Army Knife Strategy

Thursday, October 24, 2013

Recently added: BMG application notes on DLR, protein concentration and nephelometry.

BMG LABTECH recently added three new application notes two are on topics that are quite familiar to most biological scientists and one that is....not so much.

Representative DLR data.


Dual luciferase reporter (DLR) assays and using absorbance at 280 nm for protein detection are assays that many are very familiar with. DLR is the popular technique from Promega that uses two reporter constructs that allow users to monitor the regulation of gene transcription and normalize with an internal control. Since other BMG LABTECH instruments like CLARIOstar and Omega Series have been previously certified as DLR ready it was not surprising that the PHERAstar FS was also capable of passing this certification, which is described in AN #243.

BMG's LVis plate
The other common assay, protein quantification by UV absorbance at 280 nm is described in AN #242. The detection was performed using the LVis plate. The LVis plate allows the user to perform low volume detection of samples while reading up to 16 samples. In this application note the performance of the LVis plate was validated over a wide range of protein concentrations. The LVis plate is compatible with any BMG microplate reader that is equipped with our ultra-fast UV/Vis spectrophotometer such as the SPECTROstar Nano, PHERAstar FS, CLARIOstar and Omega Series of readers.


Finally we have the not so familiar. AN#238 employs an approach called nephelometry. Nephelometry is a specialized detection method that measures the amount of laser light that is scattered dependent on the size/shape of particles in a solution. The NEPHELOstar from BMG LABTECH is the only microplate reader available that employs this detection technology. In this application note the NEPHELOstar was used to monitor the amount of calcification that occurs in serum samples. When calcification takes place in blood vessels it can lead to cardiovascular disease.

These applications show that whether or your assay is usual or unusual BMG LABTECH has a microplate reader that will suit your needs.

Tuesday, October 22, 2013

RTQuIC at Expanding Prion Horizons

As the Expanding Prion Horizons Symposium wrapped up last week we at BMG LABTECH were happy that we could sponsor such a stimulating meeting. All the talks and posters were excellent and generated lively discussion. BMG LABTECH is pleased to see the Real Time Quacking Induced Conversion (RT-QuIC) assay is being used extensively and we are glad that a plate reader like the FLUOstar Omega can contribute to the prion research field. As reported at this meeting the high sensitivity RT-QuIC is capable of detecting disease prions where western blots and immunoassays can’t, such as higher through put cell culture studies where prion protein expression is low. Furthermore, its sensitivity is at least comparable to ELISA and IHC in detecting Chronic Wasting Disease (CWD) in a variety of cervid species.

Representative data from BMG application note # 232


The RT-QuIC assay is being considered for use in the early detection of prion diseases. CWD can now be detected in deer saliva in as little as 6 months, which is at least one year before deer exhibit clinical symptoms. Among the prion diseases CWD appears to be unique in the amount of disease prion that is externalized. Therefore, another study used RT-QuIC to look at which tissues are affected by prions. Finally, a more wide spread early detection application may be a blood test. Indeed, a blood test was described that is a modification of RT-QuIC and is capable of detecting prion infection at the midpoint between infection and onset of symptoms.


RT-QuIC is also being used to study routes of prion transmission. One study looked at the ability of bovine spongiform encephalopathy (BSE aka ‘mad’ cow disease) prions to infect deer and CWD prions to infect cows. Another investigated the possibility of prion transmission from mother to offspring. Clearly RT-QuIC can be used in a variety of ways and the FLUOstar Omega from BMG is an excellent tool to perform this assay.

Friday, October 18, 2013

Conference Report: Expanding Prion Horizons - Day 1

Characteristics of Alzheimer's disease
We at BMG LABTECH are happy to be able to sponsor this weeks Expanding Prion Horizons meeting at Colorado State University. The first day was certainly a success with excellent talks and stimulating discussion.

Nobel laureate Stanley Prusiner got the day started with an excellent key note speech in which he stated his hypothesis that all neurodegenerative disease can also be considered prion diseases. This includes chronic traumatic encephalopathy (CTE) which has been in the news recently due to the concerns over head trauma in football in the United States.

The morning had a number of excellent talks that discussed the various ways that protein misfolding can lead to human disease while the afternoon focused on the biology and pathology of prion diseases. One talk that BMG took particular notice of was by Martin Margittai discussing fibril growth of Tau which results in neurofibrillary tangles in Alzheimer's Disease. His talk was interesting due to the experimental approach he presented which can be used to monitor Tau filament growth. After labeling Tau monomers they were able to monitor filament growth using fluorescence spectroscopy in which they excited at a wavelength of 344 nm then performed an emission scan from 360 to 600 nm. As the Tau filament grows the emission peak shifts so they can now use this assay to test for filament growth efficiency.This approach could be easily adapted for higher throughput using the new CLARIOstar from BMG LABTECH which features an Advanced LVF Monochromator.

