Promega News and Events

Zika Virus: Another RNA Virus Emerges

Posted on by Isobel Maciver
no mosquito

Zika virus has been in the news recently due to growing concerns about its global spread. If you have never heard of Zika virus before, you are not alone. Although first discovered in the 1940s, Zika has not been the subject of much study as infection is considered rare and the symptoms mild. However, all this has changed in recent months due to the rapid spread of the virus in Latin America, where it has been associated with an increased incidence of microcephaly, a severe birth defect where babies are born with underdeveloped brains. Although the connection of Zika with microcephaly is not yet proven, the circumstantial evidence is strong, leading the World Health Organization to declare the spread of Zika virus an international public health emergency earlier this week.

Continue reading “Zika Virus: Another RNA Virus Emerges”

NanoBiT™ Assay: Transformational Technology for Studying Protein Interactions Named a Top 10 Innovation of 2015

Posted on by Michele Arduengo, PhD

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For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.

Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.

The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.

The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:

Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.

You can read the Top 10 article in The Scientist here.

Will Warmer Weather Wake the Sleeping Giant (Viruses)?

Posted on by Gary Kobs

Artist's conception of Mimivirus structure, the first of the giant viruses identified.
Artist’s conception of Mimivirus structure, the first of the giant viruses identified.

Following the discovery of Mimivirus (1) the first virus with a particles large enough to be visible under the light microscope, two additional “giant” viruses infecting Acanthamoeba have been discovered Pandoravirus (2) and Pithovirus sibericum (3), the latter from a 30,000 year old Siberian permafrost. A fourth type was recently isolated from the same sample of permafrost by Legendre et al, and named Mollivirus sibericum (4).

Mollivirus sibericum has an approximately spherical virion (0.6 µm diameter) with a 651kb GC-rich genome that encodes 523 proteins. To further characterize the virus the researchers performed transcromic- and proteomic-based time course experiments.

For the particle proteome and infectious cycle analysis, proteins were extracted and then run a 4–12% polyacrylamide gel, and trypsin digests were performed in-gel before nano LC-MS/MS analysis of the resulting peptides. Proteomic studies of the particle showed that it lacked an embarked transcription apparatus, but revealed an unusual presence of many ribosomal and ribosome-related proteins.

When the researchers explored the proteome during the course of an entire infectious cycle, the relative proportions of Mollivirus-, mitochondrion-, and Acanthamoeba encoded proteins were found to vary consistently with an infectious pattern that preserved the cellular host integrity as long as possible and with the release of newly formed virus particles through exocytosis.

In an interesting footnote, the authors of this study point out the fact that two different viruses retain their infectivity in prehistorical permafrost layers should be a concern in the context of global warming and the potential to expose humans to primeval viruses.

References

1. La Scola, B. et al.   (2003) A giant virus in amoebae. Science  299, 2033.
2. Philippe, N. et al. (2013) Pandoraviruses. Amoeba virus with genomes up to 2.5Mb reaching that of parasitic eukaryotes. Science 341,281–6.
3. Legendre, M. et al. (2014) Thirty thousand year old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc.Natl. Acad. Sci. 111, 4274–9.
4. Legendre, M. et al. (2015)  In depth study of Mollivirus sibercum, a new 30,000 year old giant virus infecting Acanthamoeba.  Proc. Natl. Acad. Sci. 112, E5327–35 (online).

Sequencing the Octopus Genome: Invertebrate Intelligence Explained?

Posted on by Kari Kenefick

In a Letter in Nature magazine last week (August 13, 2015), researchers published surprising findings from a genome analysis of the octopus. As a result, we now know that this invertebrate has more than just behavioral oddities with which to amaze.

In their publication, C. Albertin et al. report the results of genome sequencing of the California two-spot octopus, Octopus bimaculoides. They did not find the predicted whole-genome duplication, but rather an unexpectedly large genome with many rearrangements, and two gene family expansions that were previously thought to exist only in vertebrates.

