Weimer Micro Lab

Contributed by Nguyet Kong

A group at Stowers Institute in Kansas City recently published their study in PLos Genetics describing that cancer cells restructuring in the ribosomal DNA to more easily reproduce copies. The rDNA cellular functions include regulating gene expression, chromosome organization, and chromatin factors. There not much known about the stability of rDNA, but the researchers believe more data can be obtained by using digital droplet PCR to measure rDNA copies. Extracted genomic DNA from 3 mouse strains tissues samples and from different organ types. The researchers first measured the copies between the mouse strain and averaging the numbers. C57BL/6 mouse strain was 156 and DBA/2J was 123. The mean copy number for CD-1 was145 with higher individual variation. The researchers then found changed in the mtDNA copy in different tissues not equivalents to the changed reported in the rDNA copies. The researchers wanted to confirm their finding in cancer cells in human, so they obtained whole genome sequencing from 162 across eight tumor types and also analyzed rDNA copies from the sequencing data. The researchers have found a significant reduction in copies for tumor genomes in 3 of the 8 groups, suggesting low copy number at the rDNA was selected in some cancer types.

Next, the researchers wanted to see if mTOR function will suppress tumor function in PTEN could influence rDNA copies. They use a mouse model Pten leukemia to assess rDNA copies in hematopoietic stem cells (HSCs), which they did sequence and droplet PCR. This verified that the loss of PTEN is indicative of mTOR activation, and they confirm HSC cells and ribosome genesis can occur with 30 to 40 fewer rDNA repeats. The researchers suggest that instability of the genome can make the cells susceptible to chemo with DNA damaging drugs.

https://www.sciencedaily.com/releases/2017/06/170622142959.htm

Contributed by Azarene Foutouhi

While the ecological implications of climate change are well established, recent studies have pointed to changes in the microbiome as a casualty of the warming Earth. Studies testing the effects of temperature changes on the gut microbiomes of ectotherms such as lizards, temperature increases resulted in significant changes in microbiome composition. Researchers reported animals living in temperatures 3°C higher than control groups living at normal environmental temperatures suffered a 34% reduction in microbiome diversity. Resampling months later indicated this loss was driven by the extinction of individual bacterial taxa, and that the lizards with richer microbiomes tended to live a year longer. Future work will investigate the effect of increasing temperatures on the microbiomes of non-ectotherms and will help us understand previously unconsidered consequences of climate change.

http://www.the-scientist.com/?articles.view/articleNo/49368/title/Warmer-Temps-Tied-to-Altered-Microbiome-in-Lizards/

Contributed by Ning Chin

I recently went to the National Yellowstone Park for my brother’s graduation trip with family. Taking a walk in nature is always a humbling experience. We can stay in the lab and carefully control conditions to mimic nature, but the product is always less cool than what nature created.

Recognized as a UNESCO World Heritage Site, Yellowstone Park consists of lakes, canyons, rivers, and mountain rangers that have unique geothermal features. Out of all the breath-taking (the smell of rotten egg) views that the Yellowstone Park offers, my favorite one is the Grand Prismatic Spring. It is the largest hot spring in the United States, with colors matching the light dispersion by a prism – red, orange, yellow, green, and blue. The blue is due to the light scattered by particles suspended in the water. The red, orange, yellow, and green are all due to microbial growth – the temperature gradient by the edge of the hot spring created a unique niche for different microbes that can survive in that specific environment. Even the steam that’s coming out of the hot spring seemed like they’re colored because of the reflection of the spring’s light. Just think about that for a second – the majority of the colors are from microbes! It’s amazing how life can thrive even in such extreme environment. Instead of using my limited English vocabulary to describe the beauty, here’s a picture.

(link to the picture: http://img.huffingtonpost.com/asset/scalefit_720_noupscale/573bd5421300000105381fde.jpeg)

If you’re interested in learning more about the microbes, take a look at this article:

http://www.smithsonianmag.com/travel/science-behind-yellowstones-rainbow-hot-spring-180950483/

Narine Arabyan Exit Seminar June 9, 2017 2-4 pm

Contributed by Narine Arabyan

Lysozyme hydrolyzes the β-1,4-glycosidic bond of oligosaccharides. These enzymes are part of a broad group of glycosyl hydrolases that are poorly characterized. Lysozyme enzymes belong to the GH24 family of glycosyl hydrolases (GHs)¹. GHs play an important role during infection by altering the host glycan structure to gain access of the host epithelial cells by binding to terminal monosaccharides to initiate glycan degradation². Lysozyme enzymes may be new promising virulence factors due to its β-1,4-glycosidase activity and have the potential to recognize host GlcNAc containing glycans in the form of N-glycans, O-glycans, glycolipids, glycoproteins and glucosaminoglycans during infection³. These GlcNAc molecules are linked to monosaccharides in the glycan via a β-1,4-glycosidic bond⁴ that can be cleaved by enzymes from Salmonella with lysozyme activity during host association.

