Exploring the Land of the Silver Fern: South Island of New Zealand—Part I

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part I of the adventures of Sarah Theos, a client support consultant, who used her award to travel to New Zealand.

Introduction

We went to New Zealand the first two weeks in December and were surprised that there was hardly anyone there. We had long stretches of the road entirely to ourselves and felt like we were the only ones around on many of the hikes. It was a magical feeling to take in the gorgeous scenery without another soul for miles. We, of course, purchased a book that told us about all of the “off the beaten path” hikes and sights that we must do and tried to check them all off the list! The locals were also extremely friendly and eager to chat. If we did come across any locals on the trails, they always stopped to talk and were eager to hear about where we were from and how we like the island so far. It was the beginning of summer so the days were long with the sun rising before 6am and setting around 10pm. We had plenty of daylight hours to explore.

Day 1: Christchurch

Starting in Washington, DC, we traveled over 29 hours door to door to get to Christchurch, NZ. Since we arrived around 7am, we decided to fight off the jetlag and go out to explore the city. I wasn’t quite sure what to expect in Christchurch, knowing that it suffered two devastating back to back earthquakes in 2010 and 2011. My first impression was that there were a lot of gravel parking lots everywhere and boarded up high rises. It looked like a ghost town. We checked into our hotel (built after the earthquakes) and took a walk down to the City Centre. The once beautiful cathedral sat in ruins, surrounded by large buildings in various states of decay. It was a sad sight to see. We then walked further down the street to the botanical gardens. The gardens are beautiful and we spent quite a while admiring the stunning Rose Garden. After exploring the gardens, we decided to drive up to the gondola to explore the city from a different angle.

The gondola takes you up to the top of Port Hills, about 500 meters above sea level. At the top you can see a 360 degree view from the Pacific Ocean and Christchurch to the Southern Alps and the surrounding Canterbury plains. We hiked to the top of Cavendish Bluff to take in the beauty. After the gondola ride, we decided to drive a little ways up the Banks Peninsula to Governor’s Bay where we found a cute little pub. Exhaustion set in around 5pm so we decided to head to a grocery store to stock up on snacks and water in preparation for our drive to the wild, west coast the next day. We passed out around 8pm. Continue reading

High-Throughput Screening for Potential Biomarkers Using Cerebrospinal Fluid (CSF)

3240CA02_1A_rename_3Cerebrospinal fluid (CSF) is a bodily fluid present around the brain and in the spinal cord. It acts as a protective cushion against shocks and participates in the immune response in the brain. Analysis of total CSF protein can be used for diagnostic purposes, as, for instance, a sign of a tumor, bleeding, inflammation, or injury. Considering the high value of CSF as a source of potential biomarkers for brain-associated damages and pathologies, the development of robust automated platform for CSF proteomics is of great value.

The scalable automated proteomic pipeline (ASAP2)  was initially developed with the purpose of (i) discovering protein biomarkers in plasma (1). A summary of the ASAP2 process is as follows:As a first step, abundant-protein immuno-affinity depletion is performed with antibody-based columns and LC systems equipped with a refrigerated autosampler and fraction collector. This block is linked to and followed by buffer exchange performed in a 96-well plate format by manual operations that require <1 h to be completed. The rest of the process is fully automated and includes (i) reduction, alkylation, enzymatic digestion.; (ii) tandem mass tag (TMT) labeling and pooling (processing time of ); (iii) RP solid-phase extraction (SPE) purification ; and (iv) strong cation-exchange (SCX) SPE purification.

A recent reference (2) validated the use of ASAP2 for sample preparation and proteomic analysis of human CSF samples was performed. CSF samples were first depleted from abundant proteins by multiplexed immuno-affinity. Subsequently, reduction, alkylation, protein digestion (using Trypsin/Lys-C), TMT 6-plex labeling, pooling, and sample cleanup were performed in a 96-well-plate format using a liquid-handling robotic platform. Ninety-six  identical CSF samples were prepared using the highly automated ASAP2 procedure. Proteome coverage consistency, quantitative precision, and individual protein variability, were determined. Results indicated that, ASAP2 is efficient in analyzing large numbers of human CSF samples and would be a valuable tool for biomarker discovery.