Tuesday, October 15, 2013

How can non-coding mutations affect disease states?

Recent advances have made it easier to sequence the genome of an individual. With this information in hand it now becomes important to be able to use this information to make predictions about the effect that genetic mutations have on the chance that an individual will be affected by a disease. If a mutation occurs in a gene it is fairly straight forward to predict the effect that this alteration will have on the function of the protein encoded by the gene and subsequently the propensity to acquire a disease. However, genome wide studies that investigate the association of mutations with disease reveals that only 12% of the disease associated mutations occur in coding regions of the DNA. It, therefore, stands to reason that the remainder of these mutations will occur in regulatory elements, regions of DNA that bind transcription factors in order to control when and how strongly a gene is expressed. However, the mechanisms for genetic variation to affect regulation of transcription are incompletely understood.

Cartoon representation of the molecular structure of
transcription factor PU.1 residues 171-259 interacting with
a strand of DNA

by Jawahar Swaminathan





With this in mind scientists at the University of California-San Diego sought to characterize transcription factor binding across a genome. The results of their study are presented in the recent Nature article entitled: 'Effect of natural genetic variation on enhancer selection and function.' They used macrophage cells from two different mice strains and compared about 4 million DNA sequence differences. They chose the DNA sequences to analyze based on their binding to transcription factors using a technique called ChIP-Seq. This approach allows you to isolate the chromatin (DNA) which is bound to a protein by immune-precipitating the protein then perform sequencing of the DNA that is bound by the protein and therefore co-precipitates with the protein. Based on this they believe they have genomic data for lineage-determining transcription factor binding sites that can be used to study disease-associated variations in the DNA sequence.

Friday, October 11, 2013

Upcoming BMG LABTECH Events

Representative RT-QuIC data from BMG application note #232
Next week BMG LABTECH is proud to sponsor the Expanding Prion Horizons 2013 meeting at Colorado State University. Our involvement in prion research is due to the utility of the FLUOstar Omega in performing real time quaking induced conversion (RT-QuIC) assays. This fluorescence based assay monitors the accumulation of misfolded protein that is the hallmark of prion diseases. The RT-QuIC assay exhibits sensitivity equal to bio-assays that are much more time consuming and expensive.

Please stop by and meet one of the BMG LABTECH representatives during the meeting to find out more about the RT-QuIC assay and the performance of the FLUOstar Omega and other BMG LABTECH microplate readers.

For more information on this and other events which BMG will attend please visit the Events Page on the BMG LABTECH website: http://www.bmglabtech.com/events/BMG-Labtech-Events.cfm

Thursday, October 10, 2013

Key Molecule Involved in Cell Division Could be a Novel Therapeutic Target

Regulation of cell division is essential for developing organisms but can lead to harmful tumor growth when the process is dysregulated. Our expanding knowledge of how this process is regulated has lead to novel therapeutic targets based on the blockade of cell division or mitosis. A paper in the current issue of The Proceedings of the National Academy of Sciences describes the collaborative efforts of Spanish and French scientists to decipher the role of the protein Greatwall in mammalian cell division.

Cell in prometaphase
by Roy van Heesbeen

Previous to this report, Greatwall was shown to regulate cell division in invertebrates such as the Drosophilla melanogaster fly and its biochemistry has been characterized in Xenopus. The current work describes the generation of the first mammalian genetic model of this protein using mice. The authors found that cells lacking Greatwall are not capable of adequately dividing themselves even though they enter mitosis with normal kinetics. The deviation from normal cell division occurs after the nuclear envelope breaks down exposing nuclear components such as chromosomes to the cytoplasm and the enzyme activities localized there. Without Greatwall the DNA does not condense to form the correct structures and cell division is halted at prometaphase.



The potential therapeutic capacity of Greatwall is enhanced because it acts by blocking the function of PP2A, a frequently altered tumor suppressor. Therefore, inhibition of Greatwall could slow down cell division and reactivate a tumor suppressor that has been shown to be capable of inhibiting many oncogenic pathways involved in the development of cancer. The search now begins for compounds capable of inhibiting Greatwall and understanding which types of cancer would most benefit from treatment with a Greatwall inhibitor.

The title of the PNAS article is: 'Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals'


Wednesday, October 9, 2013

Three Nobel Laureates Share the 2013 Nobel Prize in Chemistry

The Royal Swedish Academy of Sciences announced today that the Nobel Prize in Chemistry for 2013 would be shared by Dr.'s Martin Karplus, Michael Levitt and Arieh Warshel 'for the development of multiscale models for complex chemical systems.' The three receive the award based on their collaborative efforts to design a computer program to reveal how chemical processes proceed.







