Author: Sara Klink

Six (and a Half) Reasons to Quantitate Your DNA

Posted on by Sara Klink

Knowing how much DNA you have is fundamental to successful experiments. Without a firm number in which you are confident, the DNA input for subsequent experiments can lead you astray. Below are six reasons why you should quantitate your DNA.

6. Saving time by knowing what you have rather than repeating experiments. If you don’t quantitate your DNA, how certain can you be that the same amount of DNA is consistently added? Always using the same volume for every experiment does not guarantee the same DNA amount goes into the assay. Each time there is a new purified DNA sample, the chances that you have the same quantity as before are lessened. Consequently, without knowing the DNA concentration of the sample you are using, the amount of input DNA cannot be guaranteed and experiments may have to be repeated.

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Revealing Time of Death: The Microbiome Edition

Posted on by Sara Klink

Forensic analysts have long sought precision when determining time of death. While on crime scene investigation television shows, the presence of insects always seems to reveal when a person died, there are many elements to account for, and the probable date may still not be accurate. Insects arrive days after death if at all (e.g., if the body is found indoors or after burial), and the stage of insect activity is influenced by temperature, weather conditions, seasonal variation, geographic location and other factors. All this makes it difficult to estimate the postmortem interval (PMI) of a body discovered an unknown time after death. One way to make estimating PMI less subjective would be to have calibrated molecular markers that are easy to sample and are not altered by environmental variabilities.

Bacterial communities called microbiomes have been frequently in the news. The influence of these microbes encompass living creatures and the environment. Not surprisingly, research has focused on the influence of microbiomes on humans. For example, changes in gut microbiome seem to affect human health. Intriguingly, microbiomes may also be a key to determining time of death. The National Institute of Justice (NIJ) has funded several projects focused on the forensic applications of microbiomes. One focus involves the necrobiome, the community of organisms found on or around decomposing remains. These microbes could be used as an indicator of PMI when investigating human remains. Recent research published in PLOS ONE examined the bacterial communities found in human ears and noses after death and how they changed over time. The researchers were interested in developing an algorithm using the data they collected to estimate of time of death.

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Preventing Viral Infection by Blocking Cellular Receptors with a Tethered Antibody

Posted on by Sara Klink

Finding a way to neutralize or block infection by HIV has long been pursued by viral researchers. Various treatments have been developed, driven by the need to find effective drugs to manage HIV in infected individuals. The ultimate goal is to develop a vaccine to prevent HIV from even taking hold in the body. With all of our knowledge about the cellular receptors HIV needs to enter the cell, there has to be a method to prevent a viral particle from binding and being internalized. Many researchers are pursuing neutralizing antibodies to the virus as one method. What about antibodies that target the cellular receptor recognized by the virus? An article published in Proceedings of the National Academy of Sciences, antibodies to cellular receptors for rhinovirus and HIV were tethered to the plasma membrane and tested for the ability to prevent infection.

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A Big Protective Step Forward for A Rare Bee

Posted on by Sara Klink
A rusty-patched bumblebee on Culver’s root in the UW–Madison Arboretum. Photo Copyright: SUSAN DAY/UW-MADISON ARBORETUM
A rusty-patched bumblebee on Culver’s root in the UW–Madison Arboretum. Photo Copyright: SUSAN DAY/UW-MADISON ARBORETUM

Bees have been in the news many times over the past several years. Much of the concern has been focused on the collapse of honey bee colonies because these bees collect nectar to create honey and can be transported for use as pollinators for farmers. Alongside the plight of the honey bee are the declines in the population of native bees in the United States. These bees include insects like the big, fuzzy bumble bees, tiny, iridescent green sweat bees and dark blue mason bees. The native bees live in different conditions. They may be solitary, have a small colony or even nest close together in a communal arrangement, but never in the numbers likely to be seen for a honey bee colony. These lower-density populations can make seeing a change in native bee numbers more difficult. While honey bees have gained the majority of bee decline attention, native bees have suffered dramatic population loss with long-term consequences for the plants they pollinate and the animals that depend upon those plants.

