Author: Ken Doyle

The NLRP3 Inflammasome: Flipping the Switch

Posted on by Ken Doyle

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.

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RAS-Targeted Drug Discovery: From Challenge to Opportunity

Posted on by Ken Doyle
cancer cell, ras-targeted drug discovery

In 1963, Jennifer Harvey was studying Moloney murine leukemia virus (MMLV) at the cancer research department of the London Hospital Research Laboratories. After routine transfers of plasma from MMLV-infected rats to mice, she made an unusual discovery. In addition to the expected leukemia, the mice that received the plasma developed solid tumors (soft-tissue sarcomas), primarily in the spleen (1). A few years later, Werner Kirsten at the University of Chicago observed similar results working with mouse erythroblastosis virus (MEV) (2).

Subsequent research, with the advent of genome sequencing, showed that a cellular rat gene had been incorporated into the viral genome in both cases (3). These genomic sequences contained a mutation later shown to be responsible for the development of sarcomas, and the word “oncogene” became a common part of the vocabulary in cancer publications during the early 1980s (4). Harvey’s discovery led to the naming of the corresponding rat sarcoma oncogene as HRAS, while Kirsten’s related oncogene was named KRAS. Several laboratories, working independently, cloned the human homolog of the viral HRAS gene in 1982 (3). The human KRAS gene was cloned shortly thereafter, as well as a third RAS gene, named NRAS (3). Additional studies showed that a single point mutation in each of these genes led to oncogenic activation, and they have been popular targets for anticancer drug discovery efforts ever since.

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COVID-19 Therapies: Are We There Yet?

Posted on by Ken Doyle

A year after COVID-19 was declared a pandemic, collaborative efforts among pharma/biotech and academic researchers have led to remarkable progress in vaccine development. These efforts include novel mRNA vaccine technology, as well as more conventional approaches using adenoviral vectors. While vaccine deployment understandably has captured the spotlight in the fight against COVID-19, there remains an urgent need to develop therapeutic agents directed against SARS-CoV-2.

COVID-19 therapeutic drugs

In the March 12 issue of Science, an editorial by Dr. Francis Collins, director of the U.S. National Institutes of Health (NIH), examines lessons learned over the past 12 months (1). Collins points out that many clinical trials of potential therapeutics were not designed to suit a public health emergency. Some were poorly designed or underpowered, yet they received considerable publicity—as was the case with hydroxychloroquine. Collins advises developing antiviral agents targeted at all major known classes of pathogens, to head off the next potential pandemic before it becomes one. A news feature in the same issue discusses the current state of coronavirus drug development (2).

The present crop of drug candidates is remarkably diverse, including repurposed drugs that were originally developed to treat diseases quite different from COVID-19. Typically, however, the mainstream candidates belong to two broad classes: small-molecule antiviral agents and large-molecule monoclonal antibodies (mAbs).

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Fighting Plant Pathogens Worldwide with the Maxwell® RSC PureFood GMO and Authentication Kit

Posted on by Ken Doyle

Among the one trillion or so species that share space on our planet, complex relationships have emerged over time. Such relationships, in which two or more species closely interact, are collectively termed symbiosis. Although it’s commonly assumed that symbiotic relationships are mutually beneficial, this example constitutes only one type of symbiosis (known as mutualism). The traditional predator-prey relationship, clearly a one-sided arrangement, is also an example of symbiosis.

Olive trees in Italy are being affected by the plant pathogen Xylella fastidiosa

The sheer diversity of microbial species has led to the development of many well-characterized relationships with plants and animals. Perhaps the best-known example of mutualism in this context is the process of nitrogen fixation. In this process, various types of bacteria that live in water, soil or root nodules convert atmospheric nitrogen into forms that are readily used by plants. On the other hand, some types of bacteria-plant relationships are parasitic: the bacteria rely on the plant for survival but end up damaging their host. Parasitic relationships can have devastating ecological and economic consequences when they affect food crops.

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Intranasal COVID-19 Vaccines: What the Nose Knows

Posted on by Ken Doyle

COVID-19 vaccine distribution efforts are underway in several countries. Recently, the Serum Institute of India celebrated the nationwide rollout of its Covishield vaccine, kicking off the country’s largest ever vaccination program. Meanwhile, many other vaccines against the coronavirus that causes COVID-19 are in either preclinical studies or clinical trials. At present, 19 vaccine candidates are in Phase 3 clinical trials, while 8 vaccines have been granted emergency use authorization (EUA) in at least one country.

intranasal covid-19 vaccine coronavirus

In the US, mRNA vaccines from Pfizer/BioNTech and Moderna are in distribution. Adenoviral vector vaccines authorized for distribution include Oxford/AstraZeneca AZD1222 in the UK (Covishield in India) and Gamaleya Sputnik V in Russia. A third type of vaccine consists of inactivated coronavirus particles, such as those developed by Sinopharm and Sinovac in China.

