Photograph of a black and white mosquito standing on a water surface, where its reflection is visible.

Excess Lipids Keep Dengue at Bay

Accumulating lipids may be Wolbachia bacteria’s secret weapon for decreasing viral transmission.

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© istock.com, doug4537

The Aedes aegypti mosquito can transmit dengue virus; however, infection with endosymbiotic bacteria from the Wolbachia genus reduces viral transmission from these insects. Consequently, Wolbachia-infected mosquitos are one biocontrol agent used in areas with endemic dengue cases. 

“We are trying to go backwards now, because the intervention works, but we don’t know how,” said Robson Loterio, a microbiologist at the Burnet Institute and author of a paper published in mBio exploring the mechanism of Wolbachias antiviral activity.1 The findings can help researchers prevent viral escape from this biocontrol method. 

Loterio and his team infected A. aegypti cells with antiviral Wolbachia strains. Using transmission electron microscopy and confocal microscopy, they showed that these bacteria predominantly clustered at the cell’s endoplasmic reticulum (ER). 

The ER produces molecules used in lipid droplet synthesis. Since both Wolbachia and dengue rely on lipid metabolism, the team investigated the effect of Wolbachia on droplet formation during viral infection by comparing antiviral Wolbachia-infected cells to Wolbachia-free cells.2 Lipid droplets accumulated in both types of cells, but cells with the bacteria had more lipid accumulation. 

Next, the team explored whether altered lipid droplet production influenced dengue virus replication by treating antiviral Wolbachia-infected and Wolbachia-free cells with an inhibitor to fatty acid synthase, an enzyme involved in lipid droplet production. The inhibitor decreased the amount of lipid droplets in both conditions. However, while this impaired dengue virus replication in Wolbachia-free cells, it enabled viral replication in antiviral Wolbachia-infected cells, supporting the role of excess lipid droplet formation in Wolbachia's antiviral activity. 

Matthew Aliota, a virologist at the University of Minnesota who was not affiliated with the study, pointed out that the study was predominantly based on cell culture work, but added that it was still a good start to understand this mechanism. “It just builds on what is known about Wolbachia pathogen blocking, in the sense that it's very complex.”

A human torso with the large bowel depicted in blue and the appendix in red.

Why Do Humans Have an Appendix?

Long believed to be purely vestigial, this troublesome organ may play an important role in gut and immune function.

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© istock.com, Ilya Lukichev

The appendix is a small, worm-like structure that, in humans at least, serves no obvious purpose beyond becoming inflamed and ruining someone’s day. In fact, this murderous little organ is responsible for more than 40,000 deaths per year.1

Eytan Wine wears a collared shirt and smiles in front of a microscope.
Eytan Wine studies factors that contribute to intestinal inflammation and is exploring the potential role of the appendix.
Eytan Wine, University of Alberta

Charles Darwin hypothesized that the appendix was an evolutionary relic leftover from humanity’s distant ancestors shifting from leaf-based to fruit-based diets. “But I don’t think the answer is that simple,” said Eytan Wine, a gastroenterologist at the University of Alberta.

One hypothesis, said Wine, is that the appendix functions as a “safe house” for beneficial microbes. “The appendix is a [blind-ended] organ, where microbes could escape different insults to the gut physiology such as infection, or antibiotics, or toxins,” he said. If some of these beneficial species get wiped out, survivors in the appendix could replace them, re-balancing the gut microbiome. “[The safe house hypothesis] is not all that well studied,” said Wine. “It’s certainly plausible and somewhat supported.”

The appendix also contains a large amount of gut-associated lymphoid tissue, populated with various types of T and B cells as well as germinal centers.2 This has led researchers to hypothesize that it may function as an immune cell priming site.

A fluorescent microscopy image of periappendicular bowel shows a group of red-labeled bacteria cells embedded in a rough oval of green-labeled mucus tissue, encased in a ring of blue-labeled epithelial cells.
Researchers think bacteria from the appendix may migrate into other regions of the digestive tract; here, bacteria (in red) are embedded in the mucus layer (green) of a periappendicular section of bowel.
Nazanin Arjomand Fard

Yet people who have undergone appendectomies are often perfectly healthy. “To me, this [indicates that] maybe the importance is more in early life…immune development is a lifelong process, but the first few years of life—those are the critical times of immune education,” said Wine. Since most appendectomies are performed between ages 10 and 30, Wine speculates that by the time it is removed, the appendix may have already served its most important function.


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A 3D render of multiple RNA strands floating around each other.

Improving RNA Sequencing with FFPE Samples

Samples are commonly stored in a way that degrades RNA. Scientists are devising new ways to overcome this obstacle for RNA sequencing. 

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modified from © istock.com, BlackJack3D

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A box containing a Twist RNA Exome kit.
Twist RNA Exome detects more targets with fewer reads.
Twist Bioscience

RNA sequencing (RNA-seq) is a powerful tool for molecular biologists looking for what drives mechanisms of disease.1 RNA-seq is routinely used for both research and clinical applications in diseases featuring high levels of genetic dynamism and heterogeneity, such as cancer.2 However, although RNA-seq is becoming more accessible and prevalent, it comes with technical obstacles that can prevent scientists from obtaining the high-quality data they need.

