5 of Our Favorite Blogs from 2018

We have published 130 blogs here at Promega this year (not including this one). I diligently reviewed every single one and compiled a list of the best 8.5%, then asked my coworkers to vote on the top 5 out of that subset. Here are their picks:

1. The Amazing, Indestructible—and Cuddly—Tardigrade

No surprises here, everyone loves water bears. Kelly Grooms knows what the people want.

The face of a creature that is nigh un-killable.

Continue reading “5 of Our Favorite Blogs from 2018”

How Prostate Cancer Cells Survive Glucose Deprivation

Illustration of energy metablism in cell.Glucose is an energy metabolite necessary for cellular survival and growth whether or not the cell is part of a tumor. Not only do cancer cells switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect) to gain more glucose, a hallmark of cancer, but they also increase the amount of glucose taken up from the surrounding extracellular space. However, the lack of glucose can have a negative effect on cells, causing them to become apoptotic in the absence of this metabolite. Cancer cells have methods to get around the requirement for glucose, including upregulating glucose transporters to improve access to the energy metabolite. In this Redox Biology article, researchers describe how activating androgen receptor in response to a lack of glucose affects the amount of GLUT1 expressed on prostate cancer cells, making the cells resistant to glucose deprivation.

To set the stage, two prostate cancer cell lines, LNCaP, an androgen-sensitive cell line, and LNCaP-R, an androgen-insensitive cell line, were deprived of glucose. Both cell lines showed signs of cell death, but LNCaP-R cells died in greater numbers. To probe how LNCaP cells died, several inhibitors (a pan-caspase inhibitor, two necroptosis inhibitors and a ferroptosis inhibitor) were added but did not change the way the cells died. However, an autophagy inhibitor enhanced cell death, suggesting the cells were necrotic not apoptotic. Teasing apart if the necrosis of LNCaP cells was due to glucose availability or merely disrupted glycolysis, the glucose analog 2DG was added to the medium with glucose. The cells survived when treated with 2DG, suggesting it was the absence of glucose that induced necrosis. When LNCaP cells were cultivated in medium that replaced glucose with mannose or fructose, the cells survived, another point in favor of sugar depletion causing cell death. Continue reading “How Prostate Cancer Cells Survive Glucose Deprivation”

Could Your Appendix Predispose You to Parkinson’s Disease?

Image of span of vagal nerve, humans.
The vagal nerve could serve as conduit for transit of alpha-synuclein from appendix to brain.

Since about 2000 we’ve learned a lot about the bacteria in our guts. We’ve learned that the right bacterial communities in our gastrointestinal system can make us feel better, think better and even help avoid obesity (1). My colleague Isobel has previously blogged about how certain gut bacteria can improve immunotherapy outcomes.

Conversely, the wrong bacteria in our guts can have negative consequences on health and cognition.

Along the way we’ve learned that gut bacterial flora can be influenced by what we eat, certain medications like antibiotics, and even stressful events. We now know that fermented foods like yogurt, sauerkraut, kombucha and that horrible-smelling stuff (kimchi) that another colleague eats are happy food for the good gut bacteria.

And you might guess that fried foods, saturated fats and certain carbohydrates can support the growth of gut bacteria that are doing us no favors when present in large quantities in our gastrointestinal system. Continue reading “Could Your Appendix Predispose You to Parkinson’s Disease?”

Nano, Nano: Tiny Lipid Particles with Big Therapeutic Potential

cell-transfection-viafect-luciferase-assayGetting DNA or RNA into cells can be a tricky business, and a variety of transfection reagents have been developed over the years to make the process easier. Lipid-based reagents are especially popular because they combine efficient transfection with relatively low toxicity.

When it comes to transfection, it pays to think small. Human cells range in volume from 20–40 µm3 (sperm cells) to as large as 4 million µm3 (mature egg cells, or oocytes). For several decades, transfection reagents have targeted this size range. However, breakthrough research involves leaving the “micro” realm and entering a world that was once the domain only of science fiction: nanotechnology. Continue reading “Nano, Nano: Tiny Lipid Particles with Big Therapeutic Potential”

A Tale of Two Toxins: the mechanisms of cell death in Clostridium difficile infections

When someone is admitted to a hospital for an illness, the hope is that medical care and treatment will help them them feel better. However, nosocomial infections—infections acquired in a health-care setting—are becoming more prevalent and are associated with an increased mortality rate worldwide. This is largely due to the misuse of antibiotics, allowing some bacteria to become resistant. Furthermore, when an antibiotic wipes out the “good” bacteria that comprise the human microbiome, it leaves a patient vulnerable to opportunistic infections that take advantage of disruptions to the gut microbiota.

