In the Literature

Blogs about published peer-reviewed scientific studies.

Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

Posted on by Leah Cronan

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

Continue reading “Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors”

To Sleep, Perchance to Clean

Posted on by Kari Kenefick

While you and I are getting some shut eye each night, things are happening in our brains. Good things. Therapeutic things.

Think of it as brainwashing of a sort. There is a multiplicity of brain activities going on during sleep, and a November 1 paper in Science shows for the first time when and where in the brain these activities occur, and how they are connected.

CSF washes through the brain.

Here’s a bit of backstory.

To assess both the progression and pathogenesis of Alzheimer’s disease (AD), as well as the efficacy of AD drugs in clinical trials, there has been interest in the concentrations of amyloid-beta (Aβ) and tau protein in cerebral spinal fluid (CSF).

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A Diamond in the Rough: New Applications of Diamond Nucleic Acid Dye

Posted on by Leah Cronan

Diamond™ Nucleic Acid Dye (Cat# H1181) is a safe, inexpensive and sensitive fluorescent dye option that binds to single-stranded and double-stranded DNA and RNA. Diamond™ Dye typically is used for staining electrophoresis gels to visualize nucleic acids in a similar to its carcinogenic counterpart, ethidium bromide. However Diamond™ Dye has several advantages: gels stained with Diamond™ Dye can be visualized using either UV or blue-light transilluminators. Also, a wash step after staining is not necessary when using Diamond™ Dye, unlike what is typically recommended for ethidium bromide.

Besides staining electrophoresis gels, there are other applications for this diamond in the rough. Highlighted below are two fascinating uses of this multifaceted tool: touch DNA localization and qPCR detection.

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All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Posted on by Sara Klink

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.

With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA, like that induced by CRISPR-Cas9, is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”

NLRP3: The New Hope for Treating Chronic Inflammatory Diseases

Posted on by Johanna Lee
Inflammasome - inflammatory diseases caused by NLRP3

Our innate immune system was meant to do good. Up until a century ago, most humans died from infectious diseases like diarrhea, tuberculosis and meningitis. Over millions of years, our immune system has evolved to fight these life-threatening infections from pathogens. As a result, we have developed a highly efficient response to these tiny invaders. But it seems that our immune system may be turning against us.

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The Surprising Life of Bones

Posted on by Kari Kenefick

Schematic of bone producing and reducing cells osteoblasts and osteoclasts.
The cells that make and degrade bone.

Standing, walking, running. When was the last time you gave your skeleton a second thought? How about when that car barely missed you in the parking lot? Or a deer ran in front of you? Maybe you just missed a car door opening on your bike ride today?

Your bones were involved in your response to that sudden shock/surprise, but not the way you think.

You may have jumped, swerved or hit the brake pedal (congratulations on the excellent reflexes) and yes, bones were involved in all of those actions. But a new article in Cell Metabolism reveals that bone is the essential component in initiation of that response.

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Striking Fear into the Heart of Cardiovascular Disease Using Zebrafish and NanoLuc® Luciferase

Posted on by Natalie Larsen
Representative images of ApoB-LP localization in zebrafish across developmental, genetic, pharmacological and dietary manipulations.
Credit: Figure 5.D of The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins by James Thierer, Stephen C. Ekker and Steven A. Farber.
Article licensed under Creative Commons Attribution 4.0 International License.

Cardiovascular diseases, or CVDs, are collectively the most notorious gang of cold-blooded killers threatening human lives today. These unforgiving villains, including the likes of coronary heart disease, cerebrovascular disease and pulmonary embolisms, are jointly responsible for more deaths per year than any other source, securing their seat as the number one cause of human mortality on a global scale.

One of the trademarks of most CVDs is the thickening and stiffening of the arteries, a condition known as atherosclerosis. Atherosclerosis is characterized by the accumulation of cholesterol, fats and other substances, which together form plaques in and on the artery walls. These plaques clog or narrow your arteries until they completely block the flow of blood, and can no longer supply sufficient blood to your tissues and organs. Or the plaques can burst, setting off a disastrous chain reaction that begins with a blood clot, and often ends with a heart attack or stroke.