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Adenoviral Vector Vaccines for COVID-19: A New Hope?

Posted on by Ken Doyle

The global war against the coronavirus that causes COVID-19 rages on, spearheaded by efforts to develop effective and safe vaccines. At the time of writing, over 100 COVID-19 vaccine clinical trials were listed in the clinicaltrials.gov database. Recent attention has focused on mRNA vaccines developed by Pfizer/BioNTech and Moderna. If licensed, they would become the first mRNA vaccines for human use.

Other vaccine development efforts are relying on more conventional techniques—using an adenoviral vector to deliver a DNA molecule that encodes the SARS-CoV-2 spike (S) protein. Examples of these adenoviral vector vaccines include the vaccines from Oxford University/AstraZeneca (the UK), Cansino Biologics (China), Sputnik V (Russia) and Janssen Pharmaceuticals/Johnson & Johnson (the Netherlands and USA).

sars-cov-2 coronavirus covid-19 infection with antibodies from a vaccine attacking the virus; several vaccines are in development including adenoviral vector vaccines
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mRNA Vaccines for COVID-19: The Promise and Pitfalls

Posted on by Ken Doyle

Updated 8/25/2021, 4/29/2024

Multiple battles are being fought in the war against the SARS-CoV-2 coronavirus that causes COVID-19. Currently, there are nearly 5,000 clinical trials listed in the World Health Organization (WHO) database, either underway or in the recruiting stage, for vaccines and antiviral drugs. The Moderna mRNA vaccine and Janssen vaccine received emergency use authorization (EUA) from the Food and Drug Administration (FDA) and have since been fully approved; the Pfizer-BioNTech Vaccine (marketed as Comiraty) received FDA approval in August 2021.

mrna vaccines and coronavirus covid-19

Both the Moderna vaccine and Comiraty are mRNA-based, as opposed to most conventional vaccines against established diseases that are protein-based. Typically, the key ingredient in viral vaccines is either part of an inactivated virus, or one or more expressed proteins (antigens) that are a part of the virus. These protein antigens are responsible for eliciting an immune response that will fight future infection by the actual virus. Another approach is to use a replication-deficient viral vector (such as adenovirus) to deliver the gene encoding the antigen into human cells. This method was used for the coronavirus vaccine developed by Oxford University in collaboration with AstraZeneca; phase 3 interim data were announced on the heels of the Pfizer/BioNTech and Moderna announcements. All three vaccines target the SARS-CoV-2 spike protein, because it is the key that unlocks a path of entry into the host cell.

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How Does Human Papillomavirus (HPV) Infection Drive the Progression of Cervical Cancer?

Posted on by Ken Doyle

Cervical cancer is a major health problem for women, and it is currently the fourth most common cancer in women globally (1). A worldwide analysis of cancer estimates from the Global Cancer Observatory 2018 database showed that cervical cancer disproportionally affects lower-resource countries, on the basis of their Human Development Index; it was the leading cause of cancer-related death in women in many African countries (1).

Global cervical cancer incidence 2018
Estimated cervical cancer global incidence rates from the GLOBOCAN 2018 database; image generated using IARC (http://go.iarc.fr/today).

Infection by human papillomavirus (HPV), a double-stranded DNA virus, is the leading cause of cervical cancer. Many types of HPV have been identified, and at least 14 high-risk HPV types are cancer-causing, according to a World Health Organization (WHO) fact sheet. Of these types, HPV-16 and HPV-18 are responsible for 70% of cervical cancers and pre-cancerous cervical lesions. HPV infection is sexually transmitted, most commonly by skin-to-skin genital contact. Although the majority of HPV infections are benign and resolve within a year or two, persistent infection in women, together with other risk factors, can lead to the development of cervical cancer [reviewed in (2)].

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NanoLuc® Luciferase: Brighter Days Ahead for In Vivo Imaging

Posted on by Ken Doyle
nanoluc in vivo imaging

The development of NanoLuc® luciferase technology has provided researchers with new and better tools to study endogenous biology: how proteins behave in their native environments within cells and tissues. This small (~19kDa) luciferase enzyme, derived from the deep-sea shrimp Oplophorus gracilirostris, offers several advantages over firefly or Renilla luciferase. For an overview of NanoLuc® luciferase applications, see: NanoLuc® Luciferase Powers More than Reporter Assays.

The small size of NanoLuc® luciferase, as well the lack of a requirement for ATP to generate a bioluminescent signal, make it particularly attractive as a reporter for in vivo bioluminescent imaging, both in cells and live animals. Expression of a small reporter molecule as a fusion protein is less likely to interfere with the biological function of the target protein. NanoLuc® Binary Technology (NanoBiT®) takes this approach a step further by creating a complementation reporter system where one subunit is just 11 amino acids in length. This video explains how the high-affinity version of NanoBiT® complementation (HiBiT) works:

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Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases

Posted on by Ken Doyle
bet proteins brd nanoluc

Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.

One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).

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