In the Literature

Blogs about published peer-reviewed scientific studies.

Cellular Senescence and Cancer Therapy: Overcoming Immortality?

Posted on by Ken Doyle

At the time of writing this post, no scientist had yet discovered the secret to immortality. In our world, we’ve come to accept that living things are born, grow old and die—the circle of life.

And yet, for many years, life scientists believed that the circle of life did not apply to our constituent cells when cultured in a laboratory. That is, cultured normal human cells were immortal, and they would continue to grow and proliferate forever, as long as they were provided with the necessary nutrients.

The animal cell cycle. Image by Kelvinsong; made available under the Creative Commons CC0 1.0 Universal Public Domain Dedication

Pioneering work published in 1961 by Leonard Hayflick and Paul Moorhead challenged that theory (reviewed in 1). Their research showed that normal cells in culture have a finite capacity to replicate. After they reach a certain number of replicative cycles, cells stop dividing. Hayflick and Moorhead made the important distinction between normal human cells and cultured cancer cells, which are truly immortal. In later years, the limit to the number of replicative cycles normal human cells can undergo became known as the Hayflick limit. Although some scientists still express skepticism about these findings, the Hayflick limit is widely recognized as a fundamental principle of cell biology.

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Neanderthal DNA and Modern Humans: Svante Pääbo Receives the 2022 Nobel Prize in Physiology or Medicine

Posted on by Ken Doyle

What makes humans “human”?

Neanderthal DNA sequencing from ancient bone samples

On October 3, 2022, the Nobel Assembly at Karolinska Institutet announced the 2022 Nobel Prize in Physiology or Medicine had been awarded to Svante Pääbo, director of the Department of Genetics at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany. The Assembly cited his “discoveries concerning the genomes of extinct hominins and human evolution”. They mentioned the highlight of his research: the seemingly impossible task, at the time, of sequencing the Neanderthal genome. The discoveries that followed from this sequencing project continue to redefine our understanding of modern human origins.

The award showcases the technological advancements made in the analysis of ancient DNA. However, Pääbo’s research had an inauspicious beginning. In 1985, he published the results of his early work, cloning and sequencing DNA fragments from a 2,400-year-old Egyptian mummy (1). Unfortunately, later analysis revealed that the samples could have been contaminated by the researchers’ own DNA (2).

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mRNA Vaccine Manufacturing: Responding Effectively to a Global Pandemic

Posted on by Ken Doyle

We’ve learned a few important lessons from the COVID-19 pandemic.

Perhaps the most significant one is the importance of an early and rapid global response to the initial outbreak. A coordinated response—including widespread use of masks and other personal protective equipment (PPE), travel restrictions, lockdowns and social distancing—could save lives and reduce long-term health effects (1). Widespread availability of effective vaccines goes hand in hand with these measures.

New Boosters to Fight Omicron

Last month, Pfizer/BioNTech announced the US Food and Drug Administration (FDA) had granted emergency use authorization (EUA) for a new adapted-bivalent COVID-19 booster vaccine for individuals 12 years and older. This vaccine combines mRNA encoding the wild-type Spike protein from the original vaccine with another mRNA encoding the Spike protein of the Omicron BA.4/BA.5 subvariants. Moderna also announced FDA EUA for its new Omicron-targeting COVID-19 booster vaccine. The Omicron variant of SARS-CoV-2 shows multiple mutations across its subvariants, and it is currently the dominant SARS-CoV-2 variant of concern across the world.

Genomic epidemiology of SARS-CoV-2 with subsampling focused globally over the past 6 months. This phylogenetic tree shows evolutionary relationships of SARS-CoV-2 viruses from the ongoing COVID-19 pandemic. Image from Nextstrain.org; generated September 20, 2022

Booster doses of vaccines have become a way of life, both due to declining effectiveness of the original vaccines especially in older adults (2), and the rapid mutation rate of SARS-CoV-2 (3). Clinical data for the new Pfizer/BioNTech booster vaccine showed superior effectiveness in eliciting an immune response against Omicron BA.1 compared to the original vaccine. Previously, Moderna published interim results from an ongoing phase 2-3 clinical trial, showing that the new bivalent booster vaccine elicited a superior neutralizing antibody response against Omicron, compared to its original COVID-19 vaccine (4).

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Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions

Posted on by Michele Arduengo

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
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Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

Posted on by Michele Arduengo
space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a “bottom-up” proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100µg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37°C. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20µg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37°C.

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The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.

Read more about ProAlanase, a new site-specific endoprotease from Promega.

Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

PROTAC Virus Vaccines: A New Approach to Vaccine Development

Posted on by Michele Arduengo

Vaccine research and development is a major area of focus for life scientists across the globe. Clinical trials have shown that vaccines that target tumors show promise for cancer treatment. Additionally, the emergence of new zoonotic diseases has revealed a need to develop vaccines quickly as the world becomes more global and human populations interact more often with each other and wild habitats. Importantly, these vaccines need to be suitable for distribution in a variety of settings, including those that do not have easy access to refrigeration.

