Understanding Knowledge Transfer at the Doctoral Level – One Pathway to Better Career Development – An Introduction

Posted on by Amy Prevost, PhD
Few studies have been conducted about knowledge transfer at the PhD level.
Few studies have been conducted about knowledge transfer at the PhD level.

Though my work is primarily in the area of biotechnology, my doctoral degree is in education. Over my next few posts, I thought that I would share some of the findings that came out of research I conducted for my dissertation.  It’s been over two years since graduation and much of this is still not published, though on my honor it’s in the works…I focused my study on understanding knowledge transfer at the doctoral level.  I was able to complete this work due to the generosity of many doctoral students, recent graduates and mentors across the country. This research was not funded in any way, but I believe it is of great importance given the socio-political context we are all functioning in as learners and professionals.  Also, many studies that are similar to the one I did are funded, with a great deal of money I might add. So, without further ado, let me start by introducing the concept of knowledge transfer and why developing knowledge transfer skills are important.

“Knowledge transfer” is a concept grounded in K-12 education and in a general sense refers to an individual’s ability to use knowledge gained in one context in another (similar or dissimilar) context. Continue reading “Understanding Knowledge Transfer at the Doctoral Level – One Pathway to Better Career Development – An Introduction”

Choosing the Right Cell Health Assay

Posted on by Promega
artists view inside a cell

Based on the Illuminations article by Dr. Terry Riss, from our Cellular Analysis group.

Choosing the most appropriate cell health assay for your experiment can be difficult.  There are several factors to consider when choosing an assay: the question you are asking, the nature of your sample, the number of samples being tested, the required sensitivity, the nature of the sample, the plates and plate readers and the reagent costs.

What question are you asking?

The first, and perhaps most important factor to consider, is the question you need answered. What do you want to know at the end of the experiment? There are cell health assays available that specifically detect the number of living cells, the number of dead cells, and for assessing stress response mechanisms or pathways that may lead to cell death. Matching the assay endpoint to the information you need is vital to choosing the appropriate cell health assay.

Continue reading “Choosing the Right Cell Health Assay”

Why We Care About Glycosyltransferases

Posted on by Promega

Today’s post is a guest blog from Michael Curtin in the cellular analysis and proteomics group at Promega.

Glycobiology is the study of carbohydrates and their role in biology. Glycans, defined as “compounds consisting of a large number of monosaccharides linked glycosidically” are present in all living cells and coat cell membranes and are integral components of cell walls (1). They play diverse roles, including critical functions in cell signaling, molecular recognition, immunity and inflammation. They are the cell-surface molecules that define the ABO blood groups and must be taken into consideration to ensure successful blood transfusions. (2).The process by which a sugar moiety is attached to a biological compound is referred to as glycosylation. Protein glycosylation is a form of post-translational modification, which is important for many biological processes and often serves as an analog switch that modulates protein activity.The class of enzymes responsible for transferring the sugar moiety onto proteins is called a glycosyltransferase (GT).

GTs can be divided into three major types based on their roles:

  • Oligosaccharide elongation for peptidoglycan biosynthesis
  • Regulation of protein activities by post-translational modification
  • Small molecule glucuronidation as means of drug metabolism

Continue reading “Why We Care About Glycosyltransferases”

Lessons From the ‘Long Goodbye’

Posted on by Ben Schmidt
Lewy Body stained with alpha-synuclein.
Lewy Body stained with alpha-synuclein.

A week ago Sunday, I walked among crowds of mothers, grandmothers, and children of all ages celebrating Mother’s Day at the Botanical Gardens in St. Louis, Missouri.  As I watched happy families, I couldn’t help being jealous.  Though I was there with my grandmother and other close relatives, I missed my mom, especially since I was in my hometown for her funeral the day before.  Had my mom been alive and well, we might have walked those same paths ourselves and enjoyed the new life teeming above the earth.  Instead, my mother lost her battle of more than six years with Lewy Body dementia the week before at the age of 61.

