Cell-free gene expression systems are a staple tool for the researcher seeking to understand the regulation of transcription and translation. Many factors can affect the efficiency of cell-free gene expression including vector sequence, reaction components and the template DNA concentration. One factor that has not been extensively studied is how DNA template length influences gene expression.
Continue reading “How Does DNA Template Length Influence Gene Expression in Cell Free Systems?”
Most of us, after we flush the toilet, don’t think twice about our body waste. To us, it’s garbage. To epidemiologists, however, wastewater can provide valuable information about public health and help save lives.
Wastewater-based epidemiology (WBE) is the analysis of wastewater to monitor public health. The term first emerged in 2001, when a study proposed the idea of analyzing wastewater in sewage-treatment facilities to determine the collective usage of illegal drugs within a community. At the time, this idea to bridge environmental and social sciences seemed radical, but there were clear advantages. Monitoring wastewater is a nonintrusive and relatively inexpensive way to obtain real-time data that accurately reflects community-wide drug usage while ensuring the anonymity of individuals.
Continue reading “From Drug Use to Viral Outbreaks, How Monitoring Sewage Can Save Lives”Oral vaccines are a great strategy and are especially beneficial in areas with poor sanitation. This form of vaccine distribution could help control the acute diarrheal disease caused by Vibrio cholera. There are an estimated 1.3 to 4 million cases and 21,000 to 143,000 deaths from cholera each year. A recent study from The Lancet Microbe finds new hope in a rice-based cholera vaccine that will fight against the diarrheal toxin without severe adverse events.
In 3D cell culture models, cells are grown under conditions that allow the formation of multicellular spheroids or microtissues. Instead of growing in a monolayer on a plate surface, cells in 3D culture grow within a support matrix that allows them to interact with each other, forming cell:cell connections and creating an environment that mimics the situation in the body more closely than traditional 2D systems. Although 3D cultures are designed to offer a more physiologically accurate environment, the added complexity of that environment can also present challenges to experimental design when performing cell-based assays. For example, it can be a challenge for assay reagents to penetrate to the center of larger microtissues and for lytic assays to disrupt all cells within the 3D system.
Earlier this week Terry Riss, a Senior Product Specialist at Promega, presented a Webinar on the challenges of performing cell-based assays on microtissues in 3D cell culture. During the Webinar, Terry gave an overview of the different methods available for 3D cell culture, providing a description of the advantages of each. He then discussed considerations for designing and optimizing cell-based assays for use in 3D culture systems, providing several recommendations to keep in mind when performing cell viability assays on larger microtissue samples.
Continue reading “3D Cell Culture Models: Challenges for Cell-Based Assays”The Delta Variant poses a unique challenge to global health. We’ve compiled answers to some of the most common questions about Delta and other SARS-CoV-2 variants.
A variant is a form of a virus that is genetically distinct from the original form.
“All organisms have mutation rates,” says Luis A Haddock, a graduate student at University of Wisconsin – Madison. “Unfortunately for us, viruses have one of the highest mutation rates of everything that currently exists. And even more unfortunately, RNA viruses have the highest mutation rates even among viruses.”
Luis works in the Friedrich Lab at UW-Madison, which has been sequencing SARS-CoV-2 genomes from positive test samples since the beginning of the pandemic. SARS-CoV-2 is constantly evolving, and sequencing can help us follow it through time and space. Most of the variants don’t behave any differently. A single nucleotide substitution might not even change the amino acid sequence of an encoded protein. However, occasionally a mutation will alter the structure or function of a protein.
Learn more about SARS-CoV-2 sequencing in the article “From Primate Models to SARS-CoV-2 Sequencing and Testing,” featuring David and Shelby O’Connor, two collaborators of the Friedrich Lab.
For decades now, peptides have been a molecule of interest for drug discovery research. Peptides offer a unique opportunity for therapeutic intervention that closely mimics natural pathways, as many physiological functions utilize peptides as intrinsic signaling molecules. Macrocyclic peptides, in particular, have recently proven to be promising candidates for targeting intracellular protein–protein interactions (PPIs), an attractive but hard-to-reach therapeutic target for conventional small molecule and biological drugs.
