Category Archives: Week 6

Not-So-Abstract Abstract

Plant development is influenced by the environment around it, and in Arabidopsis thaliana, these extra organismal development factors can be transmitted across generations. While it is known that both ancestral and offspring environment impacts development, the magnitude to which each acts is unknown. To assess the ancestral environmental impact on offspring development, the grandparental generation is grown in a warm and cool temperature, and offsprings of each respective environment are then also grown in warm and cool temperature to give 4 scenarios. Finally, for the last generation, we take the 4 different parental and self-pollinate them to give 8 possible scenarios. We expect to see the offspring environment to have the largest influence on offspring development followed by parental and grandparental, respectively.The variation in the magnitude of transgenerational environmental effects between different genotypes are also not well characterized. For these set of experiments, we grew 8 genotypes of Arabidopsis thaliana in both warm and cool temperature for the grandparental generation and ambient conditions for the parental and offspring generation. We expect the different genotypes to have different propensities to have transgenerational environmental effects.

Stage 6: I should probably have bookmarked those sources

Abnormal and excessive tau phosphorylation commonly characterizes Alzheimer’s disease (AD) neuropathology through neurofibrillary tangles (NFTs) causing axonal dysfunction and microtubular instability. Essential to DNA repair, protein modification, and gene expression, the methionine cycle generates a methyl group upon the conversion of S-adenosyl-methionine to S-adenosyl-homocysteine, which can activate protein phosphatase 2A (PP2A) and leucine carboxyl methyltransferase 1 interactions in neurons.1,2 These interactions are known to target serine/threonine phosphate groups prevalent in hyperphosphorylated tau, and prior studies have demonstrated increased aggregate formation in vivo upon PP2A inhibition.1,2 Furthermore, increased levels of homocysteine, a byproduct of the methionine cycle normally converted back into methionine or anti-oxidative glutathione, is strongly linked to the progression of AD.3 This suggests insufficient methionine resynthesis allowing the hyperphosphorylation of tau to form NFTs. Biochemical analysis of the different pathways homocysteine metabolism undertakes show many similarities to digestive reactions in the liver of the reactive intermediate imine deaminase A homolog (UK114) enzyme. We are studying the potential interactions and role of UK114 in transfected Chinese Hamster Ovary cells by recording protein expression levels via Western blot. The transfected cells are subjected to various conditions either translatable with symptoms of AD or saturated with components of the methionine cycle that are potentially catalyzed by UK114. The results of these studies will further expand our understanding of the methionine cycle in relation to AD pathology, as well as explore the antioxidant uses of UK114 in an immunological scope.

Weekly Highlights:
*LOUD THUD as Dang accidentally knocks over a giant bottle*
“Is Dang throwing things again? Bad boy.”-Joan

“Let’s go stuff some squirrels and hamsters in a fridge to induce torpor!”-Dr. Colton
“We already tried that. It didn’t work.”-Joan
“PARTY POOPER!”-Dr. Colton

“Wow, big pellets! This sample is good!
*BCA subsequently fails. negative protein concentrations returned. half the trial ruined*
“Oh no.”-Huifang

“What’s wrong with your milk? :/”-Huifang as she throws away milk and goes to her own stash

“And one day, you’ll be like these two amazing children. One graduating with distinction and the other just a lowly freshman!”-Dr. Colton

References

1.Qian, W., Shi, J., Yin, X., Iqbal, K., Grundke-Iqbal, I., Gong, C., Liu, F. (2010). PP2A Regulates Tau Phosphorylation Directly and also Indirectly via Activating GSK-3β. Journal of Alzheimer’s Disease, 19(4), 1221-1229.https://content.iospress.com/articles/journal-of-alzheimers-disease/jad01317

2.Stanevich, V., Jiang, L., Satyshur, K. A., Li, Y., Jeffrey, P. D., Li, Z., … Xing, Y. (2011). The structural basis for tight control of PP2A methylation and function by LCMT-1. Molecular Cell41(3), 331–342. http://doi.org/10.1016/j.molcel.2010.12.030

3.Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H., D’Agostino, R. B., Wilson, P. W.F., Wolf, P. A. (2002). Plasma Homocysteine as a Risk Factor for Dementia and Alzheimer’s Disease. The New England Journal of Medicine, 346, 476-483.
http://doi.org/10.1056/NEJMoa011613

