In the Literature

Blogs about published peer-reviewed scientific studies.

Optimizing Antibody Internalization Assays: pHAb Dyes

Posted on by Gary Kobs
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Promega has recently developed a method that allows antibodies to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When an antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane, the antibody-dye-antigen complex is not fluorescent, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops, and the dye becomes fluorescent.

To demonstrate the broad utility of the pHAb dye for receptor mediated antibody internalization, two therapeutic antibodies, trastuzumab and cetuximab,which bind to HER2 and EGFR respectively, were selected for a case study (1). Both the antibodies, which are known to internalize were labeled with pHAb dyes using amine or thiol chemistry.

Parameters such as the impact of dye–to-antibody ratio on the antigen–antibody binding, change in fluorescence as a function of pH of free dye and labeled dye, and labeled antibody internalization as a function of pHAb conjugated antibody concentration were evaluated.

The results indicate that pHAb dyes are pH sensitive fluorescent dyes that enable the study of receptor-mediated antibody internalization.Internalization assays can be performed in a plate-based homogeneous format and allow endpoint assays as well as real-time monitoring of internalization. They further show that internalization can be monitored even at a very low amount of antibody which is very important during the early monoclonal antibody development phase when the amount of sample is limited and the antibody concentration in the samples is low. a complimentary approach, they  also showed that a secondary antibody labeled with pHAb dye can be used instead of labeling primary antibodies.

Literature cited

Nath, N. et al. (2016) Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye J.  Immunol. Methods epub ahead of print

Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts

Posted on by Michele Arduengo

26911030-Laymans-KSPS-figure-WEB-R4Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds, and compounds that appear to have the desired potency against a specific cellular target or pathway are further evaluated. Candidate compounds that fail late in development or during clinical trials because of off-target effects are costly, and can be dangerous. Therefore drug developers not only need to ensure that a candidate compound is effective as a therapy, but also they need to predict any potential undesirable side effects due to off-target activities as early as possible in the drug discovery and development process. Continue reading “Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts”

To Meditate Perchance to Dream

Posted on by Kari Kenefick

First the disclosure: this blog is of course about Me.

But it’s also about You. And yours. Because as you know, we’ve become a culture that does not sleep.

Why don’t we sleep? I like to think that it is an evolutionary adaptation; not sleeping, after all, allows us more time for Facebook.

Or Etsy for you makers. Or Amazon for you shoppers. And let’s not forget our middle, high school and college students. Do they even have classrooms anymore, or are lectures all online (on screens)?

One tired pony. By Rachel C from Scotland (Flickr) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons
One tired pony. By Rachel C from Scotland (Flickr) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons
Honestly, the evolutionary adaptation idea comes from how we live and work today. And no, this is not another rant/lecture on the color of light emitted by whatever non-cathode ray tubes are in our phones or tablet-like devices.

It’s just that just working in our very busy online/wired world, jumping from web page to project management software, to big-screens in meetings has us adapted to being  on: capital “O” capital “N”.

This multi-multitasking has grown (for me) a new type of neurons that are not happy unless they are gleaning new information from a screen, all the time. And these neurons don’t stop working when the screen is gone; no, they continue seeking and trying to process. For me, if there’s no screen to look at, the neurons ping-pong around behind my eyeballs, looking and searching, as if to say, “Input missing! Input missing!”

The result can be hours in bed sans sleep; it seems the racket these neurons make keeps all the other neurons up. Continue reading “To Meditate Perchance to Dream”

What’s for Dinner? Mystery Meat Served at the 47th Explorer’s Club Annual Dinner Finally Identified

Posted on by Michele Arduengo
Could this be the 1951 mystery meat?
Could this be the 1951 mystery meat?

I remember one particular encounter with “mystery meat” when I was in college. I was walking along the serving line at the dining hall, and when I came to the entrée, I asked the server, “What is it?”

She replied quite succinctly, “Don’t know. Got beef in it.” I passed on the entrée that night, settling for salad and bread.

I would probably not have be a good candidate for membership in the Explorers Club.

The Explorers Club, founded in New York City in 1904, is a professional society that champions the cause of field research (1). The member list is impressive, including Teddy Roosevelt, the American President responsible for setting aside many of the most treasured public lands in the United States so that explorers have fields for research and wild places for adventures, Neil Armstrong, the first man to set foot on the moon, and Don Walsh and Jacques Piccard, the two men who descended into the Mariana’s trench to explore the deepest part of the ocean, among others.

In addition to a membership list that reads like a who’s who of science and exploration, The Explorers Club also has an annual dinner that for many years has popularized a menu of “exotic” foods (at least exotic foods from the point of view of the typical Midwest United States pallet). One of the club’s most celebrated dinners took place on January 13, 1951.