References

  1. Dayon, L et al. (2014) Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma. J of Proteome Res. 13, 3837–45
  2. Galindo, M-N. et al. (2015) Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery. Anan. Chem. 87, 10755–61

Thank a Tech or Assistant

Today’s #FridayFeeling is one of gratitude for all of those people who do the things that make our lives easier: lab techs, work-study students, undergraduate assistants. They put up with our requests and changes of mind and help keep our laboratory glassware clean, solutions sterile and experiments running. Do you have someone who helps you keep your experiments up and running?eh26

The Reality of DNA Phenotyping

Reality of DNA Phenotyping

It is easy to get excited or frightened about the predictive powers of DNA phenotyping, depending on your perspective. Knowing what genes led to higher intelligence and athletic ability was the first step towards the designer babies of GATTACA. Is this knowledge worth having given the potential for misuse? Going to such extremes with genetic selection makes for a captivating movie, but it can lead to a flawed understanding of the science. The reality of DNA phenotyping is not so scary.

How does DNA phenotyping work?

DNA phenotyping is our attempt at replicating what our bodies do naturally: translating DNA into our physical appearances. It is an attempt because there is rarely a direct correlation between a single gene and a single physical feature. Forensic scientists are currently focusing on determining facial features. Much of our understanding has been gleaned from whole genome studies where scientists compare data from over 7,000 points on participants’ faces to sections of their DNA that contain single nucleotide polymorphisms (SNPs)—that is, sections of DNA that differ by a single letter of the genetic code. Comparing facial maps to genes allows scientists to calculate the probability of physical traits based on the presence of particular SNPs. Predictive algorithms are then used to render an image of a face based on those probabilities.

There is one question that really matters to most people: how well does this all work?

What can DNA phenotyping currently predict?

  • Eye color – 77 genes identified
  • Hair color – 32 genes identified
  • Skin color – 31 genes identified

Dr. Manfred Kayser neatly summarized the specific genes and their corresponding references in a single table from his 2015 paper. These three pigment traits are a good start, but they are a far cry from generating an accurate image of a face. Determining ethnicity is currently accurate at broader levels like European, African and so on. Dr. David Ballard has more to say in this video: Continue reading

How Do Agricultural Landscapes Affect Bee Health?

Honey bee carrying pollen.Honey bees are hard-working insects. Their pollination services are in such demand, humans tow hundreds of hives carrying millions of bees around in the back of semitrucks to bring honey bees to various locations such as California almond groves. Humans are also quite partial to the bee colony winter energy storage also known as honey. So while honey bees work hard to collect pollen and nectar from blooming plants and trees and store honey for the winter, humans insist on robbing the colony’s store of delicious sweetener for their own uses. Recent reports of high mortality in honey bee colonies has caused concern in many beekeepers who manage European honey bee apiaries for honey production and pollination services. These severe depletion of honey bee colonies have been attributed to the parasitic mite Varroa destructor in the colony, not only feeding off the larvae and pupae brooding in the colony but also transmitting viruses carried by the mite. Bee nutrition is important for the pollinators especially when overwintering in the hive. Without adequate nutrition, a colony may become weak and succumb to parasite or disease pressure, unable to survive until nectar and pollen are available in the spring. A study was recently published in PLOS ONE that examined how the landscape around Midwestern honeybee hives affected the ability of bees to overwinter and assessed their health by measuring levels of Varroa mites and honey bee viruses. Continue reading

Two light stories for Friday

For this Friday blog, here’s a sampling of two recent papers highlighting use of the small, bright, NanoLuc luciferase in interesting ways.

Bioluminescence-based hormone:receptor binding studies

A review by Ya-Li Liu and Zhan-Yun Guo, published this week in Amino Acids summarizes recent work of the authors and others using NanoLuc luciferase labeled protein/peptide hormones in receptor binding assays. Typically, studies assessing binding of hormones to receptors have used radioactive tracers. The brightness of NanoLuc luciferase makes bioluminescence an attractive alternative as a sensitive and safer option. Because cell membrane receptors are difficult to purify in quantity, the amounts available for experiments are usually limited. Therefore, tracers used in binding assays need to have a high affinity for the receptor, must not interfere with binding, and must be highly sensitive. Continue reading

Shooting for the Moon: Better Assays to Hit Our Cancer Research Targets

3239CA02_1AIn his address to the clinicians, researchers, and patients at the American Association for Cancer Research meeting in April, US Vice President Joe Biden, revealed that the goal of the #cancermoonshot initiative is to accomplish 10 years of cancer research in just five years, effectively doubling the pace of cancer research (1).

