Considerations for Successful Cell-Based Assays I: Choosing Your Cells

For those of us entering the world of cell-based assays from a classical or molecular genetics background, the world of cell culture can be daunting. Yet to truly understand how the genetic mutation behind a particular phenotype works, we need to look at the biochemistry and cell biology where it all occurs: the cell.

This series of blogs will cover several topics to consider when designing your cell-based assays. In this first installment, we discuss the basics of choosing the cell type for your assay.

Continue reading “Considerations for Successful Cell-Based Assays I: Choosing Your Cells”

When the Writing Gets Tough, the Tough Write about Semicarbazide-Sensitive Amine Oxidases

These are the cranes I saw while walking and thinking about SSAOs.
When you hold a position as a scientific communication specialist at a biotech company, you never know what you are going to need to write. Most of the time I really like the fact that I have to master new subject matter on a daily basis. I’m using my degree and my brain, and articulating science in a way that connects with the reader is incredibly rewarding. It’s why I do what I do.

However, when I was asked to write about a new assay for semicarbazide-sensitive amine oxidases (SSAOs), my enthusiasm waned. This is a subject about which I know nothing, so I searched the literature to learn as much as I could. After reading several review articles I was able to write this scintillating paragraph: Continue reading “When the Writing Gets Tough, the Tough Write about Semicarbazide-Sensitive Amine Oxidases”

Avoid Multiple Freeze/Thaw Cycles: Woolly Bear Caterpillars

Recently, I was walking through a local park near Promega, when I spotted my first woolly bear of the season. As this furry brown and black caterpillar wandered along in front of me, I recalled the old wives’ tale about the width of their stripes being indicative of the upcoming winter fury. Spotting that little fellow in the sidewalk piqued my curiosity, and I decided to see what I could discover about my friend, the woolly bear. Continue reading “Avoid Multiple Freeze/Thaw Cycles: Woolly Bear Caterpillars”

A Scalable and Sensitive Assay for HDAC Activity

Dysfunction of histone deacetylases (HDACs) is associated with many diseases including cancers, asthma and allergies, inflammatory diseases and disorders affecting the central nervous system. Because of their involvement in such a wide range of pathologies, HDACs have become a target for drug discovery. Traditional HDAC activity assays are either isotopic or fluorescent assays using artificial substrates that are prone to artifacts or fluorescence interference. There is a need for a functional assay that is sensitive, accurate and amenable to drug-screening activities.

A recent paper by Halley et al. in the Journal of Biomolecular Screening describes the evaluation of a bioluminogenic HDAC assay, the HDAC-Glo™ I/II Assays,

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Identifying and Profiling Inhibitors for PI 4-Kinases Using a Luminescent High-Throughput Screen

The phosphotidylinositol 4-kinases (PI 4-kinases) generate phosphotidyl-4-phosphate (PI(4)P) from phosphotylinositol. PI(4)P is an important precursor for other phosphoinositides involved in signaling, such as PI(4,5)P2, which is the substrate of phospholipase C (PLC) and the precursor of DAG and insitol (1,4,5) triphosphate.

There are four different mammalian PI 4-kinases currently described, and these have been divided into two classes based on their sensitivities to wortmannin and adenosine. Type II PI 4-kinases (PI4K2A and PI4K2B) are not sensitive to wortmannin, but are inhibited by the nonspecific inhibitor adenosine; Type III PI 4-kinases (PI4KA and PI4KB) are sensitive to wortmannin.

The functions of the PI 4-kinases and their products are not fully understood. At least one study has shown that PI 4-kinases are important for the proper recycling of synaptic vesicles. The PI 4-kinase from Drosophila, four-wheel drive, is critical for contractile ring formation during cytokinesis. Other studies in yeasts and mammals have shown that PI 4-kinases localize to the Golgi, and in mammals might be critical for proper budding of vesicles from the Golgi. Additionally, type III PI 4-kinases appear to play a role in the replication of hepatitis C virus (HCV) and enteroviruses by participating in the formation of altered host membrane structures. Although, we have hints about their function, to really understand and dissect the precise roles of PI 4-kinases in cells, new tools, such as specific small-molecule inhibitors are required.

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How does the butterfly get its spots?

All we found were sawfly larvae.
This summer, my daughter and I have gone on several “bug-hunting expeditions”. These expeditions always begin with the same elaborate routine: donning the explorer vest, collecting the magnifying glass, bug house and butterfly net, and consulting the “bug map”. The goal is to find a caterpillar that we can capture, feed and watch as it morphs into a butterfly. So far the only thing remotely resembling a caterpillar we have found is sawfly larvae.

My daughter is fascinated by butterflies. We have at least three books on her bookshelf about butterfly life cycles, and just the other week, a trip to the library yielded yet another butterfly book for bedtime reading. Butterflies are fascinating creatures. Not just for the four-year-old who wonders in awe at their amazing life cycle, but for the biologist who marvels at the development of the intricate pattern of their wing eyespots. Wing patterns in butterflies are amazingly varied among species and between different wing surfaces (forewing and hindwing) of the same individual. How are these patterns determined? Continue reading “How does the butterfly get its spots?”

