The Simplex Things In Life: Utilizing Artificial Intelligence Models to Better Understand Autism

Autism Spectrum Disorder, or ASD, is nothing if not unique.

The way ASD manifests itself in people is unique; although it most often presents as some form of variable impairment in social interaction and communication, each individual has behaviors and habits that are as unique to them as snowflakes are to one another.

ASD has also proven itself to be a uniquely challenging disorder to study. In the past decade, de novo (new) mutations have been identified as key contributors to causality of ASD. However, the majority of these identified de novo mutations are located in protein-coding genes, which comprise only 1–2% of the entire human genome.

Up to this point, a majority of previous research has focused on identifying mutations located in the 20,000 identified genes in the protein-coding region, which would seem like a promising approach. Genes are the genetic blueprints for creating proteins, which control and perform crucial tasks in our bodies, such as fighting off infections, communicating between your organs, tissues, and cells as chemical messengers, and regulating your blood sugar levels. It seems like basic math: Genes + Mutations = Mutated Proteins. Mutated Proteins = Disrupted Protein Function.

However, it has been observed that all the known genes that are ASD-associated can explain only a minor fraction of new autism cases, and it is estimated that known de novo mutations in the protein-coding region contribute to not more than 30% of cases for individuals who have no family history of autism (better known as simplex ASD). This provides evidence to suggest mutations contributing to autism must additionally occur elsewhere in the genome. Continue reading “The Simplex Things In Life: Utilizing Artificial Intelligence Models to Better Understand Autism”

When Proteins Get Together: Shedding (Blue) Light on Cellular LOV

NanoBRETNo protein is an island. Within a cell, protein-protein interactions (PPIs) are involved in highly regulated and specific pathways that control gene expression and cell signaling. The disruption of PPIs can lead to a variety of disease states, including cancer.

Two general approaches are commonly used to study PPIs. Real-time assays measure PPI activity in live cells using fluorescent or luminescent tags. A second approach includes methods that measure a specific PPI “after the fact”; popular examples include a reporter system, such as the classic yeast two-hybrid system.

Continue reading “When Proteins Get Together: Shedding (Blue) Light on Cellular LOV”

Eight Considerations for Getting the Best Data from Your Luminescent Assays

The stage is set. You’ve spent days setting up this experiment. Your bench is spotless. All the materials you need to finally collect data are laid neatly before you. You fetch your cells from the incubator, add your detection reagents, and carefully slide the assay plate into the luminometer. It whirs and buzzes, and data begin to appear on the computer screen. But wait!

Bad data
These data are garbage!

Don’t let this dramatic person be you. Here are 8 tips from us on things to watch out for before you start your next luminescent assay. Make sure you’ll be getting good data before wasting precious sample!

Continue reading “Eight Considerations for Getting the Best Data from Your Luminescent Assays”

How Do You Solve a Problem Like Malaria?

malaria_researcher
Photo courtesy of NIH/NIAID

Malaria affects nearly half of the world’s population, with almost 80% of cases in sub-Saharan Africa and India. While there have been many strides in education and prevention campaigns over the last 30 years, there were over 200 million cases documented in 2017 with over 400,000 deaths, and the majority were young children. Despite being preventable and treatable, malaria continues to thrive in areas that are high risk for transmission. Recently, clinicians started rolling out use of the first approved vaccine, though clinical trials showed it is only about 30% effective. Meanwhile, researchers must continue to focus on innovative efforts to improve diagnostics, treatment and prevention to reduce the burden in these areas.

Continue reading “How Do You Solve a Problem Like Malaria?”

Wetlands, Water Quality and Rapid Assays

toad

The storms of the previous day had moved eastward, leaving in their wake flooded farm fields and saturated roadside wetlands. At dusk, we loaded the Ford Escort wagon and headed south. We bumped along the maze of farm roads intent upon listening for croaks and snores in the night. At one roadside wetland, I heard my first congress of Spadefoot toads. The sound was deafening, invoking everything that a “congress of snoring toads” brings to mind. Around the corner, in a low spot of a corn field, a lone Spadefoot toad called for a mate; he was joined by a rather enthusiastic Copes Gray tree frog and several chorus frogs. The congress down the road provided a rolling bass to these more melodic anurans.

Wetlands exist in many different shapes and sizes and in many different geographies: coastal margins, mountain valleys, beaches and rocky shores, estuarine wetlands where tidal saltwater and freshwater mix, and inland wetlands. Some of them are ephemeral, some of them permanent. Wetlands serve many different functions, from providing habitat and food for plants and animals to offering protection from floods and maintaining water quality. One acre of one-foot deep wetland is estimated to hold 330,000 gallons of water. Coastal wetlands are important for reducing storm erosion by decreasing tidal surge and buffering the wind. In the US alone, this benefit has an estimated value of $23.2 billion dollars each year.

Continue reading “Wetlands, Water Quality and Rapid Assays”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs.

Continue reading “A BiT or BRET, Which is Better?”

Voted Drug Discovery and Development Product for 2018: NanoBRET TE Kinase Assays

Choice Drug Discovery and Development Product 2019 award
Michael Curtin, Promega, accepting the Reviewers’ Choice for Drug Discovery and Development Product of the Year award from SelectScience.

As announced at SLAS in Washington, D.C. recently, we are excited to have NanoBRET Target Engagement (TE) Intracellular Kinase Assays awarded the SelectScience Reviewers’ Choice for Drug Discovery and Development Product of the Year 2018!