On January 11, 2017, in a landmark decision by the United States Fish and Wildlife Service, the one of the rarest native bees called the rusty-patched bumble bee (Bombus affinis) has been listed as threatened, and this designation will go into effect February 10, 2017. This is the first bee in the U.S. that has been placed on the Endangered Species list. The rusty-patched bumble bee derived its name from the rust-colored patch found on its back.

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Making It Easier to Investigate PGTs

Posted on by Sara Klink

Introduction

Studying cellular molecules can be challenging. Some processes are troublesome to study due to the lack of an assay or a complicated assay exists but lacks sensitivity. Membrane proteins in particular are difficult to isolate and characterize. Phosphoglycosyltransferases (PGTs) are transmembrane proteins that transfer phosphosugars onto phospholipids, initiating the synthesis of oligosaccharides in bacterial cell walls. This transfer creates a diphosphate link between a lipid and a sugar and generates UMP as a byproduct. Once this lipid–P–P–sugar linkage occurs, more sugars can be added by glycosyltransferases, generating membrane-based polysaccharides (e.g., peptidoglycan) used for signaling, recognition and defense.

While PGTs have been studied biochemically and an X-ray structure of one member exists, much is still unknown about these enzymes. Overexpressing and purifying membrane proteins remains a challenge, and the conventional PGT assay requires isotope labeled-UDP-sugar donors and is based on the solubility difference between substrate and product to determine enzyme turnover using extraction-based or chromatographic methods. While there are other assays that use fluorescent modified substrates or multienzyme analysis, none of the methods can be applied to all of the diverse PGT enzymes.

All PGTs generate UMP as a byproduct of the transfer of a phosphosugar to a phospholipid. Based on the principle of the luminescent UDP-Glo™ Glycosyltransferase Assay where UDP released during the glycosyltransferase reaction was quantitated, a new luminescent assay called UMP-Glo™ Assay is able to measure the activity of PGT enzymes by adding a single reagent to detect UMP. Das et al. validated this assay by testing PglC, a PGT from Campylobacter jejuni, as well as PglC from Helicobacter pullorum and WecA from Thermatoga maritime and published the results in Scientific Reports. Continue reading “Making It Easier to Investigate PGTs”

Friday Cartoon Fun: Take My Samples, Please!

Posted on by Sara Klink

Instruments can make our lives easier in the lab. Place your samples inside an instrument and let it do all the work—isolating nucleic acids or reading and analyzing a multiwell plate—while you walk away to read a new research paper or prepare for the next step in your experiment. However, with the array of machines now available to scientists worldwide, some confusion may result in the laboratory. Has this ever happened to you?

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Copyright Ed Himelblau

Digging Up More Clues in the History of the Black Death

Posted on by Sara Klink

Bubonic plague victims in a mass grave in 18th century France. By S. Tzortzis [Public domain], via Wikimedia Commons
Bubonic plague victims in a mass grave in 18th century France. By S. Tzortzis [Public domain], via Wikimedia Commons
My last blog post on the Black Death highlighted research that suggested that the reintroduction of Yersinia pestis, the causative agent of the pandemic, originated in Europe during the 14–18th centuries rather than from Asia, the hypothesized origin. In my post, I wrote about my curiosity regarding what an Asian skeleton positive for Y. pestis from that same time period would reveal about the strain or strains that were circulating. Well, a team of researchers has been exploring the issue of strain circulation and an Asian connection, and recently published what they gleaned from additional historic Y. pestis samples in Cell Host & Microbe.

Teeth from 178 individuals in three different locations (two European, one Asian) were screened for Y. pestis infection using the plasminogen activator (pla) gene. Continue reading “Digging Up More Clues in the History of the Black Death”

Make Plans Now to View a Rare Astronomical Phenomenon

Posted on by Sara Klink

Solar eclipse viewing. By Skoch3 (Own work) [CC0], via Wikimedia Commons
Solar eclipse viewing. By Skoch3 (Own work) [CC0], via Wikimedia Commons
During my college years, I witnessed an event that was new to me: A solar eclipse. I made a pinhole projector to watch the moon pass over the sun on a piece of white paper and have to admit, the darkening during midday was quite interesting. However, it was not a total eclipse so there was still some sunlight slipping around the moon. Hence using the pinhole projector to preserve my eyesight.

