Noninvasive Prenatal Genetic Testing Using Circulating Cell-Free DNA

Expectant MotherScientists have known for some time that fetal DNA can be detected in the maternal bloodstream during pregnancy (1). Up to 10% of circulating cell-free-DNA (ccfDNA) can be attributed to the fetus. Fetal ccfDNA is released from the placenta, mainly through apoptosis, and enters the maternal bloodstream, where it can be easily collected and detected by PCR amplification. This method of collection has a much lower risk of miscarriage compared to more invasive collection methods such as amniocentesis and chorionic villus sampling.

Amplification of fetal ccfDNA enables a number of prenatal genetic testing such as gender determination and detection of fetal aneuploidy and other mutations. Testing of ccfDNA also allows identification of fetuses with a higher risks of hemolytic disease of the newborn (erythroblastosis fetalis) due to expression of the Rh factor in an Rh– negative mother, who can develop antibodies against the Rh factor and mount an immune response against fetal red blood cells. Finally, ccfDNA allows prenatal paternity determination (2). However, these tests have limitations.

Continue reading “Noninvasive Prenatal Genetic Testing Using Circulating Cell-Free DNA”

Characterizing Immune-Modulating Antibodies Using Bioluminescence

Immune checkpoint pathways such as PD-1/PD-L1 and CTLA-4 are promising new immunotherapy targets for the treatment of cancer and autoimmunity. Immune checkpoint reporter-based bioassays provide a simple, consistent, and reliable cell-based assay to measure Ab function throughout the drug development pipeline.

The brief chalk talk below describes the assay principals of the reporter-based bioassay that monitors the functional blockade of PD-1/PD-L1 interactions.

Thankful it is Friday

Haven’t we all had that day where everything that could go wrong did go wrong?  Hopefully today was not one of those days, but if it was, I hope this cartoon helps.

Cartoon

And if we can help with one of those things that didn’t go quite right, remember our Technical Services Scientist are here to help. You can find out how to reach them on our website.

If you need some more laughter, you can find more cartoons here.

Happy Friday!

 

A Potential Single-Tube Multiplex Assay for Detecting Dengue Virus in the Field

In areas of the world where the electricity is intermittent, resources are limited and transporting bulky equipment and reagents that are sensitive to temperature fluctuations is difficult, diagnosis of viruses like dengue can be challenging. If you could reduce or eliminate the need for electricity dependent equipment for diagnostic assays without sacrificing sensitivity or specificity, it would be a boon to field workers. An article published in PLOS ONE describes how researchers developed a multiplex isothermal amplification method that could assess a potential dengue infection with a visual real-time or endpoint detection in a single tube.

Countries affected by dengue. By Percherie (Distribution de la dengue sur Commons) [GFDL ), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 , via Wikimedia Commons
Continue reading “A Potential Single-Tube Multiplex Assay for Detecting Dengue Virus in the Field”

Purify and Conjugate Antibodies in a Single Workflow

Isoform_Antibodies_LinkedInAntibodies labeled with small molecules such as fluorophore, biotin or drugs play a critical role in various areas of biological research,drug discovery and diagnostics. There are several limitations to current methods for labeling antibodies including the need for purified antibodies at high concentrations and multiple buffer exchange steps.

In a recent publication, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads.

Using a variety of fluorophores the researchers show that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry.

This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Literature Cited

Nidhi, N. et al. (2015) On-bead antibody-small molecule conjugation using high-capacity magnetic bead J. Immunol. Methods  http://dx.doi.org/10.1016/j.jim.2015.08.008

Rewriting the Histone Code: Searching for Treatments for Stage IV Thyroid Cancers

Chromatin fiberOften a diagnosis of thyroid cancer is associated with a good prognosis and fairly straightforward surgical treatments to remove the tumor followed by radioactive iodine ablation. Such treatment works well in tumors that have not metastasized and retain enough of their thyroid cell “identity” that they can still accumulate radioactive iodine.