As with any opportunity, there are also challenges that accompany the peptide therapeutic development. Peptide ligands typically have poor membrane permeability, so thus far the majority of peptide therapeutics predominantly target extracellular proteins and receptors. There are also multiple mechanisms for cellular uptake of peptides, including both energy-dependent routes like endocytosis, and energy-independent, like passive diffusion or membrane translocation. Multiple mechanisms of cellular uptake paired with poor permeability makes engineering enough membrane permeability into peptides in order to advance them through drug discovery pipelines extremely difficult.
There are other factors to consider in developing peptide therapeutics, such as solubility, protein/lipid binding and stability, which can also have an affect on the overall cytosolic concentration and, ultimately impact the ability of the peptide to effectively engage its desired intracellular targets.
With so many challenging factors, the ability to have a predictive, high-throughput assay to assess cell permeability, independent of the mechanism(s) of entry, would be a critical and invaluable tool to support peptide drug discovery research.
In a recent study published in ACS Chemical Biology, researchers sought to develop such a tool, and demonstrated a new application for Promega NanoBRET™ technology: the NanoClick assay.
Continue reading “Permeability Possibilities with the New NanoClick Assay”This blog was written in collaboration with our partners at Promega GmbH.
Scientists are comfortable speaking to people who know their field. Speaking to scientists outside of their field of expertise can become a little more challenging, and many find the greatest challenge of all is speaking to people who do not have a science background and are hearing about a scientific concept for the first time, such as journalists in the popular media. What can scientists and journalists do to make the most of the interface of science and journalism?
The importance of the interface between science and journalism is increasingly visible with scientific topics appearing on the national news more frequently due to COVID-19, climate change, and diseases like cancer. So, where can journalists go to learn best practices for interviewing scientists and writing about scientific topics? Promega GmbH offers a platform in which scientists and journalists come together and learn from each other in a constructive exchange. In this workshop setting, scientists speak about a certain topic, and journalists from all kinds of backgrounds can ask questions. When the journalist authors an article about what they learned in that workshop, both sides benefit. The scientists’ work becomes visible, and society learns more about scientific research and discovery that can help all of us to better understand the world and contribute to a brighter future.
Here we describe several common themes that have emerged from these science journalism workshops that may help you the next time you find yourself trying to explain your research to someone unfamiliar with your field.
Continue reading “Science and Journalism – Opposites or Not So Much?”
The Promega Corporate Responsibility Report captures a variety of stories of how we’ve supported our employees, customers and communities over the past year. For example, in 2020, 735 million samples were tested for SARS-CoV-2 using Promega reagents. We launched a new scholarship to support students from underserved backgrounds, and we completed our three largest solar arrays on our Madison, WI campus. As we look to the future, we recognize that there are always more opportunities to reduce our environmental impact. That’s why we’re setting our most ambitious sustainability goals ever.
Continue reading “2030 Sustainability Goals: Our Most Ambitious Ever”It’s been just over 10 years since the world lost a pioneering immunologist and biochemist, Dr. Jürg Tschopp. He died tragically during a hiking trip in the Swiss Alps on March 22, 2011. A host of academic journals, including Science, Nature and Cell, paid tribute to Dr. Tschopp with obituaries that highlighted his many accomplishments in the fields of apoptosis and immunology.
In 2002, a team led by Dr. Tschopp at the University of Lausanne, Switzerland, was studying the role of the proinflammatory cytokine interleukin 1 beta (IL-1β). This cytokine is produced in the cytoplasm as an inactive precursor (pro-IL-1β). It is cleaved by caspase-1 to the active form, but the exact process by which caspase-1 itself is activated was unknown at the time. Several members of the caspase family contain a conserved region known as the caspase recruitment domain or CARD, and it was proposed that this domain was essential to caspase activation.
Based on similarity to another protein containing an N-terminal CARD motif (Apaf-1) that is involved in activation of caspase-9, the researchers examined the roles of a family of proteins known as NALP1, NALP2 and NALP3 (1). In particular, they were interested in NALP1, which is involved in the immune response. Unlike Apaf-1, NALP1 contains a CARD motif at the C terminus, while the N terminus contains a related motif known as a pyrin-like domain (PYD). The research team had previously showed that the PYD region of NALP1 interacted with an adapter protein known as PYCARD or ASC, which also contains an N-terminal PYD and C-terminal CARD.
