In the Literature

Blogs about published peer-reviewed scientific studies.

Differentiating but not Mature Adipocytes Provide a Defense Against S. aureus Infection

Posted on by Kari Kenefick
http://upload.wikimedia.org/wikipedia/commons/6/65/Blausen_0012_AdiposeTissue.png
Cross-section of skin and adipose tissue enlargement. Used courtesy of Wikimedia Commons, Blausen.

A basic tenet of immunology is that antibodies produced by B cells are very important and specific immunoprotective agents, released in response to infection.

However, antibodies do not supply immediate protection. The invading organism needs to get into the host, meet up with T cells and then B cells, in order for antibody production to occur. If the host has seen this particular pathogen previously, the antibody response occurs somewhat more quickly, but we’re still talking about days. If the invading organism is a bacterium, it can multiply and double in numbers in just hours. Thus an infection could potentially gain a foothold in a body prior to an antibody response.

Fortunately we have a more rapid, first line of defense to invading pathogens, a cellular response. In the case of a puncture or skin wound, epithelial cells, mast cells and leukocytes are activated quickly in response to pathogens. Neutrophils and monocytes also aid the cellular response.

Now a recently published report demonstrates that fat cells also play a part in the cellular response to invading bacteria. R. Gallo et al. published a study on Jan. 2 in Science, providing more in depth information on the role of adipocytes in the host response to the bacterium Staphylococcus aureus (S. aureus). Continue reading “Differentiating but not Mature Adipocytes Provide a Defense Against S. aureus Infection”

Using Laser Treatment to Eliminate Blood-Borne Pathogens

Posted on by Sara Klink

One unit of fresh frozen plasma By DiverDave (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons
Keeping our blood supply safe is increasingly difficult in the face of newly discovered pathogens that can be transmitted via blood. The tests developed to ensure the blood is free of pathogens like HIV and HBV are based on known pathogens and available screening tests. What about an emerging virus? How can we ensure our blood is safe in the face of the virus we know nothing about? The PLOS ONE article by Tsen et al. explored how a nonchemical treatment using ultrashort pulsed lasers might be used to eliminate viruses from blood plasma.

The authors used ultrashort pulsed (USP) lasers in their research as this treatment is known to inactivate a spectrum of bacteria and viruses including nonenveloped viruses, a class of virus that resists inactivation. Furthermore, the laser treatment is nonionizing and does not modify proteins covalently, meaning that proteins present in blood are likely to remain active even after exposure to USP lasers. The viruses that were tested for inactivation by USP laser in human plasma were an enveloped RNA virus human immunodeficiency virus (HIV), nonenveloped RNA virus hepatitis A virus (HAV) and enveloped DNA virus murine cytomegalovirus (MCMV). Continue reading “Using Laser Treatment to Eliminate Blood-Borne Pathogens”

When DNA Is Not Enough: New Research Suggests Epigenetic Factors Play an Important Role in the High Mortality Rate of the Devi Facial Tumor Disease

Posted on by Kelly Grooms

tazThe Devil Facial Tumor Disease (DFTD) is a contagious cancer in Tasmanian Devils that is threatening the species with extinction. This disease is spread from individual to individual and has a 100% mortality rate. It is so deadly because, although the DFTF cells should be attached and killed by the host devil’s immune system, for some reason they are not—and no one is sure why. A study published in PNAS in March of last year (1) showed that DFTD cells don’t express surface MHC molecules. MHC class I and class II molecules are crucial for proper immune response, and their absence on the cell surface could explain why the DFTD cells do not stimulate an immune response.

The authors found that the loss of MHC expression is maintained as the cells divide, and is not a result of structural mutations in the genes responsible for MHC expression. Instead the authors found that this down regulation was the result of regulatory changes including epigenetic modifications to histones. Continue reading “When DNA Is Not Enough: New Research Suggests Epigenetic Factors Play an Important Role in the High Mortality Rate of the Devi Facial Tumor Disease”

Mass Spectrometry Application: Antibody Quantitation for Preclinical PK studies

Posted on by Gary Kobs

Isoform_Antibodies_LinkedInTherapeutic monoclonal antibodies (mAbs) represent the majority of therapeutics biologics now on the market, with more than 20 mAbs approved as drugs (1–3). During preclinical development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and  primates. Ligand-binding assays (LBA) are the standard technology used to perform the PK studies for mAb candidates (4). Ligand-binding assays (LBAs) are methods used  to detect and measure a macromolecular interaction between a ligand and a binding molecule. In LBAs, a therapeutic monoclonal antibody is considered to be the ligand, or analyte of interest, while the binding molecule is usually a target protein.

LBAs have certain well-documented limitations (5). Specific assay reagents are often not available early in a program. Interferences from endogenous proteins, antidrug antibodies, and soluble target ligands are potential complicating factors.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS)-based methods represent a viable and complementary addition to LBA for mAb quantification in biological matrixes. LC–MS/MS provides specificity, sensitivity, and multiplexing capability.

