In the Literature

Blogs about published peer-reviewed scientific studies.

The Role of the NanoLuc® Reporter in Investigating Ligand-Receptor Interactions

Posted on by Nicole Sandler

Luminescent reporter assays are powerful research tools for a variety of applications. Last March we presented a webinar on this topic, Understanding Luminescent Reporter Assay Design, which proved to enlighten many who registered. The webinar addressed the importance of careful experimental design when using a luminescent reporter such as Promega’s Firefly or NanoLuc® Luciferase.

Reporters provide a highly sensitive, quantifiable metric for cellular events such as gene expression, protein function and signal transduction. Luminescent reporters have become even more valuable for live, real-time measurement of various processes in living cells. This is backed by the fact that a growing number of scientific publications reference the use of the NanoLuc® Luciferase reporter and demonstrate its effectiveness as a reporter assay. Continue reading “The Role of the NanoLuc® Reporter in Investigating Ligand-Receptor Interactions”

Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology

Posted on by Kyle Hooper

Cannabinoids. What are they? Sometimes, Wikipedia can give a nice definition:

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons
Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

A cannabinoid is one of a class of diverse chemical compounds that acts on cannabinoid receptors in cells that alter neurotransmitter release in the brain. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals), the phytocannabinoids (found in Cannabis and some other plants), and synthetic cannabinoids (manufactured artificially).

Synthetic cannabinoids (SCs) were originally created for the scientific investigation of two cannabinoid receptors, CB1 and CB2, but have made their way to the streets as “safe” and “legal” alternatives to marijuana.

The problem is that these SCs engage the cannabinoid receptors more completely and with higher affinity than anything derived from marijuana. As a result, SCs can produce serious side effects that often require medical attention. In fact, you are 30 times more likely to seek emergency medical attention following the use of an SC than with natural cannabinoid sources like marijuana.

Continue reading “Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology”

To Seq, or Not to Seq

Posted on by Nicole Sandler

Seq—shorthand for “sequence”— has become a more recognizable term thanks to a novel and provocative genomics initiative called the BabySeq Project. The project, officially launched in May 2015, was designed to explore the impact of whole-exome sequencing (WES) on newborn infants and their families. A randomized, controlled trial to sequence healthy and sick infants and then provide sequencing information, it is the first of its kind. Those infants randomized to receive WES undergo genetic sequencing of all protein-coding genes and analysis of about 1,700 genes implicated in childhood health, along with 18 years of follow up genetic counseling.29813751-nov-2-blog-post-nicole-600x470-web

The project is directed by Robert C. Green, geneticist and physician at Brigham and Women’s Hospital, Harvard Medical School and the Broad Institute, and Alan H. Beggs of Boston Children’s Hospital and Harvard Medical School. Funding, totaling $25 million, comes from the National Institute of Child Health and Development and the National Human Genome Research Institute. Continue reading “To Seq, or Not to Seq”

Making It Easier to Investigate PGTs

Posted on by Sara Klink

Introduction

Studying cellular molecules can be challenging. Some processes are troublesome to study due to the lack of an assay or a complicated assay exists but lacks sensitivity. Membrane proteins in particular are difficult to isolate and characterize. Phosphoglycosyltransferases (PGTs) are transmembrane proteins that transfer phosphosugars onto phospholipids, initiating the synthesis of oligosaccharides in bacterial cell walls. This transfer creates a diphosphate link between a lipid and a sugar and generates UMP as a byproduct. Once this lipid–P–P–sugar linkage occurs, more sugars can be added by glycosyltransferases, generating membrane-based polysaccharides (e.g., peptidoglycan) used for signaling, recognition and defense.

While PGTs have been studied biochemically and an X-ray structure of one member exists, much is still unknown about these enzymes. Overexpressing and purifying membrane proteins remains a challenge, and the conventional PGT assay requires isotope labeled-UDP-sugar donors and is based on the solubility difference between substrate and product to determine enzyme turnover using extraction-based or chromatographic methods. While there are other assays that use fluorescent modified substrates or multienzyme analysis, none of the methods can be applied to all of the diverse PGT enzymes.

All PGTs generate UMP as a byproduct of the transfer of a phosphosugar to a phospholipid. Based on the principle of the luminescent UDP-Glo™ Glycosyltransferase Assay where UDP released during the glycosyltransferase reaction was quantitated, a new luminescent assay called UMP-Glo™ Assay is able to measure the activity of PGT enzymes by adding a single reagent to detect UMP. Das et al. validated this assay by testing PglC, a PGT from Campylobacter jejuni, as well as PglC from Helicobacter pullorum and WecA from Thermatoga maritime and published the results in Scientific Reports. Continue reading “Making It Easier to Investigate PGTs”

Of Elephant Research and Wildlife Crime – Molecular Tools that Matter

Posted on by Nicole Sandler

Here at Promega we receive some interesting requests…

Take the case of Virginia Riddle Pearson, elephant scientist. Three years ago we received an email from Pearson requesting a donation of GoTaq G2 Taq polymerase to take with her to Africa for her field work on elephant herpesvirus. Working out of her portable field lab (a tent) in South Africa and Botswana, she needed a polymerase she could count on to perform reliably after being transported for several days (on her lap) at room temperature. Through the joint effort of her regional sales representative in New Jersey/Pennsylvania (Pearson’s lab was based out of Princeton University at the time) and our Genomics product marketing team, she received the G2 Taq she needed to take to Africa. There she was able to conduct her experiments, leading to productive results and the opportunity to continue pursuing her work.

