The Battle for the Wall Outlet

Posted on by Alyssa Bear
student studying
Studying in the almost empty library at the beginning of the semester.

You check the clock. The time is 3:36 am and you’re barely a third of the way through the material on the 11:00 am cumulative exam. Stirring the film that has formed on top of your now-ice-cold latte, you contemplate leaving the library and heading home to a warm bed. After all, you know that the custodial staff comes around with a vacuum at 4:00 am and, like a cat, you just can’t handle the vacuum at this time of day.

You take another minute and reluctantly come to the conclusion that you should get back to work. As you pull your computer onto your lap once more, you hear the terrifying beep of a low battery signal. The battery is on 5% and you know very well there’s not a free outlet in a 2-mile radius. Without an outlet, your time in the library has come to an end.

This tiny little beep has led to my own personal defeat on multiple occasions, particularly during finals season. Continue reading “The Battle for the Wall Outlet”

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

Posted on by Gary Kobs
NanoLuc dual reporters
Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading “Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit”

Choosing a Better Path for Your NGS Workflow

Posted on by Darcia Schweitzer

Imagine you are traveling in your car and must pass through a mountain range to get to your destination. You’ve been following a set of directions when you realize you have a decision to make. Will you stay on your current route, which is many miles shorter but contains a long tunnel that cuts straight through the mountains and obstructs your view? Or will you switch to a longer, more scenic route that bypasses the tunnel ahead and gets you to your destination a bit later than you wanted?

Choosing which route to take illustrates a clear trade-off that has to be considered—which is more valuable, speed or understanding? Yes, the tunnel gets you from one place to another faster. But what are you missing as a result? Is it worth a little extra time to see the majestic landscape that you are passing through?

Considering this trade-off is especially critical for researchers working with human DNA purified from formalin-fixed paraffin-embedded (FFPE) or circulating cell-free DNA (ccfDNA) samples for next-generation sequencing (NGS). These sample types present a few challenges when performing NGS. FFPE samples are prone to degradation, while ccfDNA samples are susceptible to gDNA contamination, and both offer a very limited amount of starting material to work with.

Continue reading “Choosing a Better Path for Your NGS Workflow”

Ancient Images of Dogs Include Restraints?

Posted on by Kari Kenefick
This dog is wearing a leash.

You, like me, may occasionally find youself in need of a canine control device. While I’m not a fan of the dog tie out, I do walk dogs on leash—as is required by our county and city government here in Madison, WI.

If you have read Temple Grandin’s books about dogs, you might feel a tug at your heartstrings while enduring a tug on the leash. Aren’t dogs meant to run freely? Don’t we love to watch them run? Are leashes humane?

When walking dogs I feel the need to protect them, but also a desire to let them live like dogs, sniffing, marking and other behaviors that are all limited when the dog is leashed.

When a report in Science last week showed evidence that our ancient ancestors were using leashes 8,000-9,000 years ago I was: 1) surprised; and 2) felt vindicated from self-imposed dog owner guilt.

Continue reading “Ancient Images of Dogs Include Restraints?”

Elephant Endotheliotropic Herpesvirus—A Tiny Virus Threatens the World’s Elephants

Posted on by Kelly Grooms

My favorite ice-breaker of all time is: “List one fact about you that no one would guess”. It is my favorite because I have an awesome answer (if I do say so myself). My go-to answer is: I spent a summer working with elephants.

It was the summer before I graduated from college, and it was really only one elephant, a five-year-old African elephant named Connie. Connie was intelligent, curious and mischievous—her favorite game with me was trying to untie my shoelaces (hint: double knotting is important). Working with her was one of the most amazing experiences of my life and left me with an abiding love for these creatures.

African elephant mother and child
Young African elephant touches his mother

Understandably, I was excited last year when one of my fellow bloggers wrote about Promega helping support the work of Virginia Riddle Pearson, who was working to identify and track strains of elephant endotheliotropic herpesvirus (EEHV) in African Elephant populations. EEHV is associated with the lethal elephant hemorrhagic disease (EHD) (1). This disease is a serious threat to the captive breeding programs of these endangered creatures. Between 1962 and 2007, it accounted for 58% of the deaths of North American captive-born Asian elephants between 4 months and 15 years of age (1). These deaths include the first Asian elephant calves born at the National, Oakland and Bronx Zoos. EHD also claimed the first live-born Asian elephant calves conceived by artificial insemination in both North America and Europe.

Continue reading “Elephant Endotheliotropic Herpesvirus—A Tiny Virus Threatens the World’s Elephants”

Deubiquitinases: A Backdoor into Undruggable Targets?

Posted on by Michele Arduengo, PhD

Molecular model of the yeast proteasome.
Molecular model of the yeast proteasome.

Ubiquitin modification of a protein directs events such as targeting for proteasomal degradation. Targeting a protein for degradation through ubiquitin modification is one way to regulate the amount of time a signaling protein, such as a kinase or other enzyme, is available to participate in cell signaling events. Deubiquitinases (DUBs) are enzymes that cleave the ubiquitin tags from proteins, and they have been implicated in several diseases, including cancer.