Screening for Efficient gRNAs to Skip Exon 51 in the DMD Gene

A majority of Duchenne’s muscular dystrophy cases are caused by a frameshift mutation in the DMD gene and the deletion of exon 51 with CRISPR-Cas9 can restore the reading frame in many patients. However, there are thousands of gRNA sequences to choose from with a deletion frequency of only 2%, so which gRNAs in introns 50 and 51 are the most efficient? To test this, I designed 10 gRNAs in both introns and transfected 293T cells with these gRNAs and a CjCas9 plasmid. I harvested these cells, ran a PCR with designed surveyor primers and ran these fragments on a gel. No new bands were seen that were different from my no-gRNA control lane so the gRNAs I designed did not make an indel. I will test 2 new gRNA sequences with the same method and expect to see two bands unique to the gRNA-transfected cells. The results from my experiment will be positive controls for my mentor’s project of developing a high-throughput screening method of hDMD-targeted gRNAs. With broader and quicker gRNA screening, the CRISPR-Cas9 tool will be more efficient and used as a human medical treatment option for patients with DMD.

Regulation of System xCT (SLC7A11) by ABL Kinases in Human Lung Cancer

The ABL family of non-receptor tyrosine kinases, ABL1 and ABL2, are upregulated in non-small cell lung cancer and promote lung cancer metastasis. Recent work has shown that ABL kinases promote lung cancer brain metastasis and colonization. Global transcriptome analysis of targets controlled by ABL kinases in lung cancer cells revealed SLC7A11 as being differentially regulated in ABL inhibited cells. SLC7A11 encodes the plasma membrane antiporter system xCT which has been shown to be overexpressed in cancerous cells. System xCT exports glutamate and imports cystine, an intermediate molecule in the cellular oxidative stress response mechanism. Real-time polymerase chain reaction (RTPCR) verified a reduction in SLC7A11 mRNA levels following ABL pharmacologic inhibition or genetic knockdown. Continued work is being done to reveal a reduction in cellular protein levels through protein isolation and western blotting. FACS analysis will be deployed to determine whether membrane-localized levels of SLC7A11 decrease upon ABL inhibition. Additionally, a glutamate assay kit will be utilized to determine whether export of glutamate by xCT is reduced in ABL knockdown cells relative to control. The ultimate goal of this project is to understand if ABL kinases promote SLC7A11 expression in order to alter the brain microenvironment and promote tumor colonization.

Abstract? More like specific.

Sea urchin embryos show a great affinity to replace cells necessary for normal development through a process called transfating in which cells in the embryo express genes of other cells to compensate for the removal. Specifically, the removal of primary mesenchyme cells (PMCs) which are crucial to the development of the skeleton triggers non-skeletogenic mesoderm (NSM) cells to reprogram in order to express genes found in the PMC gene regulatory network (GRN). A possible explanation is that the PMCs provide a signal to the surrounding NSMs that inhibit NSM transfating. This study seeks to determine 1) which genes are expressed in the PMCs and 2) what signal the PMCs provide to inhibit NSM transfating. Through in situ hybridization experiments using newly synthesized probes from a candidate gene list, we hope to determine which genes are PMC specific. Using in situ hybridization and drug inhibitor treatments, we test for ectopic cell reprogramming in order to determine the specific signaling molecules from the PMCs to inhibit NSM transfating. This study can broaden our understanding of the signaling molecules crucial to normal sea urchin development.

Episode 6: The abstract

Cannabis is one of the most popular drugs consumed in the United States. While work has been done to quantify whole genome transgenerational effects of cannabis exposure, there has been little done to quantify transgenerational effects on a gene-by-gene basis. Previously, Reduced Representation Bisulfite Sequencing identified Shank1 and Dlg4, genes associated with the post-synaptic density and the post synaptic membrane, as being differentially methylated in the sperm profile of cannabis(THC) exposed rats and controls. It is however unknown that this difference in methylation is significant  under specific gene methylation analysis. It is also unknown if any differences in methylation cause significant changes in the expression of Shank1 and Dlg4. That is why in this study, potential differential methylation of Shank1 and Dlg4* were measured through pyrosequencing of the somatic tissues of F1 rats. In addition, RT-PCR** was conducted in order to quantify changes in gene expression of somatic tissues in these F1 rats. It was found that in Shank1 there was hypermethylation in both CpG sites of interests, while in Dlg4 there was hypomethylation in one CpG site and hypermethylation in the other.*** The next steps for this study would be to quantify the gene expression and methylation changes for the F2 generation, while also quantifying the phenotype in the F1 generation.

