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.