Treatments developed from cancer research have come a long way with dramatic differences in the experiences and prognoses for patients, just looking back over the last 25 years. How can we double the pace of cancer research? The #cancermoonshot will one, encourage data sharing among researchers, particularly data from clinical trials. Second, it seeks to increase collaboration across industry, academic and government scientists—each community being positioned to make unique contributions to the field. And third, the initiative looks to change the current grants award process that encourages scientists to keep data and results “quiet” until they can be published or protected legally as intellectual property.

Immunotherapy is an especially hot field in cancer research (2) that relies on the immune system to better fight cancer. Continue reading

Inflammasomes and Pyroptosis

In today’s post, guest blogger,  Martha O’Brien, PhD, provides a preview of her upcoming AAI poster and block symposium talk on the inflammasome, caspase-1 activity and pyroptosis.

Schematic of the Caspase-Glo 1 Inflammasome Assay.

Schematic of the Caspase-Glo 1 Inflammasome Assay.

Responding rapidly to microbial pathogens and damage-associated molecular markers is critical to our innate immune system. Caspase-1 is pivotal in this process leading to processing and release of essential cytokines and an immunogenic form of cell death, termed pyroptosis. Upon sensing pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), innate immune cells form inflammasome protein complexes that recruit and activate caspase-1 (canonical inflammasomes). In addition, other inflammatory caspases, 4 and 5 in humans and 11 in mice, directly bind bacterial lipopolysaccharides (LPS), triggering pyroptosis (non-canonical inflammasome). LPS-triggered non-canonical inflammasomes in mice and humans ultimately lead to canonical inflammasome engagement and caspase-1 activation (1–3).  Caspase-1 was originally termed interleukin converting enzyme (ICE) for its well-established role in processing IL-1ß and IL-18, two important inflammation cytokines. How caspase-1 mediates pyroptosis is less well understood, but is beginning to be delineated. Recently, a substrate of the inflammatory caspases, gasdermin D, was identified and its processed fragment, gasdermin-N domain, was shown to be required for pyroptosis in non-canonical inflammasome circumstances (4, 5). The precise role of gasdermin D in canonical inflammasome-triggered pyroptosis is still under investigation. Linking inflammatory caspases directly to pyroptosis is a notable step in understanding the mechanism of this important form of cell death.

Pyroptosis is clearly one means of releasing processed IL-1ß and IL-18 from the cell. However depending on the cell type and stimulus, there is evidence for inflammasome engagement, caspase-1 activation, and release of IL-1ß in the absence of cell death (6, 7). On the flip-side there is also evidence for caspase-1 mediated pyroptosis that helps clear bacteria, independent of IL-1ß and IL-18 involvement (8). To enable further studies on the inflammasome and in particular, assessing the connections between caspase-1 activation, pyroptosis, and cytokine release, Promega developed a new tool to conveniently monitor caspase-1 activation, the Caspase-Glo® 1 Inflammasome Assay.  This bioluminescent, plate-based assay is used to measure caspase-1 activity directly in cell cultures or to monitor released caspase-1 activity in culture medium from treated cells. This flexibility allows easy multiplexing to monitor all three outcomes of inflammasome stimulation; caspase-1 activity, pyroptosis, and release of IL-1ß and IL-18. Caspase-1 activation typically is monitored indirectly with western blots of processed caspase-1. Now the activity of the enzyme can be monitored directly, providing accurate information on temporal aspects of the inflammasome. The assay can be readily combined with real-time measures of cell death (e.g., CellTox™ Green Cytotoxicity Assay) and some of the culture medium can be removed for IL-1ß/IL-18 assessment, leaving the cells and remaining culture medium for caspase-1 activity measurements. At the upcoming meeting of the American Association of Immunologists (AAI) in Seattle, May 13th-17th, oral and poster presentations will highlight use of the Caspase-Glo® 1 Inflammasome Assay and its value for exploring the relationship between inflammasomes and pyroptosis.

