In the Literature

Blogs about published peer-reviewed scientific studies.

Preventing Viral Infection by Blocking Cellular Receptors with a Tethered Antibody

Posted on by Sara Klink

Finding a way to neutralize or block infection by HIV has long been pursued by viral researchers. Various treatments have been developed, driven by the need to find effective drugs to manage HIV in infected individuals. The ultimate goal is to develop a vaccine to prevent HIV from even taking hold in the body. With all of our knowledge about the cellular receptors HIV needs to enter the cell, there has to be a method to prevent a viral particle from binding and being internalized. Many researchers are pursuing neutralizing antibodies to the virus as one method. What about antibodies that target the cellular receptor recognized by the virus? An article published in Proceedings of the National Academy of Sciences, antibodies to cellular receptors for rhinovirus and HIV were tethered to the plasma membrane and tested for the ability to prevent infection.

Continue reading “Preventing Viral Infection by Blocking Cellular Receptors with a Tethered Antibody”

Making a Case for Basic Research Funding

Posted on by Darcia Schweitzer

The value of public funding for “basic” versus “applied” research has long been questioned. To address this debate, the authors of a recent report in Science performed a large-scale evaluation of the value of public investment in biomedical research. After analyzing the relationship between the U.S. National Institutes of Health (NIH) grants and private patents, they found that distinguishing research as basic or applied is not useful in determining the productivity of grant funding.

Genetic research at the laboratory

The $30 billion annual budget of the NIH makes it the largest source of life science funding in the world and provides a third of all US biomedical research and development. Although there has long been a strong argument for public investment in scientific research, attacks on the tangible benefits of this research persist. In particular, some opponents argue that “basic” research is too far removed from practical applications to be worthy of investment.

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Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

Posted on by Kyle Hooper

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal.

Continue reading “Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events”

Probing RGS:Gα Protein Interactions with NanoBiT Assays

Posted on by Kyle Hooper
gpcr_in_membrane_on_white2

When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases.

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Writing Scientific Papers: Is There More To This Story?

Posted on by Nicole Sandler
science & storytelling

The tactic of “telling a good story” is nothing new within the business of selling, marketing and even educating about science. The word itself, “storytelling,” achieved buzzword status a few years ago in the corporate world, so it’s no surprise that it now touches industry scientists.  But the importance of telling a good story within the realm of peer-reviewed scientific papers?  That is something new, and it may impact how scientists write up their results from this point forward.

In a provocative scientific study published in PLOS ONE in December 2016, researchers from the University of Washington showed that “Narrative Style Influences Citation Frequency in Climate Change Science.” Perhaps the results they report are unique to climate change science—an area of science especially susceptible to public perception. But then again, perhaps not. This paper may be worth considering no matter what field of science you call your own.

The authors—Ann Hillier, Ryan Kelly, and Terrie Klinger—used metrics to test their hypothesis that a more narrative style of writing in climate change research papers is more likely to be influential, and they used citation frequency as their measure of influence. A sample of 732 abstracts culled from the climate change literature and published between 2009 and 2010 was analyzed for specific writing parameters. The authors concluded that writing in a more narrative style increases the uptake and influence of articles in this field of science and perhaps in scientific literature across the board.

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NanoBiT Assay Applied to Study Role of SOD1 in ALS

Posted on by Isobel Maciver

NanoBiT Protein ComplementationBack in 2015 the Ice Bucket Challenge brought Amyotrophic Lateral Sclerosis (ALS) to public attention, initiating worldwide pleas for more funding of research toward a cure for this fatal disease, which is characterized by progressive degeneration of motor neurons. In spite of many efforts over the last few decades, the precise cause of ALS is still unknown.

The complexity of the problem of ALS pathogenesis is highlighted in the review “Decoding ALS: from genes to mechanism”  published in Nature in November 2016. The review highlights a long list of genetic factors implicated in ALS, grouping them into genes affecting protein quality control, RNA stability/function, and the cytoskeletal structure of neuronal cells.

Mutations in the antioxidant enzyme superoxide dismutase (SOD1) were the first to be associated with ALS. According to the review, more than 170 SOD1 mutations causing ALS have since been identified. Many of these mutations are thought to result in misfolding of SOD1, contributing to toxicity when the misfolded protein accumulates within the cell.

A paper by Oh-hashi et al., published in Cell Biochemistry and Function in October 2016 used the NanoBiT protein complementation assay to investigate the effect of two common ALS-associated SOD1 mutations on dimerization of the SOD1 protein. Continue reading “NanoBiT Assay Applied to Study Role of SOD1 in ALS”

Cell Free Expression Application: In vitro degradation assay

Posted on by Gary Kobs

A protein chain being produced from a ribosome.
A protein chain being produced from a ribosome.