Continue reading “What’s for Dinner? Mystery Meat Served at the 47th Explorer’s Club Annual Dinner Finally Identified”

Inflammasomes: Peeking Inside the Inflammatory Process

Posted on by Kelly Grooms

Most of us have experienced an inflammatory response at some point in our lives. Fever, achy joints, swelling around a scrape or cut, all of these are forms of inflammatory response. Inflammation is the body’s response to infection or tissue damage and acts to limit harm to the rest of the body. A key player in the inflammation process is a group of protein complexes call inflammasomes. The term “inflammasome” was first used in 2002 by researchers in Switzerland (1) to refer to a caspase-activating protein complex. We now know that inflammasomes are cytosolic multiprotein platforms that assemble in response to pathogens and other signals. Inflammasome assembly results in the processing of the inactive procaspase-1 into the active cysteine-protease enzyme, caspase-1. Caspase-1, in turn, activates the proinflammatory cytokines Interleukins IL-1β and IL-18. In addition, caspase-1 is also required for pyroptosis, which is an inflammatory form of cell death that combines the characteristics of apoptosis (fragmented DNA) and necrosis (inflammation and cytokine release) and is frequently associated with microbial infections.

Inflammasome complexes are made up of scaffolding sensor proteins (NLR, AIM2, ALR), and an adaptor protein containing a caspase activation and retention domain (CARD) and inactive procaspase-1. Most inflammasomes are formed with one or two NLRs (NOD-like receptors). However, non-NLR proteins such as AIM2 (absent in melanoma 2) and pyrin can also form inflammasomes. The different sensor proteins are activated by different types of outside stimuli, and inflammasomes are loosely sorted into families based on the protein forming these sensors.26061534-Inflamasome-Assay-Blog-figure-WEB Continue reading “Inflammasomes: Peeking Inside the Inflammatory Process”

The Black Death: World Traveler or Persistent Homebody?

Posted on by Sara Klink

Spread of the Black Death. By Timemaps (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons.
In the last six years, researchers have untangled the origins of devastating human plagues, sequenced the genome of a Yersinia pestis strain responsible for the Black Death and explored how long this bacterium has been with humans. However, the information arising from this research begs more questions. How many variations of Y. pestis occurred during the 14–17th centuries, the second pandemic that began with the Black Death? Did these differences reflect the location in which the Y. pestis-positive skeletons were found? What were the geographic source or sources of these plagues? A recent PLOS ONE article examined Y. pestis found in German remains separated by 500km and 300 years to answer to some of these questions. Continue reading “The Black Death: World Traveler or Persistent Homebody?”

Dino Protein: New Methods for Old (Very) Samples

Posted on by Gary Kobs
Hadrosaurus skeleton vintage engraving.
Hadrosaurus skeleton vintage engraving.

Brachylophosaurus was a mid-sized member of the hadrosaurid family of dinosaurs living about 78 million years ago, and is known from several skeletons and bonebed material from the Judith River Formation of Montana and the Oldman Formation of Alberta. Recent fossil evidence indicates structures similar to blood vessels in location and morphology, have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty  network after degradation of original organic components (i.e., bacterial, slime mold or fungal in origin).  Cleland et al. (1) tested the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence.

Continue reading “Dino Protein: New Methods for Old (Very) Samples”

Unexpected connections: Gut bacteria influence immunotherapy outcomes

Posted on by Isobel Maciver

Over the last few years, human microbiome studies have revealed fascinating connections between our colonizing microorganisms and ourselves—including associations between gut bacterial populations and obesity, disease susceptibility, and even mood. The relationship between us and our microbial colonists—once considered completely benign, is now being revealed as an intricate, complicated partnership with the potential to redefine who “we” are in fundamental ways.

Two papers published back-to-back in the November 27 issue of Science add further to this growing body of knowledge—reporting a new and unexpected connection between gut bacterial species and the effectiveness of cancer immunotherapies in mice. The work suggests one reason why such treatments are effective in some circumstances, but not others. Both papers report that the presence of specific bacterial populations may be required for the efficacy of certain treatments, and raise the intriguing question “Could the composition of bacteria in the gut be manipulated to enhance the effectiveness of cancer treatments?”

Continue reading “Unexpected connections: Gut bacteria influence immunotherapy outcomes”

ProteaseMAX: A Surfactant for the Most Complex Mixtures

Posted on by Gary Kobs
Alternate Proteases Cover

Here we provide two examples of “atypical” experiments that take advantage of the properties of the ProteaseMAX™ Surfactant to improve studies involving digestion of complex protein mixtures.

Example 1
Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. A recent publication (1) illustrated a novel strategy using three different approaches  to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores.

Continue reading “ProteaseMAX: A Surfactant for the Most Complex Mixtures”

Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity

Posted on by Michele Arduengo
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Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.

Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.

In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.

Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment?

Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”