RNA is more subject to degradation than DNA, whether chemical, physical, or enzymatic. Complicating the matter, scientists performing disease research rely on clinical tissue samples as RNA sources. These samples are commonly formalin-fixed and paraffin-embedded (FFPE), a process that preserves sample integrity over the long-term for immunohistochemical analysis but can result in highly degraded RNA.2

Scientists are designing FFPE sample-tailored RNA library preparation protocols, reagents, and kits to remove this bottleneck. Twist Bioscience, for example, offers a rapid library preparation kit designed to work with FFPE and other low input samples that takes as little as 4.5 hours to complete from start to finish. The kit protocol includes an optional depletion step for removing ribosomal and globin RNA, leaving only messenger RNA. It also incorporates dUTP nucleotides during second-strand synthesis, allowing scientists to trace amplified sequences back to originating strands.

The Twist RNA Library Preparation Kit is intended to be a strong first step in the RNA-seq process, and therefore is designed to pair with their other target enrichment solutions such as the RNA Exome panel (enriching across the whole transcriptome) or RNA Fusion (targeting specific cancer-relevant fusion genes) panel. Finally, Twist Bioscience can also design custom panels for scientists with a unique application and goal in mind.

Learn more about the available solutions for maximizing RNA sequencing using FFPE samples. 

 

What RNA sequencing applications do you perform in your research?

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Two scientists look at microscopy data, and one thinks about her own images.

Right Protein, Wrong Pattern

Julia Darby’s tagged chimeric proteins told a convincing story. Later, she learned that they distorted some of the details.

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© istock.com, Tatiana Sozonova, Wanlee Prachyapanaprai, Nadezhda Deineka; Designed by Ashleigh Campsall

As a doctoral student in Ann Marie Craig’s laboratory at the University of Illinois, Urbana-Champaign, I studied what directed two glutamate receptors to their positions for functions in hippocampal neurons. Suspecting that the cytosolic end of the protein may be responsible, I swapped the C-terminal tails of these proteins to create chimeras. To facilitate the detection of both chimeras and the two original proteins in my different cell cultures, I inserted a sequence for a three amino acid tag into the N-terminus of all my proteins that I could detect with an antibody. 

Photograph of a Julia Darby, currently a private tutor for school districts in Iowa and Illinois, learned that her convenient protein tags may have done more than she bargained for. She is wearing a red shirt and smiles at the camera with her hands folded under her chin. 
Julia Darby, today a private tutor for school districts in Iowa and Illinois, was a graduate student studying glutamate receptors at the University of Illinois, Urbana-Champaign. She learned after she defended her dissertation that some of her methods may not have worked like she thought. 
Julia Darby 

When I expressed the different protein constructs in neuron cells in culture, I demonstrated that these C-terminal tails determined the localization of the glutamate receptors. I also got beautiful images that illustrated one receptor was found in the axons and the second one localized along the axon and in the dendrites, and they were so stunning that Neuron selected them for the issue cover when we published the data. 

I defended my PhD with this success story and moved to Washington University in St. Louis to pursue a postdoctoral position. Roughly a year later, a postdoctoral researcher in Craig’s lab followed up on my project to study the native receptors. Their data showed that my receptor that appeared homogenously along the axon in fact clustered in its places on the axon and synapse. It turned out my introduced tag interrupted this activity, so the proteins didn't interact with each other like they should have. 

When I learned about the discrepancy, my first thought was that I was glad I already had my PhD completed. We didn’t retract the paper, because although the arrangement we observed was wrong, our findings regarding the role of the C-terminus were correct. 

The incident serves as a reminder for me that in science, we ask a lot of questions, but how we answer them is often more important than the results themselves. 

This interview has been edited for length and clarity.


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Photo of the Capitol Building in Washington DC.

From Lab Coat to Legislation

Following graduate school, Sarah Carter headed to Washington, DC to carve out a career in science policy.

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© istock.com, Joecho-16

Early in her graduate studies at the University of California, San Francisco, Sarah Carter knew that a career in research wasn’t for her. She was interested in science policy but coming from an academic laboratory with little experience to draw from, she had to forge her own path. Now, Carter works as an independent science policy consultant, advising nonprofit organizations and think tanks on biotechnology and the bioeconomy.

Photo of Sarah Carter
For the last 15 years, Sarah Carter has worked on science policy projects related to emerging biotechnology, the bioeconomy, synthetic biology, and biosecurity. 
Sarah Carter

How did you transition to science policy?

After a fellowship with the National Academies during graduate school, I embarked on a science and technology policy fellowship with the American Association for the Advancement of Science, where I worked with the Environmental Protection Agency on energy and climate change. These opportunities kickstarted my career—I learned a lot about policy and the inner workings of government. 