A recent reference (6) illustrates an automated method to perform LC–MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard,and anti-human Fc enrichment. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. The interesting finding of the difference between ELISA and LC–MRM-MS data indicated that those two methods can provide complementary information regarding the drug’s PK profile.

Literature Cited

  1. Mao, T. et al. (2013) Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry. Anal.Chem. 85, 4239–46.
  2. Weiner, L. M. et al. (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–27.
  3. Nelson, A. et al. (2010) Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discovery. 9, 767–74.
  4. DeSilva, B. et al. (2003) Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of Macromolecules. Pharm. Res. 20, 1885–00.
  5. Ezan, E.et al. (2009) Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 1, 1375–88.
  6. Zhang, Q. et al. (2014) Generic Automated Method for Liquid Chromatography–Multiple Reaction Monitoring Mass Spectrometry Based Monoclonal Antibody Quantitation for Preclinical Pharmacokinetic Studies. Anal.Chem. 86, 8776–84.

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?”

DNA Amplification Using Body Heat, No Instrument Required

Posted on by Sara Klink

Cartoon by Ed Himelblau
Cartoon by Ed Himelblau Copyright Ed Himelblau.
When I was in the lab, there was more than a few times I held tubes in my hand (maybe even under my arm) to make them thaw faster, especially reaction buffers. However, I never considered whether this could be a strategy for actual incubation although humans run at about 37°C and many restriction enzyme reactions proceed most efficiently at 37°C. But research published in PLOS ONE by Crannell, Rohrman and Richards-Kortum took this idea and decided to experiment with the possibility of eliminating an instrument-based DNA amplification. Continue reading “DNA Amplification Using Body Heat, No Instrument Required”

Insights into the Function of P7C3 Compounds in Neuroprotection

Posted on by Kari Kenefick
The multiple Lombardi trophies won by Pittsburgh Steelers. Image used under Wikimedia Creative Commons, and attributed to daveynin.
The multiple Lombardi trophies won by Pittsburgh Steelers. Image used under Wikimedia Creative Commons, and attributed to daveynin.

It is fall and the season for American football. For this football fan, watching the game is a bit less enjoyable than it used to be, as more and more information is available about the serious and permanent brain injuries suffered by football players.

In the introduction to a recent paper in the journal Cell, “P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage”, not a word about American football is mentioned.

However, the paper begins, “No substantive therapeutics are available for the treatment of almost any form of disease entailing nerve death” (1). The authors list a range of neurodegenerative disorders such as Huntington’s, Alzheimers and Parkinson’s diseases, as well as ALS  or Lou Gherig’s disease. They also note that there are currently no effective treatments for trauma to the brain or peripheral nervous system.

The authors note that a chemical treatment that could interfere with nerve cell death would have a “transformative impact in modern medicine”. Continue reading “Insights into the Function of P7C3 Compounds in Neuroprotection”

Telepathy and Mind Control: From Science Fiction Movies to Reality?

Posted on by Terri Sundquist

BrainInterfaceThere is something very futuristic, and perhaps scary, about the idea of nonverbally transferring one person’s thoughts to another person, especially for the purpose of controlling or influencing a person’s actions and behaviors. Maybe that’s why telepathy and mind control are favorite topics of many science fiction movies. However, there are times when direct, nonverbal transfer of thoughts would be advantageous, for example when communicating complex concepts or feelings that are difficult to convey. Direct transfer also would circumvent the need to translate information from one language to another. For these reasons, scientists are currently developing technologies to allow such thought transfers. A recent PLOS ONE article describes a simple brain-to-brain interface in humans and shows how this interface can be used to capture a thought generated by one person and communicate that information directly to the brain of a second person and elicit a physical response (1).

Continue reading “Telepathy and Mind Control: From Science Fiction Movies to Reality?”

Genome Editing and Reporter Technologies Enable Endogenous Pathway High-Throughput Assays

Posted on by Promega

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;
ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;


This article review was written by guest author, Amy Landreman, in the Cellular Analysis and Proteomics Group at Promega.

Charcot-Marie Tooth (CMT) disease is one of the most common inherited neurological disorders affecting approximately 2.8 million people worldwide. The most common form of CMT, CMT Type 1A, is caused by a 1.5Mb genomic duplication on Chr17 that results in trisomy of the critical myelin gene Peripheral Myelin Protein 22 (Pmp22). The extra copy of Pmp22 results in excessive PMP22 protein causing the neurophathy associated with CMT type 1A. Although there is no way to remove the extra copy of the gene, even subtle decreases in Pmp22 expression have shown promise against this inherited neuropathy in laboratory models.

In a recent paper, Inglese et al. 2014, describe an interesting new approach used to identify compounds that effectively decrease Pmp22 expression using a novel gene editing strategy and reporter-based screen. Their challenge was to create an assay that accurately represented endogenous Pmp22 expression including both transcriptional and post-transcriptional regulatory mechanisms, while maintaining the sensitivity required to detect subtle changes in expression in a loss of signal assay in a format compatible with microtiter 1536-well quantitative high-throughput screening (qHTS). Continue reading “Genome Editing and Reporter Technologies Enable Endogenous Pathway High-Throughput Assays”

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”