Califonia two spot octopus. Image by Jeremy S. Taken at Santa Monica Aquarium. Used via Creative Commons license, Wikimedia.
Califonia two spot octopus. Image by Jeremy S. Taken at Santa Monica Aquarium. Used via Creative Commons license, Wikimedia.

The Research
Albertin et al. sequenced the O. bimaculoides genome using a whole-genome shotgun approach, and then annotated it using extensive transcriptome sequences from 12 tissues. They estimate that the genome assembly incorporated 97% of protein-coding sequences, and 83% of the entire 2.7gigabase genome. The remaining sequence was composed largely of repetitive elements. Continue reading “Sequencing the Octopus Genome: Invertebrate Intelligence Explained?”

A Reason for Ribonuclease: From Laboratory Nuisance to Cancer Therapeutic

Posted on by Kari Kenefick

"RNase A". Licensed under CC BY-SA 2.5 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:RNase_A.png#/media/File:RNase_A.png
“RNase A”. Licensed under CC BY-SA 2.5 via Wikimedia Commons – https://commons.wikimedia.org/wiki/File:RNase_A.png#/media/File:RNase_A.png

RNase, back in the early 1990s, posed a serious threat to laboratories working with RNA isolation. My graduate work involved isolating RNA from the tissues of Lyme disease-infected mice and hamsters. We struggled to DEPC-treat glass and plasticware, or autoclave anything that could be autoclaved, kept tissues cold during RNA harvest and held our breaths (truly, as aerosol could be another source of ribonuclease) until PAGE proved us successful in RNA isolation.

Ribonuclease (RNase) was omnipresent and the arch rival of our work, across several species, due to its RNA destroying abilities.

Now, a July 13, 2015 publication by researchers at the University of Wisconsin-Madison provided both a catch-up for this former lab rat on modern day research with and knowledge of RNase, as well as an exciting look at what may be a real purpose for this RNA-destroying molecule: RNase has moved to clinical trials due to the discovery of it’s cytotoxicity for cancer cells.

Raines’ group in the Department of Chemistry at UWI-Madison published in ACS Central Science their findings on the ligand that RNase 1 uses to attach to human cancer cells, in the article, “Human Cancer Antigen Globo H is a Cell-Surface Ligand for Human Ribonuclease 1”. Continue reading “A Reason for Ribonuclease: From Laboratory Nuisance to Cancer Therapeutic”

Designer Bacteria Detect Cancer

Posted on by Isobel Maciver

Every day scientists apply creative ideas to solve real-world problems. Every so often a paper comes up that highlights the creativity and elegance of this process in a powerful way. The paper “Programmable probiotics for detection of cancer in urine”, published May 27 in Science Translational Medicine, provides one great example of the application of scientific creativity to develop potential new ways for early detection of cancer.

The paper describes use of an engineered strain of E.coli to detect liver tumors in mice. The authors (Danino et al) developed a potential diagnostic assay that uses a simple oral delivery method and provides a readout from urine, all of which is made possible by some seriously complex and elegant science. Continue reading “Designer Bacteria Detect Cancer”

Your Health has a Season

Posted on by Kari Kenefick

Photo of pasque flowers
Pasque flowers in a northern hemisphere garden in spring.

As the seasons change so does the general state of health for many of us. The further from the equator we live, the more pronounced these effects are. For instance, did you know that blood pressure elevation for many people increases with the distance they live from the equator, an effect most pronounced during the low sunlight season (winter in the northern hemisphere)?

A report published online in Nature Communications May 12, shows evidence of changes in cellular physiology with the seasons. Todd et al. published a study entitled: “Widespread seasonal gene expression reveals annual differences in human immunity and physiology”, where they note,

“Here we find more than 4,000 protein-coding mRNAs in white blood cells and adipose tissue to have seasonal expression profiles, with inverted patterns observed between Europe and Oceania.”