Next year on August 21, the United States will experience a total solar eclipse. While I will be able to see the solar eclipse in Wisconsin, I will not experience a total eclipse. In fact, I will need to head south and west to states like Nebraska, Kentucky and Missouri to reach part of the US where the moon will fully block the sun. Why is everyone talking about the 2017 Solar Eclipse in 2016? So you can plan your vacation of course!

Have a relative or friend you haven’t seen in a while conveniently located in the total eclipse zone? Ask if they would be willing to cohost a Solar Eclipse party. Alternatively, just ask to stay with friends or family and join up with any public observations of the solar eclipse. You need to plan a family vacation anyway, right? Why not conveniently plan to stay in a location where hey, there’s a total solar eclipse today. Let’s watch! Fun and educational for everyone.

Don’t forget your eclipse viewing glasses (so attractive in cardboard chic) or add filters to telescopes and binoculars for magnified viewing pleasure. Bonus to a total solar eclipse? You can gaze at the moon-blocked sun with your naked eyes for up to 2.5 minutes, depending on location. Just don’t look too long to preserve your retinas.

So if you need an excuse to plan a unique vacation (and maybe appease some rarely seen friends and relatives), consider placing yourself in the swath of the country where the moon will obliterate your view of the sun (for less than three minutes). And if these locations don’t appeal to you, just wait until 2024 when the eastern portion of the US will be treated to a total solar eclipse. Different cities in which to vacation and other relatives to visit!

Improving the Success of Your Transfection

Posted on by Sara Klink
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Not every lab has a tried and true transfection protocol that can be used by all lab members. Few researchers will use the same cell type and same construct to generate data. Many times, a scientist may need to transfect different constructs or even different molecules (e.g., short-interfering RNA [siRNA]) into the same cell line, or test a single construct in different cultured cell lines. One construct could be easily transfected into several different cell lines or a transfection protocol may work for several different constructs. However, some cells like primary cells can be difficult to transfect and some nucleic acids will need to be optimized for successful transfection. Here are some tips that may help you improve your transfection success.

Transfect healthy, actively dividing cells at a consistent cell density. Cells should be at a low passage number and 50–80% confluent when transfected. Using the same cell density reduces variability for replicates. Keep cells Mycoplasma-free to ensure optimal growth.

Transfect using high-quality DNA. Transfection-quality DNA is free from protein, RNA and chemical contamination with an A260/A280 ratio of 1.7–1.9. Prepare purified DNA in sterile water or TE buffer at a final concentration of 0.2–1mg/ml.

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microRNA: The Small Molecule with a Big Story

Posted on by Sara Klink

Introduction

miR-34 precursor secondary structure. The colors indicate evolutionary conservation. Ppgardne [GFDL (http://www.gnu.org/copyleft/fdl.html)

RNA molecules have become a hot topic of research. While I was taught about messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA), many more varieties have come into the nomenclature after I graduated with my science degrees. Even more interesting, these RNAs do not code for a protein, but instead have a role in regulating gene expression. From long non-coding RNA (lncRNA) to short interfering RNA (siRNA), microRNA (miRNA) and small nucleolar RNA (snoRNA), these classes of RNAs affect protein translation, whether by hindering ribosomal binding, targeting mRNA for degradation or even modifying DNA (e.g., methylation). This post will cover the topic of microRNAs, explaining what they are, how researchers understand their function and role in metabolism, cancer and cardiovascular disease, and some of the challenges in miRNA research.

What are microRNAs? MicroRNAs (miRNAs) are short noncoding RNAs 19–25 nucleotides long that play a role in protein expression by regulating translation initiation and degrading mRNA. miRNAs are coded as genes in DNA and transcribed by RNA polymerase as a primary transcript (pri-miRNA) that is hundreds or thousands of nucleotides long. After processing with a double-stranded RNA-specific nuclease, a 70–100 nucleotide hairpin RNA precursor (pre-miRNA) is generated and transported from the nucleus into the cytoplasm. Once in the cytoplasm, the pre-miRNA is cleaved into an 18- to 24-nucleotide duplex by ribonuclease III (Dicer). This cleaved duplex associates with the RNA-induced silencing complex (RISC), and one strand of the miRNA duplex remains with RISC to become the mature miRNA.

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