As a biologist, I was well-aware of Alzheimer disease in the abstract, and tau proteins, beta-amyloid, and genetic predisposition.  But until my mom was diagnosed in 2008, I was painfully ignorant of dementias other than Alzheimer disease.  Once we knew what mom was fighting, I learned that Alzheimer disease and Lewy Body are hardly unique.  The number of other dementias that exist is long and includes vascular dementia, mixed dementia, Parkinson’s disease, frontotemporal dementia, Creutzfeldt-Jakob disease, Huntington disease, and many others.[1]

Continue reading “Lessons From the ‘Long Goodbye’”

His-Tagged Fusion Proteins: Application Update

Posted on by Gary Kobs

Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;
Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;

Researchers often need to purify a single protein for further study. One method for isolating a specific protein is the use of affinity tags. Affinity purification tags can be fused to any recombinant protein of interest, allowing fast and easy purification following a procedure that is based on the affinity properties of the tag.

The most commonly used tag to purify and detect recombinant expressed proteins is the polyhistidine tag. Protein purification using polyhistidine tags relies on the affinity of histidine residues for immobilized metal such as nickel, which allows selective protein purification. The metal is immobilized to a support through complex formation with a chelate that is covalently attached to the support.

Polyhistidine tags offer several advantages for protein purification. The small size of the polyhistidine tag renders it less immunogenic than other larger tags. Therefore, the tag usually does not need to be removed for downstream applications following purification.

A large number of commercial expression vectors that contain polyhistidine are available. The polyhistidine tag may be placed on either the N- or C-terminus of the protein of interest.

And finally, the interaction of the polyhistidine tag with the metal does not depend on the tertiary structure of the tag, making it possible to purify otherwise insoluble proteins using denaturing conditions. The resulting purified protein can be used for a variety of applications.

The following references illustrate examples of some of the most common post purification applications with fusion proteins containing a polyhistidine tag:

Enzymatic assays

  1. Negi, V-S. et al. (2014) A carbon nitrogen Lyase from Leucaena leucocephala catalyzes the first Step of mimosine degradation. Plant Physiol. 164,  922–34.
  2. Yu, S. et al. (2013) Syk Inhibits the activity of protein kinase A by phosphorylation tyrosine of the catalytic subunit. J. Biol. Chem. 288, 10870-81.
  3. Rusconi, B. et al. (2013) Discovery of catalases in members of the Chiamydiales order. J. Bact. 195, 3543–51.

Structural analysis

  1. Araiso, Y. et al. (2014) Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB a novel subunit assembly in tRNA –dependent amidotransferases. Nucl.Acids. Res.(available only online).
  2. Someya, T. et al. (2012) Crystal Structure of Hfq from Bacillus subtilis in complex with SELEX-dervived RNA aptamer: insight into RNA-binding properties of bacterial Hfq. Nucl. Acid Res. 40, 1856-67.

Protein pulldowns

  1. Yun, S-C. et al. (2010) Pmr a Histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. J.Bact. 192, 4720–31.
  2. Haim, H. et al. (2010)  Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target. Microbiology 156, 3710-21.

Additional Resources
His-tagged Protein Purification Systems

Plant Biologists Take the Lead on Elucidating Zombie Genetics

Posted on by Michele Arduengo, PhD

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Biology is full of stories that read like a modern day zombie apocalypse. For instance, the parasite Toxoplasma gondii has been in the news for its ability to infect the brains of rats, and reprogram their normal behavioral responses such that they lose their innate fear of cats. Previously, we reviewed the research about the parasitic fungus that infects ants, causing drastic changes in typical ant behavior to aid in distribution of the fungal spores.

In April of this year, MacLean and colleagues published research in PLOS Biology describing interactions between a phytoplasma parasite and Arabidopsis thaliana. What is nice about this particular “zombie” biology story is that the researchers present the beginnings of the genetics that underlie the plant-parasite-insect relationship, moving beyond a description of the phenotypic changes that occur to describing an actual mechanism for those changes.

