Author: Isobel Maciver

NanoBiT Assay Applied to Study Role of SOD1 in ALS

Posted on by Isobel Maciver

NanoBiT Protein ComplementationBack in 2015 the Ice Bucket Challenge brought Amyotrophic Lateral Sclerosis (ALS) to public attention, initiating worldwide pleas for more funding of research toward a cure for this fatal disease, which is characterized by progressive degeneration of motor neurons. In spite of many efforts over the last few decades, the precise cause of ALS is still unknown.

The complexity of the problem of ALS pathogenesis is highlighted in the review “Decoding ALS: from genes to mechanism”  published in Nature in November 2016. The review highlights a long list of genetic factors implicated in ALS, grouping them into genes affecting protein quality control, RNA stability/function, and the cytoskeletal structure of neuronal cells.

Mutations in the antioxidant enzyme superoxide dismutase (SOD1) were the first to be associated with ALS. According to the review, more than 170 SOD1 mutations causing ALS have since been identified. Many of these mutations are thought to result in misfolding of SOD1, contributing to toxicity when the misfolded protein accumulates within the cell.

A paper by Oh-hashi et al., published in Cell Biochemistry and Function in October 2016 used the NanoBiT protein complementation assay to investigate the effect of two common ALS-associated SOD1 mutations on dimerization of the SOD1 protein. Continue reading “NanoBiT Assay Applied to Study Role of SOD1 in ALS”

Magnetic Bacteria Carry Drugs into Tumors

Posted on by Isobel Maciver
cancer cell

At first glance, the biology of magnetic, underwater-dwelling, oxygen-averse bacteria may seem of little relevance to our most pressing human health problems. But science is full of surprises. A paper published in Nature Nanotechnology presents an inspired use of these bacteria to deliver anti-cancer drugs to tumors, specifically targeting the oxygen-starved regions generated by aggressively proliferating cells.

Continue reading “Magnetic Bacteria Carry Drugs into Tumors”

Previewing ISHI 27: Mitochondrial DNA Analysis in Forensic Investigations

Posted on by Isobel Maciver
Credit: National Institutes of Heath, USA
Mitochondrial DNA has important implications for forensic analysis. Image Credit: National Institutes of Heath, USA

mtDNA heteroplasmy was key to identifying the remains of Tsar Nicholas II
mtDNA heteroplasmy was key to the identification of the remains of Tsar Nicholas II

Heteroplasmy is the presence of more than one mitochondrial genome within an individual. Perhaps the most famous example of the effect of mtDNA heteroplasmy on a forensic investigation is the identification of the remains of Tsar Nicholas II. mtDNA from bones discovered in a mass grave in 1991, was identical in sequence to known relatives of the Tsar except at one position, where there was a mixture of matching (T) and mismatching (C) bases. Lingering doubt caused by this result meant that confirmation of the authenticity of the remains was delayed. Ultimately mtDNA analysis provided the needed evidence for identification, showing that the same heteroplasmy was present in mtDNA extracted from bones of the Tsar’s brother, confirming the Tsar’s identity (Ivanov et al., (1996) Nature Genetics 12(4), 417-20).

Here is what Dr. Holland had to say about the work he will present at ISHI:

Continue reading “Previewing ISHI 27: Mitochondrial DNA Analysis in Forensic Investigations”

Zika Virus: Another RNA Virus Emerges

Posted on by Isobel Maciver
no mosquito

Zika virus has been in the news recently due to growing concerns about its global spread. If you have never heard of Zika virus before, you are not alone. Although first discovered in the 1940s, Zika has not been the subject of much study as infection is considered rare and the symptoms mild. However, all this has changed in recent months due to the rapid spread of the virus in Latin America, where it has been associated with an increased incidence of microcephaly, a severe birth defect where babies are born with underdeveloped brains. Although the connection of Zika with microcephaly is not yet proven, the circumstantial evidence is strong, leading the World Health Organization to declare the spread of Zika virus an international public health emergency earlier this week.

Continue reading “Zika Virus: Another RNA Virus Emerges”

Unexpected connections: Gut bacteria influence immunotherapy outcomes

Posted on by Isobel Maciver

Over the last few years, human microbiome studies have revealed fascinating connections between our colonizing microorganisms and ourselves—including associations between gut bacterial populations and obesity, disease susceptibility, and even mood. The relationship between us and our microbial colonists—once considered completely benign, is now being revealed as an intricate, complicated partnership with the potential to redefine who “we” are in fundamental ways.