However, aggressive thyroid cancers, which often metastasize and recur, do not respond to standard treatments because they are generally too dedifferentiated to accumulate iodine, so alternative treatments are needed.

One approach is to look for compounds that will reverse dedifferentiation, making tumor cells more likely to take up and concentrate radioactive iodine regardless of their location in the body. One possible target to effect dedifferentiation is epigenetic modification of histone proteins.

Histone proteins are more than the structural components of the nucleosome that organizes the chromatin inside cells. Histone proteins are subject to a host of protein modifications on their N-terminal tails such as acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation. These various modifications are seen as creating a “histone code” that is read by other proteins and protein complexes (1). This code regulates patterns of gene expression and activity for a cell—in part resulting in a differentiated phenotype. Previous studies have suggested that some histone deacetylase (HDAC) inhibitors (e.g., valproic acid) can reverse some of the dedifferentiation associated with aggressive cancers (2).

Jang, et al. in a recent paper (3) published in Cancer Gene Therapy synthesized a group of HDAC inhibitor analogs (AB1–AB13) and tested them for their ability to inhibit growth of three aggressive human thyroid cancer cell lines and induce partial re-differentiation to the thyroid cell phenotype. Continue reading “Rewriting the Histone Code: Searching for Treatments for Stage IV Thyroid Cancers”

A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation

Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.

mirna

The accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.

Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.

Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control.

Continue reading “A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation”

Friday Cartoon Fun: Is It Drawn or Is It Real?

I always enjoy Ed Himelblau’s cartoons, but one that makes me chuckle every time I see it is the following:

Copyright Ed Himelblau.

I am sure our readers that enjoy coffee can empathize.

Recently, our Swiss branch had fun with a number of the cartoons from our Cartoon Lab archive and recreated the cartoon in real life:
Real-life recreation of Ed Himelblau's cartoon.
What do you think?

Back to school!

Kindergarten teacher and children looking at bird's nest in librBack to school! We’re experiencing a cold snap this week, and my kids are complaining that it shouldn’t be this cold on the last week of summer! I agree, but I’m so excited that school is back! I’m a full-time working mom, but the stresses of summer are hard. My kids aren’t on schedules, there is always some party/event/BBQ, and trying to fit in a summer vacation. I’m tired just thinking about it! Especially in Wisconsin, where we don’t have the best weather in the winter so we try to soak it all up in the summer.

As we are getting back into routines, I’m working on setting up some ground rules so our school year isn’t as hectic this year as previous years. Summer has been a free for all at our house, so the adjustment might be rough, but my kids do much better on routines. Continue reading “Back to school!”

The Volcano Beneath our Feet

Photo credit: Kelly Grooms
Firehole River, Yellowstone National Park. Photo credit: Kelly Grooms

This summer my family and I vacationed on one of the world’s largest active volcanoes. We weren’t alone. Every year over three million people visit this super volcano.

Yellowstone National Park covers almost 3,500 square miles in the northwest corner of Wyoming (3% of the park is in Montana and 1% in Idaho). The park is famous for its hydrothermal features, including the Old Faithful Geyser and vivid hot springs such as the Grand Prismatic Spring.

The park’s hydrothermal system is the visible expression of the immense Yellowstone volcano; they would not exist without the underlying partially molten magma body that releases tremendous heat. National Park Service website

Photo Credit: Nathan Grooms
Grand Prismatic Spring, Yellowstone National Park. Photo Credit: Nathan Grooms

These features are all visible reminders of the immense volcano that exists beneath the surface.  Recently, a team of seismologists discovered a reservoir of partly molten rock 12–28 miles beneath Yellowstone National Park. This video from Science 360 describes the discovery and why scientists are interested in it. It is important to note that this discovery does not mean that there is new activity or that the volcano under the park is closer to erupting. It does mean that scientist now have a better picture of the underground “plumbing”.