Let’s Take a Look at the Research

Todd et al. looked at ethnically and geographically distinct populations, including subjects from Australia, The Gambia (Africa), Germany, the UK and Iceland. Individuals from the various studies were infants, adults with type 1 diabetes and asthmatics in the range of 18-83 years of age. The authors analyzed RNA from peripheral blood mononuclear cells and subcutaneous adipose tissue biopsies, as well as examining peripheral blood cell counts and circulating levels of proinflammatory cytokines. Continue reading “Your Health has a Season”

Avoid False Hits During Compound Screening for Drug Discovery

Posted on by Promega

One goal of drug discovery and research programs is to reduce false hits as early as possible in the process. Follow-up on false hits is costly in terms of time and resources, and the longer the false hits remain in the drug development pipeline, the more costly they are. So methods that can easily reduce the number of false hits during compound screening early in the discovery process are particularly sought after.

Reporter assays have proven to be invaluable tools for elucidating the mechanisms of action of small molecules or other agents on signaling pathways within cells, and the luciferase reporter assay has become a standard research tool in the biological research laboratory.

However, one caveat of using standard luciferase-based reporter assays for larger-scale compound screening efforts is the frequency of false hits that result from direct interaction of compounds with the luciferase reporter. This issue can be mitigated with a “coincidence reporter” system where two independent reporter proteins are produced from a single transcript. In this type of assay, a bicistronic transcript is stoichiometrically translated into two nonhomologous reporters by means of a 2A “ribosomal skipping” sequence. Since it is unlikely that compounds will interact with two distinct types of reporter, “coincident” responses will indicate on-target activity. Such a coincident reporter system provides an important control against costly false hits early in drug discovery research programs.

A paper published online in ACS Chem Biol in February describes the first successful application of the firefly/NanoLuc luciferase coincidence reporter system to identify new pathways that up-regulate PARK2 expression.

Continue reading “Avoid False Hits During Compound Screening for Drug Discovery”

Immune Checkpoint Inhibitors: Has Cancer Met its Match?

Posted on by Kari Kenefick
The cover of S. Mukerjee's book, The Emporer of All Maladies: The Biology of Cancer. Used courtesy of Wikimedia and WLU.
The cover of S. Mukerjee’s book, The Emporer of All Maladies: The Biology of Cancer. Used courtesy of Wikimedia and WLU.

Dr. Drew M. Pardoll, Johns Hopkins University School of Medicine in Baltimore, in his 2012 review, “The blockade of immune checkpoints in cancer immunotherapy” published in Nature Reviews Cancer (1) writes:

“The myriad of genetic and epigenetic alterations that are characteristic of all cancers provide a diverse set of antigens that the immune system can use to distinguish tumour cells from their normal counterparts.”

Tumors have antigens, so we should be able to address/attack these antigens with our immune system, right?

Various immune mediators as therapeutic agents against cancer have entered and mostly flopped in clinical trials over the past 30 or more years. As a graduate student in the 1980s I remember IL-2 and interferon raising many hopes. More recently, drugs against chronic myeloid leukemia and CLL have shown early promise. However, so far cancer cells have mostly won against these therapies. Yet recent news points to some exciting new therapeutic agents, that over the past 15 years or so, and in and out of clinical trials, are getting a leg up in the cancer battle. These drugs are immune checkpoint inhibitors.

Continue reading “Immune Checkpoint Inhibitors: Has Cancer Met its Match?”

Culturing the Unculturable Bacteria

Posted on by Isobel Maciver
Culturing bacteria is't always this easy.
Culturing bacteria is’t always this easy.

It is estimated that all the bacterial species so far described represent only a tiny fraction of the total. The rest remain unknown to science because they are “unculturable” in standard (or known) laboratory media. Given that many antibiotics were first isolated from environmental bacteria, it seems likely that these as yet unknown organisms could also be a rich source of potential new drug candidates. The desperate need for new strategies to combat multi-drug resistant infections gives impetus to studies investigating how we can culture some of these “unculturable” bacteria and uncover their potential as a source of much-needed new treatments. 

Continue reading “Culturing the Unculturable Bacteria”