Two papers published back-to-back in the November 27 issue of Science add further to this growing body of knowledge—reporting a new and unexpected connection between gut bacterial species and the effectiveness of cancer immunotherapies in mice. The work suggests one reason why such treatments are effective in some circumstances, but not others. Both papers report that the presence of specific bacterial populations may be required for the efficacy of certain treatments, and raise the intriguing question “Could the composition of bacteria in the gut be manipulated to enhance the effectiveness of cancer treatments?”

Continue reading “Unexpected connections: Gut bacteria influence immunotherapy outcomes”

Designer Bacteria Detect Cancer

Posted on by Isobel Maciver

Every day scientists apply creative ideas to solve real-world problems. Every so often a paper comes up that highlights the creativity and elegance of this process in a powerful way. The paper “Programmable probiotics for detection of cancer in urine”, published May 27 in Science Translational Medicine, provides one great example of the application of scientific creativity to develop potential new ways for early detection of cancer.

The paper describes use of an engineered strain of E.coli to detect liver tumors in mice. The authors (Danino et al) developed a potential diagnostic assay that uses a simple oral delivery method and provides a readout from urine, all of which is made possible by some seriously complex and elegant science. Continue reading “Designer Bacteria Detect Cancer”

Choosing Primary and Control Reporters for Dual-Luciferase Assays

Posted on by Isobel Maciver

Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”

This infographic outlines the various NanoDLR dual-reporter assay choices and the situations where you would choose one format over another. Continue reading “Choosing Primary and Control Reporters for Dual-Luciferase Assays”

Culturing the Unculturable Bacteria

Posted on by Isobel Maciver
Culturing bacteria is't always this easy.
Culturing bacteria is’t always this easy.

It is estimated that all the bacterial species so far described represent only a tiny fraction of the total. The rest remain unknown to science because they are “unculturable” in standard (or known) laboratory media. Given that many antibiotics were first isolated from environmental bacteria, it seems likely that these as yet unknown organisms could also be a rich source of potential new drug candidates. The desperate need for new strategies to combat multi-drug resistant infections gives impetus to studies investigating how we can culture some of these “unculturable” bacteria and uncover their potential as a source of much-needed new treatments. 

Continue reading “Culturing the Unculturable Bacteria”

Christensenellaceae—A Natural Way to Stay Thin?

Posted on by Isobel Maciver

microbiome studies show how bacterial colonists influence healthA study published in the Nov 6 issue of Cell outlined results suggesting that an obscure family of bacteria colonizing the human gut may be inherited and may also have a direct influence on body weight. The paper is the first to identify such an association and to link a particular microbial colonist with lower BMI. Continue reading “Christensenellaceae—A Natural Way to Stay Thin?”

Detecting Inhibition of Protein Interactions in vivo

Posted on by Isobel Maciver
Protein Interactions with NanoBRET

In a paper published in the September 2014 issue of ACS Medicinal Chemistry Letters, researchers from GlaxoSmithKline in the UK and Germany report on the discovery, binding mode and structure:activity relationship of a potent BRPF1 (bromodomain and PHD finger containing protein family) inhibitor. This paper came to our attention as it is one of the first publications to apply Promega NanoBRET technology in an vivo assay that reversibly measures the interaction of protein partners. The technology enabled the identification of a novel inhibitor compound that disrupts the chromatin binding of this relatively unstudied class of bromodomain proteins.

What exactly are bromodomains and why do they matter?
Bromodomains are regions (~100 amino acids) within chromatin regulator proteins that recognize and “read” acetylated lysine residues on histones. These acetylated lysines act as docking stations for regulatory protein complexes via binding of the bromodomain region. Because of their role in chromatin binding and gene regulation, bromodomains have attracted interest as potential targets for anti-cancer treatments. Although some bromodomain-containing proteins (e.g., those in the bromodomain and extraterminal domain (BET) subfamily) are well characterized and have been identified as potential therapeutic targets, others are less well understood.

Continue reading “Detecting Inhibition of Protein Interactions in vivo”