Smart Gloves (and catheters): Tracing a Beating Heart May Now Be at Your Fingertips

A paper recently published in Nature Materials by Kim and colleagues highlights exciting work in materials science to improve the mechanical balloon catheter, that staple of cardiology used to open stenotic arteries, insert and expand stents, and find and ablate tissues in the heart that are the source of dangerous arrhythmias.

In this study, the researchers designed a “smart” balloon catheter specifically for cardiac ablation therapy, a technique that is used to treat sustained arrhythmias such as atrial fibrillation (A-fib). Time is often an important factor in treating such conditions, and using conventional mechanical catheters to find the malfunctioning heart tissue then inserting a second catheter to perform the ablation is a time-consuming process that takes a great deal of art and skill on the part of the practitioner. New catheters are improving some of the mechanics, but they still do not provide important sensory information to the physician such as local blood flow, temperature, pressure, etc.

Here the researchers sought to integrate semiconductor devices and sensor technologies directly into the membranes of balloon catheters. Continue reading “Smart Gloves (and catheters): Tracing a Beating Heart May Now Be at Your Fingertips”

PCR-Based Diagnosis Wins by a Mile In the White-Nose Syndrome Race

Affected bats in a cave in MA. Image courtesy of the U.S. Geological Survey.
Affected bats in a cave in MA. Image courtesy of the U.S. Geological Survey.

Since the first photograph of bats with white muzzles in Albany, NY, was published, hibernating bat populations in the northeastern U.S. have been devastated by an emerging disease, White-Nose Syndrome (WNS), which continues to spread throughout the United States and now has been found in two Canadian provinces. Bats suffering from WNS are emaciated with little or no body fat and have a characteristic white fungal growth on their wing membranes, ears and muzzles. Instead of hibernating all winter, these bats can be seen active in the snow, when there is virtually no food available.

The infectious agent that responsible for WNS is a new species of the cold-loving fungus, Pseudogymnoascus destructans (formerly, Geomyces destructans) . Currently, WNS is confirmed either through histological analysis or by fungal culture. Both of these techniques have significant limitations. First, they have turnaround times of at least one week. Secondly, they require large amounts of tissue sample from affected bats, more than can be reasonably taken from live bats. Histological analysis is a laborious process that requires highly skilled and trained personnel. Fungal culture can be difficult because bats harbor many bacteria and fungi, and getting a pure culture of a causative organism is not simple. Furthermore, researchers need a quick method for assessing spread of the disease that can provide results quickly.

Polymerase Chain Reaction (PCR), already used for a host of diagnostic tests in humans, plants and animals, is a logical choice. In a paper published in the Journal of Veterinary Diagnostic Investigation, Lorch and colleagues design and evaluate a PCR-based diagnostic method for WNS. They compare the PCR method to a fungal culture method and the “gold standard” traditional histological analysis. Continue reading “PCR-Based Diagnosis Wins by a Mile In the White-Nose Syndrome Race”

Knitting Needles, Balls of Yarn and the First Molecular Model

ball-and-stick model of a molecule

One day while reading a knitting blog I discovered in 1883 a Scottish chemist created the first “ball-and-stick” model of a molecule using knitting needles and balls of yarn. This initial ball-and-stick molecule represents the structure of sodium chloride and is constructed of knitting needles, representing the bonds, and alternating balls of blue and red yarn, representing the atoms of sodium and chloride. It was displayed as part of the International Year of Chemistry 2011 activities.

The chemist who created this model was Alexander Crum Brown, distinguished chemistry and professor at the University of Edinburgh, and one of his particular interests was the arrangements of atoms in molecules and the depiction of these structures. Those of us who spent countless hours poring our organic chemistry books and molecular model sets trying to understand nucleophilic attacks and SN1 and SN2 reactions have Alexander Crum Brown to thank. Those students who now use computer 3D modeling programs to accomplish the same studies (without the delight of chasing down the last nitrogen atom that has rolled off the desk and under the dresser) are also indebted to Dr. Brown.

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Are we finally smart enough to learn from the gloomy octopus?

Test octopus responding to stimulus. See blog for link to videos.

The octopus is a fascinating creature and probably the smartest invertebrate known. It has an extremely well developed visual system with high acuity, and although it cannot distinguish color, it can see polarized light. Scientists have proposed that octopuses communicate with each other by polarizing the light that reflects from their scales, creating a messaging system that could not be observed, much less understood by other creatures in their realm who have much less sophisticated visual systems (1).

Many animals rely on visual stimuli for recognizing predators, food and other members of their species. And scientists have used pictures, models and videos to attempt to understand how visual cues affect animal behavior. Pictures and models lack the movement aspect of live stimuli, so video playback presents the best, controllable method, for presenting visual stimuli. However, video has been designed for the human visual system, not that of the octopus, and until recently no video study has managed to elicit a biologically appropriate response from cephalopods (octopus, cuttlefish or squid). Continue reading “Are we finally smart enough to learn from the gloomy octopus?”