However, I wanted to get back to my biology roots, so I joined the policy center at the J. Craig Venter Institute. It was a great fit—I worked alongside scientists at the forefront of synthetic biology to consider the broader societal effects of emerging technologies. I examined the successes and limitations of regulatory systems and advised policymakers on ways to strengthen the guidance to limit biosecurity threats while supporting scientific progress.1,2 

What can scientists bring to the science policy landscape?

Scientists are essential to policy decision-making. At the bench, I felt like I was only an expert on my specific research project, but when I started engaging in policy issues, I realized that I had a broader grasp of biology. Policymakers often receive conflicting reports about the risks associated with a new technology; scientists can provide comprehensive assessments that outline the relative risks of different biosecurity scenarios. 

This interview has been edited for length and clarity

  1. Carter SR, et al. J Craig Venter Institute; 2014.
  2. Carter SR, Friedman RM. J Craig Venter Institute; 2015.
A gel with dye-labeled bands indicating proteins separated by electrophoresis.

SDS-PAGE Technology for the 21st Century

New innovations streamline and simplify SDS-PAGE from a multi-hour workflow to a matter of minutes.

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© istock.com, extender01

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) uses an electric current to separate proteins in a heterogeneous sample according to molecular weight and is one of the most commonly used techniques in the life sciences.1,2 The polyacrylamide gel is an integral part of SDS-PAGE. However, despite the technique being over 50 years old, most researchers still cast gels by hand via a time-consuming process that can take 90 minutes or longer. Furthermore, scientists create these gels using the toxic compounds ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) to catalyze the polymerization of acrylamide and bisacrylamide into polyacrylamide.

          A scientist using the mPAGE® Lux Casting System.
The mPAGE® Lux Casting System uses three pre-made reagents and a curing station to produce gels in a process that takes about three minutes. 
MilliporeSigma

Pre-cast gels are commercially available, but they are expensive and their packaging tends to generate considerably more waste than hand-casting. Moreover, researchers cannot modify the composition of pre-cast gels to fit their experiments.2 All of this ultimately creates a dilemma where scientists must choose between time, resources, and flexibility. As such, pre-cast gels have not superseded hand-casting as the preferred method among the scientific community.

Clearly, new innovations are needed for this old workhorse technique, and the mPAGE® Lux Casting System provides a novel solution. Designed to provide flexibility and reliability without incurring costs in terms of time, resources, or waste, the mPAGE® Lux Casting System uses three pre-made reagents and a curing station to produce gels in a process that takes about three minutes. The system generates Bis-Tris gels as opposed to commonly used Tris-Glycine gels, enabling shorter run times and longer shelf lives. The system further removed the need for APS and TEMED, which are toxic compounds. Finally, because gels can be rapidly made on-demand, researchers no longer have to risk wasting resources on pre-preparing gels that will go unused.

Learn more about how a new system lets researchers produce SDS-PAGE gels on-demand and to their own specifications in three minutes. 


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A microscopy image of a mouse embryo expressing the red fluorescent protein mCherry in the central nervous system.

Dynamic Enhancers Orchestrate Development

Evgeny Kvon leverages transgenic models and genomic techniques to uncover the ways enhancers control the transcription of genes.

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Joshua Alcantara

Evgeny Kvon, a geneticist at the University of California, Irvine, fell in love with molecular biology when he learned about the experiments that led to the discovery of DNA as the carrier of genetic information in high school. Now, as a group leader, Kvon explores how enhancers, which are non-coding regulatory DNA elements, orchestrate the precise spatiotemporal transcription of genes during mammalian development.

A group of people is surrounded by desert plants. 
Evgeny Kvon (right) and his team investigate how enhancers regulate gene expression during development.  
Sandra Jacinto

How does your team study enhancer function?

We use transgenic mouse embryos to show that a DNA sequence is an enhancer. In one assay, we clone the enhancer sequence upstream of a promoter and lacZ reporter gene and then inject this construct into the mouse egg. By looking at lacZ activity, which appears as a blue color in the mouse embryo, we can track the location and activity of that potential enhancer. We also use high-throughput chromosome conformation capture techniques to study enhancers and their chromatin interactions. Using both methods, we recently mapped the enhancer-promoter interactions of almost 1000 known enhancers in different mouse embryo tissues and found that around 60 percent of them skip the promoters of neighboring genes and contact more distant genes.1

What are some of the unanswered questions that you would like to explore in the coming years?

How enhancers know which genes to ignore and which to activate is an interesting question that needs further investigation. It is also unclear how remote enhancers can act on their target genes even though they are located far away from one another. We started to tackle this question in a recent preprint, and we found that a long-range enhancer has an element, which we named range extender (REX), right next to it.2 When we added REX to a short-range enhancer, it gained the ability to act long range. We believe we identified a genetic signature that allows remote enhancers to work that way.     

This interview has been edited for length and clarity.

Photo of the Capitol Building in Washington DC.

Science Crossword Puzzle

Put on your thinking cap, and take on this fun challenge.

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© istock.com, Joecho-16

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