With their roles in the stabilization of proteins involved in cell cycle progression and other critical processes, DUBs are promising targets for small molecule inhibitors, particularly since they may provide a “back door” for targeting otherwise intractable, undruggable proteins by modulating their half lives. However, finding small molecule inhibitors of the ubiquitin proteases to date has not been trivial. Here we highlight two papers describing the identification and characterization of small molecule inhibitors against the DUB USP7. Continue reading “Deubiquitinases: A Backdoor into Undruggable Targets?”

Using CellTiter-Glo® Luminescent Cell Viability Assay to Assess Cell Viability in Cancer Cells Treated with Silver Nanoparticles and DNA-PKcs Inhibitor

Posted on by Johanna Lee

Silver nanoparticles (Ag-np) are commonly used in many consumer products, including cosmetics, textiles, electronics and medicine, largely due to their antimicrobial properties. More recently, Ag-np are being used to target and kill cancer cells. It has been known for years that silver nanoparticles (Ag-np) can induce cell death and DNA damage. Studies have also shown that Ag-np inhibit cell proliferation and induce apoptosis in cancer cells. However, cancer cells are able to fight back with DNA repair mechanisms such as non-homologous end joining repair (NHEJ). The NHEJ pathway requires the activation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus DNA-PKcs may protect against the Ag-np-induced DNA damage in cancer cells.

Could inhibition of DNA-PKcs increase the ability of Ag-np to kill cancer cells? In a 2017 study, Lim et al. wanted to test whether inhibition of DNA-PKcs can increase the cytotoxic effect of Ag-np in breast cancer and glioblastoma cell lines. To effectively determine cell viability in these cancer cell lines, the authors used the CellTiter-Glo® Luminescent Cell Viability Assay. The CellTiter-Glo® Assay determines the number of viable cells in culture based on quantitation of ATP, an indicator of metabolically active cells. A major advantage of this assay is its simplicity. This plate-based assay involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. This generates a luminescent signal proportional to the amount of ATP present, which is detected using a luminometer. Cell washing, removal of medium and multiple pipetting steps are not required. Another advantage of the CellTiter-Glo® Assay is its high sensitivity. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing, making it ideal for automated high-throughput screening, cell proliferation and cytotoxicity assays.

The authors first confirmed that Ag-np treatment reduced proliferation and induced cell death/DNA damage in two breast cancer cell lines and two glioblastoma cell lines. The cytotoxic effect of Ag-np is specific to cancer cells, as minimal cytotoxicity was observed in non-cancerous human lung fibroblasts used as control. Next, they pre-treated the cancer cells with a DNA-PKcs inhibitor for 1 hour before adding Ag-np. Inhibition of DNA-PKcs increased Ag-np-mediated cell death in all four cancer cell lines. This suggests that DNA-PKcs may be protecting the cells from Ag-np cytotoxicity. The authors further showed that DNA-PKcs may repair Ag-np induced DNA damage by NHEJ and JNK1 pathways. In addition, DNA-PKcs may help recruit DNA repair machinery to damaged telomeres.

This study suggests that a combination of Ag-np treatment and DNA-PKcs inhibition may be a potential strategy to enhance the anticancer effect of Ag-np.

Reference: Hande M.P., et.al. (2017) DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells. Mutat Res Gen Tox En. 824, 32

Determination of Antibody Mechanism of Action Using IdeS Protease

Posted on by Gary Kobs

Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)

In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.

Learn more about IdeS and IdeZ Protease available from Promega.

Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.

The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.

  1. Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells  26, 1454.
  2. Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.

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Hot Wings and Snow Birds: A Study of Genetic Selection in Chickens

Posted on by Sara Klink
African chicken breed Boschvelder. Image copyright ICBH GROUP.

This past summer, I visited the county fair and stopped by the animal barn to look at some of the poultry on display. Specifically, I wanted to see examples of the breeds of chickens available that I may be interested in adding to my flock. Rather than each chicken in their display cage being labeled with a bird’s breed, each cage listed the geographic origin of the chicken within such as Asiatic, Continental or American. This did not benefit my search for potential new members of my flock, but intrigued me enough that I wanted to find out how my flock of 19 hens and pullets would be characterized. Using the classes delineated by the Wisconsin State Fair, my feathered ladies break down to 12 American, 4 English and 3 Continental chickens. There are also classes for Mediterranean and Asiatic (and Other). I live in a part of the United States that gets cold, snowy weather for what seems like six months out of the year, weather that my chickens seem to take in stride. But in other places in the world, heat is the name of the game for the poultry strutting there. In a Genes, Genomics, Genetics publication, Fleming et al. wanted to know if there were genetic differences in Northern European and African chickens that might be caused by their environment.

Continue reading “Hot Wings and Snow Birds: A Study of Genetic Selection in Chickens”

Honoring Caregivers

Posted on by Promega

This blog post is contributed by guest blogger Diana Clark, Benefits Manager, Promega Corporation

November is National Family Caregivers Month, first proclaimed by President Clinton in 1997, the proclamation has been renewed by every U.S. President since. When President Obama proclaimed this designation in 2012, he commented, “The unselfish devotion of family caregivers affirms the importance of respecting the dignity of life in all stages and underscores the importance of the family unit.”

Hearing these words, I felt even prouder to be a part of the Promega family. You see, we are already in the process of rolling out Caregiver Leave for 2018. Caregiver Leave will provide Promega employees with an additional three weeks of paid time off annually to care for a sick parent, spouse or child, or to welcome a new child into their family via birth, adoption or foster placement. Continue reading “Honoring Caregivers”