Under normal conditions chemical reactions occur very quickly as electrons move between atomic nuclei. This makes it virtually impossible for every step in the process to be mapped experimentally. When the 3 Nobel Laureates began their collaboration in the 1970's suitable computer programs were being developed based on classical Newtonian physics, which were useful in showing how atoms are positioned in a large molecule, such as a protein, and  programs based on quantum physics which were useful for simulating chemical reactions. The work that earned Karplus, Levitt and Warshel the Nobel Prize was the program they developed that allowed these two fundamentally different types of physics to work side by side. They used quantum theoretical calculations to describe the interface between a protein and its smaller substrate while the rest of the large protein was simulated using classical physics. Their breakthrough made the computer just as important to chemists as traditional experiments.

Information for this blog was obtained from:

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2013/press.html


Tuesday, October 8, 2013

2013 Nobel Prize in Physics Awarded Today

The Royal Academy of Sciences gave the Nobel Prize in Physics jointly to Dr.'s François Englert and Peter W. Higgs “for the theoretical discovery of a mechanism that contributes to our understanding of the origin of mass of subatomic particles, and which recently was confirmed through the discovery of the predicted fundamental particle, by the ATLAS and CMS experiments at CERN’s Large Hadron Collider”. The Noble Laureates independently proposed the theory for how particles acquire mass in 1964 and saw their ideas confirmed last year with the discovery of the Higgs particle.

This represents the Standard model of elementary particles:
You can see representations for 12 fundamental particles
that make up matter (orange and green boxes) and
4 fundamental force carriers (purple boxes).

Dr.'s Higgs and Englert are both Professors Emeritus at University of Edinburgh, UK and Université Libre de Bruxelles, Belgium respectively. Their theory is central to the Standard Model of particle physics which describes the construction of the world based on the interaction between matter particles and force particles. The Standard Model relies on the existence of the Higgs particle which originates from an invisible field. The theory for which Higgs and Englert received this award describes the process where particles acquire mass when they interact with the invisible field.

Congratulations Dr. François Englert and Dr. Peter W. Higgs! Thank you for your contribution to our understanding of the physical world!



Information for this blog was obtained from:

 http://www.nobelprize.org/nobel_prizes/physics/laureates/2013/press.html

Monday, October 7, 2013

Winners of The 2013 Nobel Prize in Physiology or Medicine Announced.

The Nobel Assembly decided to jointly award the 2013 Nobel prize in Physiology or Medicine to Dr.'s James E. Rothman, Randy W. Scheckman and Thomas C. Südhof 'for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells'. Vesicles are the sub-cellular packages surrounded by membranes that are responsible moving molecules, such as hormones, enzymes or neurotransmitters which are produced by cells, from their site of production to the their eventual release either inside of outside of the cell. Each of the Nobel Laureates was instrumental in uncovering the molecular principles that govern the appropriate delivery of these packages.

Exocytosis, one of the possible endpoints of vesicular
trafficking,  is the process by which a cell directs secretory
vesicles to the cell membrane. These membrane-bound
vesicles contain soluble proteins to be secreted to the
extracellular environment, as well as membrane proteins
and lipids that are sent to become components of the
cell membrane.
by Mariana Ruiz
Dr. Südhof, a German born scientist, currently a Howard Hughes investigator and Professor at Stanford University, revealed how signals precisely regulate the release of cargo from vesicles. Dr Scheckman, is also a Howard Hughes and is a Professor at University of California at Berkley. His contribution was the discovery of a set of genes that are required for the cargo carrying vesicles to move through the cell. Meanwhile, Dr. Rothman, currently a Professor and Chariman of the Department of Biology at Yale, discovered the protein-protein interactions necessary for vesicles to fuse with their correct targets.

Without the appropriate function of vesicle transport a variety of physiological processes can be disrupted such as the release of hormones and cytokines. The result of these disruptions are a variety of disease states such as diabetes as well as neurological and immunological disorders. The contributions of these scientists have expanded our understanding of a vital process and are clearly worthy of the honor that is The Noble Prize!

Information from this blog was obtained from:
http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/press.html


Friday, October 4, 2013

New Small-Molecule Catalyst Could Greatly Speed Therapeutic Discovery Process

To create new therapeutics scientists identify lead compounds which have the desired effect. In order to make these compounds more efficacious, modifying this lead compound is often desirable to improve biological function. Currently, this usually involves either a long synthetic process or engineering an enzyme that is specific to that molecule. However, a recent report in the Journal of the American Chemical Society describes the work of University of Illinois chemists that shows the utility of a human-made catalyst that is an 'enzyme-mimic'.