Researchers and clinicians are fairly certain that all cervical cancers are caused by Human Papillomavirus (HPV) infections, and that HPV16 and HPV18 are responsible for about 70% of all cases. HPV16 and HPV18 have also been shown to cause almost half the vaginal, vulvar, and penile cancers, while about 85% of anal cancers are also caused by HPV16.

E6 is a potent oncogene of HR-HPVs, and its role in progression to malignancy continues to be explored. The E6 oncoprotein of HPV can promote viral DNA replication through several pathways. It forms a complex with human E3-ubiquitin ligase E6-associated protein (E6AP), which can in turn target the p53 tumor-suppressor protein, leading to its ubiquitin-mediated degradation. In particular, E6 from HR-HPVs can block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV-infected cells.

In a recent publication a new procedure generated a stable, unmutated HPV16 E6 protein (1). Continue reading “Cell Free Expression Application: In vitro degradation assay”

Weaving Tangled Webs with Cell-Free DNA

Posted on by Michele Arduengo

The ability to isolate and assay circulating cell-free DNA from plasma holds promise for improved diagnostics and treatment in the clinic. The use of blood-based non-invasive prenatal testing (NIPT) has been well described. Such testing is based on circulating cell-free fetal DNA in blood of a pregnant woman for diagnosis and screening  of chromosomal anueploidy (e.g. Trisomy 21, Down Syndrome), sex-linked diseases, and genetic diseases that are known to result from a specific mutation in a single gene (1). Additionally, most cancers carry somatic mutations that are unique to the tumors, and dying tumor cells release small pieces of their DNA into the blood stream (2). This circulating cell-free tumor DNA can be used as a biomarker to “follow” cancer progression or regression during treatment, and diagnostic methods also are being developed to detect even early stage cancers from circulating tumor DNA (3). Further, increases in circulating cell-free DNA have been well documented after intense exercise, trauma, sepsis and even associated with autoimmune diseases such as system lupus erythematosus (SLE; 1,4). In these latter examples increases in extracellular DNA are associated with evolutionarily conserved innate immune responses involving the production of neutrophil extracellular traps (NETs). Monitoring the circulating cell-free DNA of NETs has implications for treatment and diagnosis of autoimmune diseases, cardiovascular events and traumatic injuries (4–7).

How Neutrophils Weave a Defensive Web

Blood smear showing two prominent neutrophils in the field of view
Blood smear showing two prominent neutrophils in the field of view

Neutrophils are the most abundant type of white blood cell and are part of the innate immune response, participating in non-specific immune responses to injury or pathogens. They are one of three types of granuolcytes, and can be recognized by their multi-lobed nucleus and the prominent granules that fill their cytoplasm. Generally they are first to the scene of injury or infection. Early in my scientific career, I was taught that neutrophils fought disease via phagocytosis and occasionally by firing a barrage of toxic enzymes and molecules at invaders. Mostly though they released cytokines that recruited the “important” cells of the specific immune system to the area.

For these reasons, I never really thought much about neutrophils. That is until recently, when I learned about Neutrophil Extracellular Traps (NETs). It turns out that neutrophils are pretty awesome, sacrificing themselves in a cloud-like explosion of DNA, chromatin, and granule proteins

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Top Science Books of 2016

Posted on by Nicole Sandler

While I planned to write about New Year’s resolutions for the first Promega Connections blog of 2017, I was sidetracked by some “best of 2016” lists—in particular, best science books. I realized though that these seemingly unrelated ideas overlapped at some level because every year I resolve to find time to read more books. What was once an easy and natural escape for me, like for so many others, reading for fun now requires a bit of effort and prioritization. With the continual distractions of Netflix, social media and online news stories, it’s a challenge to find time to read books the way I once did.

So, in honor of a new year’s resolution do more of what I like and less of what I don’t like, here is a list of what has been deemed the best science books of 2016. I culled through the lists of several of the most reputable science blogs and publications and looked for overlap among them. Between the Science Friday blog, New York Magazine’s blog, The Science of Us, Smithsonian Magazine, NPR, and the New York Times’ best of 2016 lists there are loads of suggestions to keep you reading until the start of the next decade. Below are eight recommendations that appeared on several “best of” lists. Continue reading “Top Science Books of 2016”

Plumage Revealed: A 99 Million Year Old Feathered Coelurosaur Tail Trapped in Amber

Posted on by Kelly Grooms

Touching a Dinosaur—Almost

Imagine holding a 99 million year old feathered dinosaur tail in the palm of your hand. The only thing keeping you from actually touching its feathers? A few centimeters of petrified resin. This was reality for the group of scientists who published their findings about this discovery in the December issue of Current Biology (1).

It all began roughly ninety-nine million years ago when a young coelurosaur met an untimely death. Continue reading “Plumage Revealed: A 99 Million Year Old Feathered Coelurosaur Tail Trapped in Amber”