References

  1. Schmid-Burgk et al. (2015) Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells. J. Immunol. 45, 2911–7.
  2. Baker et al. (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. J. Immunol. 45, 2918–26.
  3. Ruhl, S. and P. Broz (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ Eur. J. Immunol. 45, 2927–36.
  4. Shi et al. (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–5.
  5. Kayagaki et al. (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–71.
  6. Gaidt et al. (2016) Human monocytes engage an alternative inflammasome pathway. Immunity 44, 833–46.
  7. Chen et al. (2014) The neutrophil NLRC4 inflammasome selectively promotes IL-1ß maturation without pyroptosis during acuteSalmonella Cell Reports 8, 570–82.
  8. Miao et al. (2010) Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nature Immunology 11, 1136–42.

Festival of Genomics: Not Your Typical Scientific Conference

27849820-May-6-FOG-LOGOAs a global life sciences company, Promega participates in scientific conferences and trade shows around the world, all year long. ISHI, ASHG, SLAS, ISBER, PAG, SOT, ESHG—the alphabet of conference names may be hard to keep straight, yet preparation for each involves a strong collaboration between Promega R&D scientists, product managers and the marketing services team. A new conference on the calendar caught our attention recently, as it’s billed as a “Festival.” That’s right, the name Festival of Genomics hints at its unique nature, and true to its title, it offers a novel approach in its organization, focus and objectives.

Created and organized by a London-based media company, Front Line Genomics, the Festival is described as a “three day celebration of genomics across the spectrum from the lab to the clinic, taking in new research, technology and advances in medicine.” The young event is intended to provide an environment for scientists to gather, connect and share with their peers. The hope is that new ideas will flourish and ultimately lead to more progress in the field of genomics research.

Also somewhat novel, a different “flavor” of festival is offered in three different cities worldwide. The Boston Festival will be held June 27-29, the California (San Diego) Festival September 19-21, and the London Festival January 30–February 1, 2017. By offering it three times a year in various locations, more members of the genomics community have the opportunity to participate. And the festivals are open not only to research scientists, but to anyone who considers themselves part of that community, including academia, industry, healthcare organizations, patient organizations, and investment firms. Continue reading

Familial DNA Searching for Criminal Forensics: Q&A

When DNA evidence is collected at a crime scene, submitting the sample for a search within a DNA database does not always identify a profile match. There is a way to extend that search and generate leads, called familial searching (FS). FS is used to identify close biological relatives of an unidentified DNA profile obtained as evidence. The basic premise is that DNA profiles of immediate family members, such as siblings, parents, or children, are likely to have more alleles in common than unrelated individuals. These familial profile matches can generate new investigative leads for law enforcement.

Currently, a few states are using FS under their state database laws, although none explicitly permit FS. Many agencies have yet to adopt policies related to FS, even though it has been found to be as effective as CODIS for identifying sources of evidence. The absence of clear ethical guidelines and policy regarding how to properly utilize FS prevents many local and state jurisdictions from adopting this investigational tool.

In order to address concerns and existing policies related to FS and to guide policy decisions by agencies implementing FS, the National Institute of Justice (NIJ) issued the report Familial DNA Searching: Current Approaches in January 2015. The goal of the report was to provide information to policy makers, law enforcement officials, forensic laboratory practitioners, and legal professionals about how FS is being applied within the criminal justice realm.

Mr. Rock Harmon, former prosecutor

Mr. Rockne Harmon, former prosecutor

 

Answers to the following questions about FS were provided by Mr. Rockne Harmon, a retired former prosecutor and member of the team that produced the report for the National Institute of Justice.

 

What is familial DNA searching?

Familial searching (FS) is an additional search of a DNA profile in a law enforcement DNA database that is conducted after a routine search fails to identify any profile matches. The FS process attempts to provide investigative leads to agencies engaged in the pursuit of justice by identifying a close biological relative of the source of the unknown forensic profile obtained from crime scene evidence. Continue reading