The NanoBRET™ Target Engagement (TE) Kinase Assay, first available in the fall of 2017, has been getting great reviews on the SelectScience site for more than a year now. Continue reading “Voted Drug Discovery and Development Product for 2018: NanoBRET TE Kinase Assays”

Executing a NanoBRET™ Experiment: From Start to Data

This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Meet Měnglà Virus: the newest cousin in the Ebola and Marburg virus family tree

Ebola virus (EBOV) and Marburg virus (MARV) are two closely-related viruses in the family Filoviridae. Filoviruses are often pathogenic, causing hemorrhagic fever disease in human hosts. The Ebola outbreak of 2014 caught the world by surprise by spreading so quickly and severely that public health organizations were unprepared. The devastating outcome was a total of over 11,000 deaths by the time the outbreak ended in 2016. Research that provides further understanding of filoviruses and their potential for transmission is important in preventing future outbreaks from occurring. But what if the outbreak comes from a virus we’ve never seen before?

fruit_bat
Měnglà virus was discovered among filoviruses isolated from Old World fruit bats (Rousettus)

All in the viral family

A recent study published in the journal Nature Microbiology provides evidence of a newly identified filovirus species. Using serum samples taken from bats, a well-known host for filoviruses, Yang et al. isolated and identified viral RNA for an unclassified viral genome sequence using next generation sequencing analysis. This new virus genome sequence was organized with the same open reading frames as other filoviruses, encoding for nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L). This new genome sequence shared up to 54% of the nucleotide sequences for the filovirus species Lloviu virus (LLOV), EBOV and MARV, with MARV being the most similar. Their analysis suggested that this novel virus should be classified within the Filoviridae family tree as a separate genus, Dianlovirus, and was named Měnglà virus (MLAV).

Continue reading “Meet Měnglà Virus: the newest cousin in the Ebola and Marburg virus family tree”
Light enters eyes and is transmitted to SCN and PHb.

Light: A Happy Pill for Dark Days?

Have you ever had a day where you feel exceptionally good? As intake on the world kind of good? You feel so much better than the previous couple of days that you stop to wonder why.

Then it dawns on you.

The sun is out. It’s been cloudy for the past week but now—SUNSHINE.

You go out to lunch or for a walk just to take in those rays. Sure, it feels warmer than your darkened office space, but it’s the light rather than warmth that’s making a difference.

You purposely don’t wear sunglasses and it feels like the light is coming in through your eyes and massaging that part of your brain that is your happy zone. Are you imagining it or is the sun really affecting how you feel?

In a study reported in the September 2018 issue of Cell we learn that this is not a figment of your or my imagination (1). There is, in fact, a type of retinal cell that transports sunlight directly to the part of our brains that affects mood.

Eyes and the Body’s Master Clock

Circadian rhythms are innate time-keeping functions found in all multicellular organisms. This subject of the 2017 Nobel prize in Physiology or Medicine, circadian rhythms are fueled by daily light-dark cycles and are critical to the function of neurologic, immune, musculoskeletal and cardiac tissues (2). Nearly every mammalian cell is affected by circadian rhythms.

The human body has a circadian master clock, the suprachiasmatic nucleus or SCN. The SCN is a highly innervated tissue located in the hypothalamus (see image). It is connected directly to the retina by the optic nerve, and thus is influenced by external light and dark.

Light enters eyes and is transmitted to SCN and PHb.
Light enters the eyes and affects the SCN (physiologic effects), and as discussed in recent research, Fernandez et al. here, the perihabenular nucleus (behavioral effects). (Image in public domain.)

The retina of the eye is the light-gathering instrument for this organ. Historically, it’s been understood that the retina is composed of two cell types, rods and cones, that function in transmitting light and images to the optic nerve, which sends those signals to the brain.

Drawing of the retina with rods and cones, some nervous tissues.
Some parts of the retina. Light enters the eye (from left) and passes through to the rods and cones. Here a chemical change converts the light to nerve signals. Image-based on drawing by Ramón y Cajal, 1911 and licensed under Wikimedia commons.

Studies by Hattar et al. in the early 2000s identified another cell found in the retina, the melanopsin-containing intrinsically photoactive retinal ganglion cells (ipRGCs) as the transmitter of circadian light signals (3). Through this direct connection to the SCN, the circadian master clock, the ipRGCs can influence a wide range of light-dependent functions independent of image processing (4).

Now Fernandez et al. have identified multiple types of ipRGCs. They showed that ipRGCs that mediate the effects of light on learning work via the SCN, while the pathway for light influencing emotions is different.

They discovered a new target of ipRGC cells, the perihabenular nucleus (PHb). The PHb is a newly recognized thalamic region of the brain. The authors showed that the connection between light and mood is regulated by ipRGCs through the PHb versus the SCN. They show that the PHb is integrated into other mood-regulating centers of the thalamic region.

In Conclusion

Daylight, and lack thereof, does affect both our mood and our ability to learn. In this 2018 report, we have learned that the pathways for these effects are distinct, and gain an understanding of a new thalamic region by which the light and mood actions occur. This information could influence the development of better drugs and/or therapies for major depressive disorders.

For those of us with seasonal affective disorder, the evidence is undeniable—lack of light can cause issues, from sleep-wake problems, to mood and learning issues.

And while we can’t create sunshine, a special lamp or lightbox may help to gain some full-spectrum light. To learn more about how to choose such a lamp and when to use it, see this Mayo clinic article for details.

References

  1. Fernandez, D.C. et al. (2018) Light affects mood and learning through distinct retinal pathways. Cell 175, 71–84.
  2. Ledford, H. and Callaway, E. (2017) Circadian clock scoops Nobel prize. Nature 550, 18.
  3. Hattar, S. et al. (2002) Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, 1065–70.
  4. Hattar, S. et al. (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424(6944)76–81.