The results of the team’s in vitro binding, activation and immunodetection studies showed that a multi-unit protein complex is responsible for caspase activation, and they called this complex the “inflammasome” (1). It is composed of caspase-1, caspase-5, PYCARD/ASC and NALP1.
Continue reading “The NLRP3 Inflammasome: Flipping the Switch”The Brazilian state of Amazonas experienced two distinct waves of COVID-19 infections in 2020. After the first wave, a team from the University of Sao Paolo projected that the city of Manaus would reach the theoretical threshold for herd immunity by the end of the summer. However, a second COVID-19 wave erupted in December 2020, coinciding with the rise of Variant of Concern (VOC) P.1.
New research published in Nature Medicine examined the different lineages of COVID-19 present in Brazil over time and determined that the two waves were driven by different variants. The first wave was driven by the variant B.1.195, which was imported from Europe in the spring. The second wave was largely driven by VOC P.1. The Nature Medicine study is the first to use viral sequences from samples collected throughout 2020 to explore the epidemiological and virological factors behind the two distinct COVID-19 waves.
The researchers started by generating whole-genome sequences of 250 SARS-CoV-2 samples collected between March 2020 and January 2021. The survey showed that 20% of the sequences belonged to the B.1.195 lineage, and these mostly corresponded with the first exponential growth phase. 24% of the samples belonged to the P.1 lineage, and all of these samples corresponded with the rise of the second exponential growth phase. The largest share belonged to B.1.1.28 (37%), which replaced B.1.195 as the dominant variant in Brazil shortly after the first wave until the rise of VOC P.1.
The team also used real-time RT-PCR to analyze 1,232 positive samples collected in Amazonas between November 1, 2020 and January 21, 2021. The assay was designed to detect a deletion in NSP6, which is a signature mutation of VOC P.1. None of the samples collected before December 16 showed the NSP6 deletion, but it was common in samples starting in mid-December. Combining the two analysis methods, the team found the P.1 lineage in 0% of samples collected in November 2020, but by January 1-15 it was present in 73.8% of samples.
This data supports the theory that VOC P.1 first emerged in December 2020 and was the dominant lineage driving the second wave in Amazonas.
In addition to tracking the prevalence of lineages throughout the pandemic, the researchers also offered suggestions for how Amazonas experienced two distinct waves of COVID-19 infections.
Using computer modeling, the team found a significant reduction in reproductive efficiency (Re) of lineages B.1.195 and B.1.1.28 in April-May 2020, around the same time that Amazonas increased social distancing measures. Transmission rates remained low until the interventions were relaxed in September 2020. This suggests that the reduction in cases was not a result of herd immunity. Instead, nonpharmaceutical interventions (NPI) limited the first wave and contained the spread through the summer.
Using real-time RT-PCR, the researchers found that the viral load of P.1 infections was nearly ten times the viral load of non-P.1 infection. They also referenced other research that found that VOC P.1 has a stronger affinity for the human receptor ACE2 than B.1.195 and B.1.1.28. P.1 is clearly a highly transmissible VOC, and it evolved in an ideal environment for rapid spread. Amazonas had relaxed social distancing measures by late 2020, P.1 was able to quickly reach extremely high infection rates.
The study did not directly address theories that P.1 evades immunity developed from prior infections, but they concluded that a combination of epidemiological and virological factors allowed P.1 to drive a second wave of COVID-19 in Amazonas starting in December.
The paper includes a supplementary note suggesting that NPIs instituted in Manaus in January 2021 significantly reduced transmission rates of VOC P.1. The team ends the paper by reiterating the importance of adequate social distancing measures to limit the spread of COVID-19 and prevent the emergence of new Variants of Concern.
Read the entire paper here.
This study used the Maxwell® RSC Viral Total Nucleic Acid Purification Kit to extract viral RNA from samples. Learn more about the kit and its uses during the COVID-19 pandemic here.