Given the global prevalence and magnitude of this problem, there is a significant and urgent demand for better ways to treat CVDs. In a recent study published in Nature Communications, researchers at the Carnegie Institution for Science, Johns Hopkins University and Mayo Clinic are taking the fight to CVDs through the study of low-density lipoproteins (LDLs), the particles responsible for shuttling bad cholesterol throughout the bloodstream.

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Delving into the Diversity of The Plague of Justinian

Posted on by Sara Klink

Wayson stain of Yersinia pestis showing the characteristic
Yersinia pestis. U.S. Center for Disease Control [Public domain], via Wikimedia Commons.
Human teeth play a key role in our understanding of how organisms evolve. Whenever a possible new member of the hominid family is uncovered, the shape and number of teeth are used to place that individual in the family tree. Teeth also harbor information about pathogens that have plagued humans for millennia. Because bacteria use our bloodstream as a transport system, protected places that can preserve DNA—like the pulp of teeth—are a rich medium for uncovering information about humans and the microbes that infected them.

Teeth have been the choice for identifying the infectious agent behind the Plague of Justinian in the sixth century and the Black Plague in the 14th century. In fact, Yersinia pestis, the bacterium responsible for these plagues, has infected humans as far back as the Neolithic. But what can we learn about the pandemic strain or strains of Y. pestis described in historical records? A team of researchers from Europe and the US, many of whom have been delving into the history of Y. pestis for the last decade, wanted to further investigate the Plague of Justinian. They studied bacterial DNA extracted from human remains found in Western European communal graves that were dated to around 541–750, the period of the historically documented Plague of Justinian. Their investigation examined the bacteria’s diversity and how far it spread during this “First Pandemic” of plague. Continue reading “Delving into the Diversity of The Plague of Justinian”

CRISPR/Cas9, NanoBRET and GPCRs: A Bright Future for Drug Discovery

Posted on by Ken Doyle

GPCRs

G protein-coupled receptors (GPCRs) are a large family of receptors that traverse the cell membrane seven times. Functionally, GPCRs are extremely diverse, yet they contain highly conserved structural regions. GPCRs respond to a variety of signals, from small molecules to peptides and large proteins. Many GPCRs are involved in disease pathways and, not surprisingly, they present attractive targets for both small-molecule and biologic drugs.

In response to a signal, GPCRs undergo a conformational change, triggering an interaction with a G protein—a specialized protein that binds GDP in its inactive state or GTP when activated. Typically, the GPCR exchanges the G protein-bound GDP molecule for a GTP molecule, causing the activated G protein to dissociate into two subunits that remain anchored to the cell membrane. These subunits relay the signal to various other proteins that interact with or produce second-messenger molecules. Activation of a single G protein can result, ultimately, in the generation of thousands of second messengers.

Given the complexity of GPCR signaling pathways and their importance to human health, a considerable amount of research has been devoted to GPCR interactions, both with specific ligands and G proteins.

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Activating the Inflammasome: A New Tool Brings New Understanding

Posted on by Michele Arduengo

Innate immunity, the first line of immune defense, uses a system of host pattern recognition receptors (PRRs) to recognize signals of “danger” including invariant pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These signals in turn recruit and assemble protein complexes called inflammasomes, resulting in the activation of caspase-1, the processing and release of the pro-inflammatory cytokines IL-1ß and IL-18, and the induction of programmed, lytic cell death known as pyroptosis.

Innate immunity and the activity of the inflammasome are critical for successful immunity against a myriad of environmental pathogens. However dysregulation of inflammasome activity is associated with many inflammatory diseases including type 2 diabetes, obesity-induced asthma, and insulin resistance. Recently, aberrant NLRP3 inflammasome activity also has been associated with age-related macular degeneration and Alzheimer disease. Understanding the players and regulators involved in inflammasome activity and regulation may provide additional therapeutic targets for these diseases.

Currently inflammasome activation is monitored using antibody-based techniques such as Western blotting or ELISA’s to detect processed caspase-1 or processed IL-1ß. These techniques are tedious and are only indirect measures of caspase activity. Further, gaining information about kinetics—relating inflammasome assembly, caspase-1 activation and pyroptosis in time—is very difficult using these methods. O’Brien et al. describe a one-step, high-throughput method that enables the direct measurement of caspase-1 activity. The assay can be multiplexed with a fluorescent viability assay, providing information about the timing of cell death and caspase-1 activity from the same sample. Continue reading “Activating the Inflammasome: A New Tool Brings New Understanding”