Characterizing Unique Protein: DNA Interactions Using Cell-Free Protein Expression

Molecular model of human telomere DNA

Molecular model of human telomere DNA

The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5′-TTAGGGTTAG-3′ and forms a dimer with the TPP1 protein. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5′-TTAGGG whereas OB2 binds to the downstream TTAG-3′ (1,2). Several recent studies from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected (3). A recent reference reexamined the sequence-specificity of the Human POT1 protein (4).
SELEX (Systematic Evolution of Ligands through Exponential Enrichment) was used  to re-examine the DNA-binding specificity of human POT1 (5). Continue reading

Travelogue Iceland: Visiting the Home of the Hellisgerði

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular feature on the Promega Connections Blog.

Today’s travelogue brings the adventures of Rebecca Hartsough Brentin , a Senior Client Support Specialist, who used her award to travel to Iceland.

The direct flight from Denver to Iceland was only 6.5 hours, which was the main reason we chose Icelend. It was never on the bucket list, but I’m so glad we visited.Iceleand 1

The culture was something I could get used to… coffee shops didn’t open until 7 or 8 am, no traffic outside our window in Reykjavik until 8am.

Iceland 2

We stayed in the old part of town directly above a heritage museum, which featured the foundation of a longhouse from the year 872! Continue reading

Five Interview Responses Recruiters Can See Right Through

20708189_lRecruiters aren’t pessimists, but throughout the years we have become more cautious and maybe a little suspicious. Many of us interviewed enough candidates that we have come to approach each new person with a “trust but verify” mentality. I’m very trusting in my personal life, but at work, my job is to be a detective. I follow clues to dig up the good, the bad, and the ugly.

During a recent talk with fellow recruiters, we realized there are some things many candidates say that perk up our sleuth ears every single time. These answers may be coming from a truthful and benign place, but they raise suspicions in any good recruiter. The average candidate has no idea what other candidates are saying, so I’m here to share. Recruiters hear these answers often and, take it from us, you’ll come off better in your interview if you avoid them. Continue reading

Returning the Stolen: A Preview of the ISHI 27 Keynote Lecture

Abuelas-de-Plaza-de-Mayo-feature

Grieving Abuelas de Plaza de Mayo who have lost their children and grandchildren. Daniel Garcia / Agence France-Presse – Getty Images

Argentina is probably not the first place that comes to mind when you think of dictatorships, yet the “Dirty War” of the late 1970s killed 10,000–30,000 citizens in an act of political repression by the Argentine Anticommunist Alliance (AAA). Among this figure includes some 13,000 people who disappeared overnight, sent to a network of hundreds of concentration camps.

Dirty-War-arrests

Citizen arrests made by the AAA. Click for full article from targina.net (Spanish)

The political landscape of Argentina was hardly stable at the time, supporting the idea that this was a civil war between the AAA and guerrilla militants. However it soon became clear that countless human rights violations were being conducted on anyone who held a contrary political ideology. Left-wing activists, trade unionists, students and journalists were subject to abduction, torture and assassination. Continue reading

Finding a Connection Between Glucose Metabolism and Macrophage Activation

Introduction to Glucose Metabolism

Macrophages. By NIAID (https://www.flickr.com/photos/niaid/17380707492/) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Many think of glucose as something diabetics have to test each day using a blood monitor, or a quick energy boost for someone exercising intensely. However, the simple sugar glucose, a monosaccharide, fuels most of the cells in our bodies. Disaccharides that contain glucose (e.g., sucrose is comprised of glucose and fructose) and glucose polymers (e.g., starch and glycogen) are carbohydrates that are consumed by organisms from bacteria to humans to produce energy. These carbohydrates are broken down into component monosaccharides like glucose and lactose. The process of glycolysis generates the energy currency of cells, ATP, as well as precursor molecules for nucleotides, lipids and amino acids. Because glucose is the cell fuel source, the uptake of glucose and its subsequent metabolism is increased by cells that divide rapidly like cancer cells. The more energy and precursor molecules the cancer cell can create for itself, the more rapidly the tumor can grow.

Because glucose metabolism is central to cellular functioning, changes that decrease glucose uptake or increase glycolysis have a widespread effect on on both the cells and organism. How does a simple sugar molecule create such broad effects on health? For example, diabetes results from the inability to store glucose because of a lack of insulin, a hormone that draws glucose from the blood and stores it as glycogen in the liver, muscles and adipose tissue. High levels of sugar in the blood negatively affect the body over the long term, damaging blood vessels and eyesight, making the kidneys work harder to excrete the excess sugar and increasing the risk of stroke and coronary artery disease. Because cancer cells have such a high metabolic demand for glucose, many of the mutations in cancers affect pathways that regulate glucose uptake and glucose breakdown, allowing the cancer cells to survive and grow, crowding out nearby normal cells.