Continue reading “Of Elephant Research and Wildlife Crime – Molecular Tools that Matter”

Protein:DNA Interactions—High-Throughput Analysis

Posted on by Gary Kobs
Copyright: molekuul / 123RF Stock Photo
Copyright: molekuul / 123RF Stock Photo

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Coupled Reticulocyte Lysate). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.

The Cell Line Identity Crisis

Posted on by Nicole Sandler
29981164-Whistle

If you work with cell lines you may have paid attention to the dramatic headline published last month in the online journal STAT, Thousands of studies used the wrong cells, and journals are doing nothing.” In their column The Watchdogs (“Keeping an eye on misconduct, fraud, and scientific integrity”), Ivan Oransky and Adam Marcus call out the fact that scientists continue to publish research using cell lines that are contaminated or misidentified. Recent estimates have found that the percentage of misidentified cell lines used by scientists is as high as 20 to 36. The blame here is being placed on the peer reviewed journals for not blowing the whistle. The authors call for journals to put some “kind of disclaimer on the thousands of studies affected.”

This is not a new claim. The continuing problem of cell line misidentification, of lack of authentication, has been covered before in various channels. It’s easy to find news publicizing yet another retracted publication. In May 2015 the journal Nature required authors of all submitted manuscripts to confirm the identity of cell lines used in their studies and provide details about the source and testing of their cell lines.

Continue reading “The Cell Line Identity Crisis”

Digging Up More Clues in the History of the Black Death

Posted on by Sara Klink

Bubonic plague victims in a mass grave in 18th century France. By S. Tzortzis [Public domain], via Wikimedia Commons
Bubonic plague victims in a mass grave in 18th century France. By S. Tzortzis [Public domain], via Wikimedia Commons
My last blog post on the Black Death highlighted research that suggested that the reintroduction of Yersinia pestis, the causative agent of the pandemic, originated in Europe during the 14–18th centuries rather than from Asia, the hypothesized origin. In my post, I wrote about my curiosity regarding what an Asian skeleton positive for Y. pestis from that same time period would reveal about the strain or strains that were circulating. Well, a team of researchers has been exploring the issue of strain circulation and an Asian connection, and recently published what they gleaned from additional historic Y. pestis samples in Cell Host & Microbe.

Teeth from 178 individuals in three different locations (two European, one Asian) were screened for Y. pestis infection using the plasminogen activator (pla) gene. Continue reading “Digging Up More Clues in the History of the Black Death”

Magnetic Bacteria Carry Drugs into Tumors

Posted on by Isobel Maciver
cancer cell

At first glance, the biology of magnetic, underwater-dwelling, oxygen-averse bacteria may seem of little relevance to our most pressing human health problems. But science is full of surprises. A paper published in Nature Nanotechnology presents an inspired use of these bacteria to deliver anti-cancer drugs to tumors, specifically targeting the oxygen-starved regions generated by aggressively proliferating cells.

Continue reading “Magnetic Bacteria Carry Drugs into Tumors”

An Epizootic for the Ages: Revisiting the White-Nose Syndrome Story

Posted on by Michele Arduengo
Map showing the spread of WNS across North America
Map showing the spread of WNS across North America

In March 2016, two hikers on a trail east of Seattle, WA, found a little brown bat lying on the ground in obviously poor condition. The bat was taken to an animal shelter where it died two days later from White-Nose Syndrome (WNS).

This bat was the first case of WNS found west of the Rocky Mountains. It represented a jump in the spread of WNS, and a troubling one. WNS was first detected in a cave in Albany, New York, and since then it has been moving slowly westward at a rate of about 200 miles per year, according to David Blehert of the United States Geological Survey, the laboratory that confirmed the WNS diagnosis for the Washington bat. Before this year’s discovery outside of Seattle, the westward-most case detected was in eastern Nebraska.

WNS, caused by a cold-loving fungus, Psuedogymnoascus destructans (Pd), can kill 100% of the hibernating bats in a colony, and in the ten years since it has been detected and monitored has killed over 6 million bats in the United States and Canada. As of July 2016, bats infected with the fungus have been found in 29 states and 5 Canadian provinces.

According to Blehert, this is probably the “most significant epizootic of wildlife” ever observed; never before have we seen hibernating mammals specifically affected by a skin fungus. What does that mean? Are we looking at extinction for some bat species? What are the ecological consequences of rapidly losing so many individuals to disease so quickly? And, what, if anything, can be done to combat the disease and help bat populations recover?

Continue reading “An Epizootic for the Ages: Revisiting the White-Nose Syndrome Story”