One such bacteria, Clostridium difficile, is of growing concern world-wide since it is resistant to many different antibiotics. When a patient is treated with an antibiotic, C. difficile can thrive in the intestinal tract without other bacteria populating the gut. C. difficile infection is the leading cause of antibiotic-associated diarrhea. While symptoms can be mild, aggressive infection can lead to pseudomembranous colitis—a severe inflammation of the colon which can be life-threatening.

C. difficile causes disease by releasing two large toxins, TcdA and TcdB. Understanding the role these toxins play in colonic disease is important for treatment strategies. However, most published research data only report the effects of the toxins independently. A 2016 study demonstrated a method of comparing the toxins side-by-side using the same time points and cell assays to investigate the role each toxin plays in the cell death that leads to disease of the colon. Continue reading “A Tale of Two Toxins: the mechanisms of cell death in Clostridium difficile infections”

Automated Approach for Multiomic Analysis

With the use of a suite of “-omics” technologies you can examine the way in which complex cellular processes work together across all molecular domains (i.e., proteomics, metabolomics, transcriptomics) in a single biological system. Several studies have been published across a wide range of fields illustrating the power of such a unified approach (1,2). Most studies however did not focus on the development of a high-throughput, unified sample preparation approach to complement high-throughput “omic” analytics.

A recent publication by Gutierrez and colleagues presents a simple high-throughput process (SPOT) that has been optimized to provide high-quality specimens for metabolomics, proteomics, and transcriptomics from a common cell culture sample (3). They demonstrate that this approach can process  16−24 samples from a cell pellet to a desalted sample ready for mass spectrometry analysis within 9 hours. They also demonstrated that the combined process did not sacrifice the quality of data when compared to individual sample preparation methods.

Literature Cited

1. Roume, H. (2013) Sequential Isolation of Metabolites, RNA, DNA, and Proteins from the Same Unique Sample. Methods Enzymol. 531, 219−236.
2. Lo, A. W. et al. (2017) ‘Omic’ Approaches to Study Uropathogenic Escherichia Coli Virulence. Trends Microbiol. 25, 729−740.
3. Gutierrez, D. et al. (2018)  An Integrated, High-Throughput Strategy for Multiomic Systems Level Analysis J. Proteome Res.

How Autophagy Feeds Cancer’s Need for Metabolites

Illustration of energy metablism in cell.Metabolism underpins numerous cellular processes. Without it, cells would not grow, divide, synthesize or secrete. Another pathway, autophagy, degrades unwanted cellular materials, helping to maintain cell health. With these opposing roles, is there a connection between autophagy and metabolism? As it turns out, the answer is yes. Because molecules degraded by autophagy are recycled and fed into metabolism pathways as precursor compounds. There are interesting implications as a result of this connection, ones that affect cancer cells as described in a recent Cell Metabolism review article.

Autophagic flux, the process by which molecules and organelles are directed to the autophagosome, fuse with the lysosome and are degraded, involves a selective process that determines the cargo carried within the autophagosome. Autophagy-related genes (ATGs) direct the process and particular receptor proteins bind the cargo. What is interesting about the connection among cancer, autophagy and metabolism is the complexity of the role that autophagy plays in cancer. While autophagy was thought to act in a more tumor suppressive manner as shown when one copy of an ATG6 analogous gene in mice was deleted and the other left unaltered, and malignant tumors developed, but in mice mosaic for ATG5 deletions, the inhibition of autophagy resulted in benign tumors in the liver. This latter experiment suggested autophagy was needed for cancer progression, a hypothesis reinforced by the lack of ATG mutations in human cancers. Continue reading “How Autophagy Feeds Cancer’s Need for Metabolites”

Autophagy: The Poem

Roberta A. Gottlieb, MD, is the Director of Molecular Cardiobiology at Cedar-Sinai, a nonprofit academic healthcare organization. She is interested in the role of autophagy in myocardial ischemia, a kind of heart disease in which blood flow to the heart is blocked. (Studies have shown that autophagy is upregulated during myocardial ischemia, but why this happens is not entirely clear.) Her ultimate goal is to understand and mitigate ischemic injury, with the hope of developing therapeutics for humans.

And—she’s a poet. Continue reading “Autophagy: The Poem”

All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.
With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA like that induced by CRISPR-Cas9 is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”