Lysozyme enzymes with β-1,4-glycosidase activity are also involved during the secretion of proteins which is central for the virulence of all pathogenic bacteria¹. Gram-negative organisms translocate proteins across the peptidoglycan that is composed of linear chains of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) and the alternating sugars are connected by β-1,4-glycosidic bonds^5-7. The peptidoglycan structure is a physical barrier for the assembly of macromolecular complexes and for the transport of proteins. For this reason, all bacterial lysozymes degrade the peptidoglycan to allow the assembly of type III or type IV secretion systems essential in virulence, flagella, or conjugation^8,9. This remodeling creates gaps in the peptidoglycan necessary for the assembly of these macromolecular systems. Intracellular pathogenic bacteria, such as Brucella abortus, use lysozyme during its early stages of intracellular replication⁸. Lysozyme enzymes are important for growth and are being recognized as emerging virulence factors.

1          Mushegian, A. R., Fullner, K. J., Koonin, E. V. & Nester, E. W. A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals. Proc Natl Acad Sci U S A 93, 7321-7326 (1996).

2          Arabyan, N. et al. Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling. Sci Rep 6, 29525, doi:10.1038/srep29525 (2016).

3          Jacobs, H. et al. Characterization of membrane N-glycan binding sites of lysozyme for cardiac depression in sepsis. Intensive Care Med 31, 129-137, doi:10.1007/s00134-004-2487-y (2005).

4          Stanley, P., Schachter, H. & Taniguchi, N. in Essentials of Glycobiology   (eds A. Varki et al.) (2009).

5          Allaoui, A., Sansonetti, P. J. & Parsot, C. MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins. Mol Microbiol 7, 59-68 (1993).

6          Kirby, A. J. Mechanism and stereoelectronic effects in the lysozyme reaction. CRC Crit Rev Biochem 22, 283-315 (1987).

7          Pei, J. & Grishin, N. V. COG3926 and COG5526: a tale of two new lysozyme-like protein families. Protein Sci 14, 2574-2581, doi:10.1110/ps.051656805 (2005).

8          Del Giudice, M. G., Ugalde, J. E. & Czibener, C. A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication. Infect Immun 81, 956-964, doi:10.1128/IAI.01158-12 (2013).

9          Koraimann, G. Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria. Cell Mol Life Sci 60, 2371-2388, doi:10.1007/s00018-003-3056-1 (2003).

Contributed by Carol Huang

On Friday, May 5^th, 2017, the Yolo County Health & Human Services Agency advised residences to take precautions to limit spread of a GI Illness Outbreak.

County health authorities are investigating whether reports of illness at UC Davis and elsewhere are the result of Norovirus.

Norovirus are a group of related viruses that can cause gastroenteritis, inflammation of the stomach or intestines or both. This leads to diarrhea, throwing up, nausea, stomach pain; other symptoms like fever, headache and body aches. These symptoms can be serious for some people, especially young children and older adults. A person usually develops symptoms 12 to 48 hours after being exposed to norovirus. Most people with norovirus illness get better within 1 to 3 days.

Norovirus is a very contagious virus that can infect anyone. It also spreads quickly. It can be found in stool (feces) even before a person starts feeling sick. The virus can stay in stool for 2 weeks or more after infected person feel better. One can be infected by person to person contact, contaminated food or water, or by touching contaminated surfaces.

Norovirus is the leading cause of illness and outbreaks from contaminated food in the United States. Most of these outbreaks occur in the food service like restaurants. Infected food workers are frequently the source of the outbreaks, often by touching ready-to-eat foods, such as raw fruits and vegetables, oysters, with their bare hands before serving them. However, any food served raw or handled after being cooked can get contaminated with norovirus

Noroviruses are relatively resistant. They can survive temperatures as high as 140°F and quick steaming processes that are often used for cooking shellfish.

According to CDC, each year Norovirus causes 19 to 21 million illnesses, 56,000 to 71,000 hospitalizations and 570 to 800 deaths, mostly among young children and the elderly

It is estimated that a person will get norovirus about 5 times during their lifetime. Many people usually get sick with norovirus in cooler months, especially from November to April.

There is no specific medicine to treat people with norovirus illness.

For preventing Norovirus infection: practice proper hand hygiene; carefully wash fruits and vegetables before preparing and eating them. Cook oysters and other shellfish thoroughly before eating them.