Ball-and-stick model of the camphoric acid molecule,
 an oxidation product of camphor.

(Black: Carbon(C), White: Hydrogen(H), Red: Oxygen (O)
Unlike enzymes which are large proteins that act on only select molecular targets the new catalyst can oxidize specific carbon hydrogen (C-H) bonds on many different targets. The hope is that catalysts like this new one (called iron CF-3 PDP) and a previously reported catalyst (called iron PDP) will represent a new tool-box that will allow therapeutic designers to alter any C-H bond on any molecule quickly and efficiently.

If these catalysts realize their potential it will allow new therapeutics to be made in less than an hour rather than weeks. The new compounds can then be used in high throughput screening, something at which the PHERAstar FS from BMG LABTECH excels.

The original article from JACS is entitled: Catalyst-Controlled Aliphatic C–H Oxidations with a Predictive Model for Site-Selectivity.

Tuesday, October 1, 2013

New High-Throughput Screen for Hepatitis C inhibitors


Simplified diagram of the structure of Hepatitis C virus
by Graham Colm
RNA-dependent RNA polymerases (RdRp) play an essential role in replication of hepatitis C virus (HCV). As such it is a key target for novel antiviral therapies and inhibitors are in clinical trials for HCV genotype 1 (G1). However there are 6 distinct genotypes and the inhibitors for G1 exhibit a poor efficacy for non-G1 genotypes.

It was because of this scenario that a group from Australia sought to identify potential inhibitors for genotype 3a. The result of their work is described in the recent JBS article entitled: 'A Fluorescence-BasedHigh-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3aHepatitis C Virus RNA Polymerase’.

Their assay design was quite straight-forward in that they sought to assess RdRp activity by detecting synthesis of double-stranded RNA from a single-stranded template. To do this they took advantage of the fact that the commercially available fluorescent dye PicoGreen binds double stranded RNA preferentially over single-stranded RNA. Production of double-stranded RNA was quantified by measuring fluorescence intensity on a POLARstar Omega microplate reader from BMG LABTECH.


We at BMG LABTECH are happy that we can contribute in some small way to the assay development and performance of HTS necessary for drug discovery such as that described in this JBS article. Our new CLARIOstar provides even more flexibility when designing a FI based assay due to its Advanced Linear Variable Filter Monochromator. Furthermore, the PHERAstar FS continues to be an excellent choice for HTS. Whatever your microplate reader needs, BMG LABTECH has a product that will help you make the most of your application.

Wednesday, September 25, 2013

Conference Report: Discovery on Target

For the past couple of days representatives from BMG LABTECH have been in attendance at Discovery on Target in Boston, MA, USA. Here, we have learned more about epigenetics, which continues to grow in importance as the scientific community understands more about how epigenetics occurs and is regulated. Central to epigenetics is the post-translational modification of histones which plays a role in determining what genes will be transcribed into RNA and eventually translated into protein.
Cartoon diagram of an epigenetic reader binding to an
acetylated lysine in a histone tail

The proteins that are involved in epigenetics based on histone modification are broadly categorized into three groups. Writers and erasers which add and remove the post-translational modifications as well as readers which detect the modification and are often part of a multi-protein complex which interpret the modification and lead to a change in gene expression. These readers have been studied more avidly in recent years and many presentations and discussions at this conference have indicated the importance of readers in normal function. Furthermore, alterations of readers are associated with several disease states and they have emerged as new druggable targets.

In the simplest of terms the interaction between a modified histone tail and an epigenetic reader is a protein-protein interaction. As such it is not surprising that approaches that have been previously successful in elucidating protein-protein interactions have been applied to this field. This includes, but is not limited to fluorescence polarization and AlphaScreen. As BMG microplate readers, such as the PHERAstar FS and the new CLARIOstar, have been shown to perform these types of analyses well, they should be considered excellent tools for investigating the function of epigenetic readers.

One talk of particular note was by Danette Daniels, PhD, from Promega. Dr. Daniels described the combination of NanoLuc labeling of a bromodomain protein (an epigenetic reader) and fluorescent labeling of histone. In this way they can tell when the epigenetic reader is bound to histone in live cells through the use of BRET. This data was also recently shared in a BMG hosted webinar which showed that the new CLARIOstar is the only microplate reader with a monochromator that was sensitive enough to detect the NanoLuc BRET signal.

We are very glad that we have had the opportunity to attend Discovery on Target and meet with so many great scientists. We look forward to the remainder of the conference and hope that BMG can help you in your drug discovery program!