Continue reading “Plant Biologists Take the Lead on Elucidating Zombie Genetics”

Grays On the Move: Whale Watching in San Diego

Posted on by Robert Deyes

As a boy, one of my favorite childhood books was without a doubt, Herman Melville’s Moby Dick. For those not familiar with the story, it tells of the obsessive quest of one Captain Ahab to kill a white whale in revenge for an attack that left him with a severed leg. The story is told by the character Ishamel who accompanies Ahab and provides the reader with a front row seat on the doomed saga of Ahab and his crew. After reading the book, I set myself the task of learning more about whales and how we can do more to protect the lives of these magnificent ‘leviathans of the deep’. My parents bought me Jacques Cousteau’s Whales one Christmas. From that moment on, my heart and mind were transfixed. This year I was privileged to see Gray whales for the first time, following their migratory path down the west coast of the United States. Together with a handful of other excited tourists, I went on a 3 hour cruise outside of San Diego bay, organized by the Scripps Institute Birch Aquarium. Below are several of the many pictures I took on that memorable day.

All Aboard Skipper!
All Aboard Skipper!

Continue reading “Grays On the Move: Whale Watching in San Diego”

Biology of Overeating and the Weight-Gain Cycle

Posted on by Anupama Gopalakrishnan

ScaleA person needs to browse through any health related journal, magazine or website to find new and novel ways to reduce weight. While the options range from bariatric surgery to good old “eat-less-exercise-more” concepts, it is intriguing how the more weight a person gains, the harder it is to shed the extra calories. Losing weight is an uphill battle for majority of us. And that got me thinking about how much our biology cooperates while we try to lose weight. I came across these two elegant studies that explain why this is indeed an uphill battle. Continue reading “Biology of Overeating and the Weight-Gain Cycle”

CheckMate™ Mammalian Two-Hybrid System: Application Update

Posted on by Gary Kobs

Assay principle for CheckMate™ Mammalian Two-Hybrid System.
Assay principle.

In the CheckMate™ Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA-binding domain upstream of a multiple cloning region, and the pACT Vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The two genes encoding the two potentially interactive proteins of interest are cloned into pBIND and pACT Vectors to generate fusion proteins with the DNA-binding domain of GAL4 and the activation domain of VP16, respectively. The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn, is upstream of the firefly luciferase gene (luc+). The pGAL4 and pVP16 fusion constructs are transfected along with pG5luc Vector into mammalian cells. Interaction between the two test proteins, as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression over the negative controls. Traditionally mammalian two hybrid analysis was used to confirm initial data obtained from yeast two hybrid experiments.

Due to enhanced bioinformatics information and the development of improved co-immunoprecipiation/pulldown procedures/technology, there is a growing trend to use only mammalian cells to characterize protein:protein interactions. The following references illustrate the use of the CheckMate™ system to complement  other techniques to characterize protein;protein interactions using only mammalian cells .

Bagchi, P. et al. (2013) Molecular Mechanism behind Rotavirus Nsp-1 Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85Subunit of PI3 kinase. J. Vir. 87, 2358-62.

Greninger, A. et al. (2013) ACBD3 Interaction with TBC1 domain 22 protein is differentially affected by Enteroviral and Kobuviral 3A protein binding.  mBio 4, 00098-13.

Patki, M. et al. ( 2013) The ETS Domain Transcription Factor ELK1 Direct a critical component of growth signalling by Androgen Receptor in prostrate cancer cells. J.Biol. Chem. 288 11047-65

Konig, H-G. (2012) Fibroblast growth factor homologous factor 1 interact with NEMO to regulate NF-kappaB signaling in neurons J. Cell Sci. 125, 6058-70

Can Fruit Flies Glow in the Dark?

Posted on by Sara Klink

Fruit fly. Image from morguefile.
Question: How is a fruit fly like a firefly? No, this is not an obvious answer (their names start with the letter “f”) or the beginning of a bad entomology joke. These two organisms may both be winged insects, but as it turns out, what makes the firefly light show such a special treat on summer evenings is something that fruit flies, the bane of the kitchen in the summertime and annoyance for labs near Drosophila researchers, can mimic with a little help from a synthetic luciferin substrate as reported in PNAS. Continue reading “Can Fruit Flies Glow in the Dark?”