Ref: https://www.cdc.gov/norovirus/

http://www.yolocounty.org/Home/Components/News/News/4974/26

Contributed by Ning Chin

Polymerase Chain Reaction (PCR) is one of the most important technique in the field of molecular biology. The idea of PCR came to Kary Mullis in 1983 when he was driving from Berkeley to Mendocino. 34 years later, PCR technique is used heavily and taught to students as a basic molecular biology technique. The invention of PCR catalyzed the advancement in the field of molecular biology, which also benefited PCR the medical field and the forensic field, to name a few.

The theory of PCR is simple yet effective: you increase temperature to denature double-stranded DNA, lower temperature to anneal primers and then increase temperature again to allow polymerase to make the DNA based on your DNA template. However, as simple as it sounds, there are many details that we can tweak to improve the PCR technique. The details in PCR condition are especially important when dealing with low template DNA. My instructor from the forensic science program once jokingly said that PCR is the short-form of Pure Chance Reaction. How true is this when dealing with low template DNA? When you have very low amount of template DNA, the PCR reaction result become a statistical problem: what is the probability that you will pipette the amount of DNA needed for your PCR reaction?

In forensic science, a stochastic effect is often solved by increasing the cycle number—the strictly controlled nature of forensic science does not allow for a lot of personal creativity to ensure the quality and standards of the analysis. In research settings, there are many other factors that you can change to increase your PCR efficiency. Many of these were explored by other researchers, such as primer design, salt concentration, polymerase type, and others. However, I haven’t seen any research on how secondary structure of template DNA, more specifically hairpin structures, can affect PCR. Theoretically, double-stranded DNA denatures completely at 95C – but when the temperature cools down to allow primer annealing, would single-stranded DNA form a hairpin and block primer from binding? Is this the reason why I couldn’t amplify my low template PCR? A simple experiment will solve this mystery – adding dithiothreitol into the PCR master mix to denature hairpin formation.

And no. My PCR is still unsuccessful.

Contributed by Azarene Foutouhi

Susceptibility to bacterial infections depends on many things including the individual’s immunologic condition and the virulence of the bacteria. After preliminary data showed prolonged macrophage activation during gamma-Herpesvirus latency, researchers wondered if this state might be associated with resistance to infection. Mice five weeks post infection with HV68 or MCMV challenged with L. monocytogenes showed nearly complete resistance, while mice previously exposed to viruses incapable of latency exhibited bacterial titers identical to control. Mice reacted similarly to Y. pestis challenge. Interestingly, this protection is not provided by acute gamma or beta-Herpesvirus infection and wanes by latency week 12. Because approximately 90% of humans are herpesvirus positive, there is a great need to re-examine interactions between immune function and possibly symbiotic microbes.

Barton, Erik S., Douglas W. White, and Herbert W. Virgin. “Herpesvirus Latency and Symbiotic Protection from Bacterial Infection.” Viral Immunology 22.1 (2009): 3–4. PMC. Web. 18 Apr. 2017.

Weis, Allison M., Kristen A. Clothier, Bihua C. Huang, Nguyet Kong, and Bart C. Weimer. 2017. Multidrug-resistant abortive Campylobacter jejuni draft genome from Northern California. Genome Announcements 5(15):e 00171-17. (DOI:10.1128/genomeA.00171-17)

Contributed by Poyin Chen

The premise for the video game, Assassin’s Creed, is based on the technological ability to re-live our ancestors’ experiences by accessing their memories inherited through their DNA. While this is actually scientifically impossible, the concept of heritable “memories” through epigenetic programming is currently an active area of study.

A publication by Fang et. al. studies the transgenerational effect of in utero caffeine exposure in the cardiac tissue male mice. Embryonic development is marked by a rapid demethylation event, followed by a re-methylation event. This demethylation event serves to remove any epigenetic imprints that the parental DNA possessed, however, this event can be influenced by many factors, one of which being nutrition.

Caffeine exposure in utero is known to result in low birth weight in F1 progeny but Fang et. al. sought to explore the effects of this exposure on F2 and F3 progeny who are not directly exposed to caffeine at any point in development.

The study found that depending on the stage of embryonic development at which caffeine is introduced, F1 progeny would have differential cardiac phenotype and function as well as differential expression of genes involved in cardiac development. In addition, exposure of F1 embryos to caffeine resulted in a significant change in cardiac phenotype and function in F2 and F3 progeny.

While we may not be able to relive our ancestors’ memories as extensively as this video game portrays, in the future, we may be able to genetically identify how our parents’ actions (such as ingesting too much caffeine) shaped who we are today.

Reference: Fang X, Poulsen RR, Rivkees SA, Wendler CC. In utero caffeine exposure induces transgenerational effects on the adult heart.2016 Scientific Reports.

DOI: 10.1038/srep34106