Influenza Virus. Si et al used influenza as a model to engineer and test PROTAC Virus vaccines

There are many ways to classify the different types of vaccines that are currently available. The National Institute of Allergy and Infectious Diseases in the United States, categorizes vaccines as: whole pathogen vaccines, subunit vaccines, and nucleic acid vaccines—based on how the antigen that stimulates the immune response is delivered to the host.

Whole-pathogen vaccines, which include many of vaccines used in clinical settings, use the entire pathogen (organism that causes the disease) that has been either weakened or killed to elicit a protective immune response. Killed vaccines are what their name implies: the pathogen has been killed so that it cannot cause disease, but enough of its structure remains to generate antibody response. Often, the immune response generated with killed vaccines is not as robust as that generated with other kinds of vaccines. 

Weakened or attenuated vaccines use whole pathogens that have been weakened in the laboratory through long-term culture or other means. Our modern MMR (measles, mumps and rubella) vaccine is an example of an attenuated vaccine. These vaccines tend to generate strong, long-lasting immune responses, but have increased risk for immunocompromised individuals.

Engineering an Influenza A PROTAC Virus Vaccine

A recent paper by Si et al published in Nature Biotechnology describes a new type of live-attenuated whole pathogen vaccine: the PROTAC virus. PROTAC viruses are prevented from replicating by targeting critical viral proteins for degradation using the host cell protein degradation pathway. The vaccine is live-attenuated by the host cells that degrade critical proteins.

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The Human Cell Atlas: Mapping a Cellular Landscape

Posted on by Jordan Nutting

From macrophages that seek out and destroy infectious agents to fibroblasts that hold tissues and organs together, cells give form and function to our bodies. However, despite their foundational roles in our biology, there is still much we don’t know about cells—like where different cell types are localized, what states a given cell type may take on, how the molecular characteristics of cells change over a person’s lifetime and more. Addressing these questions will provide a deeper understanding about the cellular and genetic basis of human health and disease.

Image contains several cells with a hazy outline of a DNA molecule in the background and one cell is highlighted
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COVID-19 Intranasal Vaccines: Right on the Nose?

Posted on by Promega

Last updated April 28, 2023

covid-19 intranasal vaccines

COVID-19 is still a global pandemic. Around the world, as of 5:40pm CEST, 26 April 2023, there have been 764,474,387 cumulative confirmed cases of COVID-19, including 6,915,655 deaths, reported to the World Health Organization. As of 24 April 2023, a total of 13,325,228,015 vaccine doses have been administered. The adoption of vaccines worldwide continues to increase, yet periodic spikes and surges in infection rates continue to occur with new SARS-CoV-2 variants, such as that observed in Australia in Jan 2022. Vaccine booster doses provide effective protection against developing severe disease and hospitalization, but vaccine adoption and distribution face ongoing challenges in low- and middle-income (LMIC) countries (1). The development of intranasal vaccines could help alleviate some of the challenges in these areas. Therapeutic interventions for those already infected are in development, with one (Paxlovid) currently available under emergency use authorization (EUA) in the US.

Cumulative COVID-19 statistics by country: WHO COVID-19 Dashboard. Geneva: World Health Organization, 2020. Available online: https://covid19.who.int/ (last cited: April 28, 2023).

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PD-1 Blockade Treatment shows 100% Tumor Resolution in Mismatch Repair Deficient Rectal Cancer Patients

Posted on by Kelly Grooms

Rectal cancer cases are rising in young adults (1). Typically, these cancers are treated with a multipronged approach that includes chemotherapy, radiation and surgery. These treatments show complete response in approximately 25% of patients and come with a long list of toxic side effects and life-altering complications including negative effects on bladder and bowel function, sexual health and fertility issues (2). 

artistic image of cancer cell and immunotherapeutic agents such as PD-1 blockade

Approximately 5–10% of rectal cancers have deficiencies in their mismatch repair mechanisms (dMMR), and these cancers tend to be less responsive to standard chemotherapy treatments (2). Tumors are identified as dMMR using either immunohistochemistry (IHC) to detect the presence or absence of the major mismatch repair proteins, or by molecular testing for high-frequency microsatellite instability (MSI-H), the functional evidence of dMMR . These tumors often have somatic mutations that produce “foreign” proteins that can be detected by the immune system. As a result, these tumors are effective at priming an immune response and tend to respond well to immune checkpoint therapies such as PD-1 blockade treatments. Immune checkpoint blockade, or immune checkpoint inhibitor, therapies are a revolutionary, and relatively new, approach to treating cancer. Some tumors express immune checkpoints to prevent the immune system from producing a strong enough immune response to kill the cancer cells. Immune checkpoint blockade therapies work by blocking immune checkpoint proteins that act to negatively regulate the immune system through the PD-1 pathway. When these checkpoint proteins are blocked, the body’s T-cells can recognize and kill the cancer cells.

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Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Posted on by Michele Arduengo
polypeptide chain showing protease cleavage sites
Learn about the new Trypsin Platinum Protease in this blog.

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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