Glucose metabolism is altered by processes other than mutations or an reduced production of a hormone. Throughout its life cycle, a cell will vary its requirements for glucose. For example, the cells that comprise our innate immune response are typically in a quiescent or steady state. However, when these immune cells encounter an foreign invader, they become activated and increase their demand for glucose. To respond to a potential pathogen, the activated cells need glucose to fuel cell proliferation and the production of cytokines, chemicals that activate other immune cells and initiate an inflammatory response. The typical signs of inflammation are red inflamed area that may be painful to the touch, such as a cut that becomes infected. Most inflammation resolves when the infection is eliminated, leaving behind whole skin in the instance of a cut, and the activated immune cells become quiescent again.

An Interesting Observation about Glucose Metabolism in M2 Macrophages

Glucose uptake, immunity and metabolism are cellular pathways that are intertwined such that understanding how glucose is utilized in macrophages illuminates gene induction and regulation in activated macrophages. In a recently published eLife article, Covarrubias et al. studied how activation of murine bone marrow-derived macrophages (BMDMs) by interleukin-4 (IL-4), a signaling cytokine, altered glucose metabolism in the cells and regulated a subset of genes involved in macrophage activation. Continue reading

Vitamin D: Power in Cancer Prevention?

This and vitamin D should get your attention.

This and vitamin D should get your attention.

Have you ever noticed that after a good long day outdoors, maybe hiking, at the beach or even working in the yard, you feel really strong and healthy, maybe even more relaxed than after an indoor session in front of the telly or computer? Maybe a February trip to someplace sunny like Mexico or the Canary Islands has given you renewed zest for your normal tasks?

While rest and a change of scenery is never a bad thing, time outdoors and in the sunshine might have gained for you something more than rest and relaxation. If it included a little UVB irradiation, your time outdoors may have increased your serum vitamin D level. And though it’s been presumed for years, we now have proof that higher serum vitamin D3 levels correlate with a decreased incidence of certain cancers. Continue reading

For Protein Complementation Assays, Design is Everything

Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use  a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.

A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.

Continue reading

Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.

Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.

The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading

Promega Tech Tour 2016: Preview of a Fascinating DNA Crime Story

“Is your life just like CSI?”

That is the prevailing question I’m asked when someone learns of my occupation as Deputy Sheriff Criminalist for the Contra Costa County (CA) Office of the Sheriff. Alas, my life is not quite so glamorous. It actually often entails entering formulas into an excel spreadsheet while being placed on hold as I order some pipette tips.

But, why does it have to be that way?

crime sceneI have attended my fair share of professional conferences and workshops and written numerous journal articles. As a forensic scientist I do believe in the importance of sharing data, new techniques, and new methodologies with my colleagues. Yet what I think is not highlighted enough is the one element that differentiates our field from any other scientific field—our involvement with the criminal justice system.  Every case we work on involves a mystery, a crime, a victim(s), and a suspect(s).  And while scientists in other fields typically only speak to other scientists, in my world, forensic scientists usually interact with a person in a black robe who has the power to strongly influence the outcome of a case.  These wildly frustrating, invigorating, and challenging cases are the most interesting things about our field, and yet we hardly share our stories.

I aim to change that.

I have been fortunate enough to be invited to speak at the 2016 Promega Tech Tour on April 12 at the CA Department of Justice Jan Bashinski DNA Laboratory in Richmond, CA.  The story I plan to share is about the small part I played in the case against Joseph Naso, the serial killer who preyed upon his victims from the 1950’s through the 1990’s in California. Continue reading

Better, Faster, Cheaper: Measuring the Speed of Science

Are we better off now than we were 10 years ago? Often times this question is answered subjectively and will vary from person to person. We can empirically show how life expectancy has increased over the centuries thanks to advances in the fields of agriculture and medicine, but what about quality of life? Science affects our lives every day, and the general notion is that better science will (eventually) translate into better lives. There is a burning curiosity shared by myself and others to quantify how we have progressed in science over the years:

publication-growth

Click for full article. Source: Bornmann, L. & Mutz, R. (2015). Growth rates of modern science: a bibliometric analysis based on the number of publications and cited references. Journal of the Association for Information Science and Technology, 66(11), 2215–2222.

Bornmann and Mutz demonstrate in the image shown above how we have been doubling scientific output every nine years since the 1940s. That is not to say that we have become twice as smart or efficient; this phenomenon could be partially fueled by a desire to gain prestige through a high number of publications. To better assess the topic of efficiency, we can measure how long it takes to perform specific procedures and how much they cost. This article compares the rate of improvement for DNA sequencing, PCR, GC-MS and general automation to the rate of improvement for supercomputers and video game consoles.

Continue reading