*Biotin labeled primer for this gene(necessary for pyrosequencing) may not arrive due to inconsistencies with Dlg4’s primers in PCR

** RT-PCR may not be done at the time of this poster presentation

*** This data is from the RRBS whole genome methylation, not from specific gene methylation from pyrosequencing

Potential Abstract?

How does the ability to do phagocytosis of a new cell population compare to other dendritic cells?

The intestines are home to immune system cells whose role is to monitor the microbiota within the gut; one group of these cells being dendritic cells. Recently, a population of dendritic cells that are CD14+, a marker that was associated with macrophages, were found in the colon. The role of these CD14+ DCs in the colon and their ability to do phagocytosis is unknown. We hypothesize that that these novel CD14+ DCs will be better at phagocytosis compared to other DCs. We extracted cells from the colon and incubated them with a pseudopathogen with a colored marker under multiple different conditions. Afterwards the cells were stained with antibodies and analyzed using a flow cytometer. Under multiple conditions, we found a higher median fluorescence for the novel DCs when compared to the other DCs in the colon. At the same time, the novel DCs did not have a median fluorescence intensity as high as the macrophages. Our data suggests that the novels DCs have an increased ability to perform phagocytosis compared to other DCs but not at the level of the macrophages.

Abstract Initial Draft

Dystonia is a neuromuscular disorder in which muscles make involuntary contractions, leading to abnormal and often times repetitive movements. It has been found that a common mechanism for several types of dystonia—each characterized by the gene adversely affected—was an impairment in ISR signaling. (Rittiner, et al,) Furthermore, the alleles causing dystonia have incomplete penetrance. Thus, individuals who have the dGAG mutation for dystonia could either be manifesting—individuals who exhibit the disorder—or non-manifesting—individuals who do not have the disorder but have the mutation. This project will focus on this idea of manifesting and non-manifesting patients with the dGAG mutation through the examination of exosomes and other extracellular vesicles. Specifically, this research explores the potential for exosomes to be biomarkers for the difference in manifesting and non-manifesting disease states, because exosomes are known for their role in intracellular communication and disease progression. To test this possibility, ultracentrifugation is being utilized to isolate exosomes from patient fibroblasts grown in conditioned media. Then, a bicinchoninic acid assay was run to examine protein content in both cell lysates and extracellular vesicle samples. For further testing, the plan is to run a western blot utilizing antibodies that will indicate whether or not exosomes were enriched, and then to send these samples to another lab for further testing. Potential results would be that there may be a difference in the RNA and protein composition of the exosomes between the manifesting and non-manifesting. There may also be a difference in the production of exosomes between the manifesting and non-manifesting. Finally, there could be no significant difference between these states.

Process and Abstract…

So far during my summer of research there have a combinations of highs and lows. Leading up to now, much of my research has been spent in trying to troubleshoot the issues with my project and trying countless different approaches to attempt to successful express and purify my protein of interest. Needless to say, there were definitely some frustrating days where after a week of work and anticipation resulted in a negative result. I can proudly say I have found many ways to NOT express my protein of interest. Yet even in frustrating times there were many lessons to be learned and interesting topics to understand. Often times scientists talk about how they learn the most from their failures, and this concept has never been more evident to me.

However, not too long ago I experienced the opposite of such valleys. Having tried a new bacterial strain and additive of glucose to my cultures, I was able to express and purify my protein. It was definitely a great feeling to see positive results and to see the product of countless hours of work. This result now allows me to continue moving further into the project and approach the ultimate goal of solving a structure. With the protein expressed, I can now run different assays and experiments to further test the reactivity and nature of this particular protein.

Seeing both sides of the story in research, in failure and success, has definitely made me appreciate how much there is to learn in both regards. Failure brings knowledge and information that success can’t, and same vice versa. I look forward to continuing my project and experiencing the joys and woes of research to come.

ABSTRACT:

***This is a draft, revised version emailed***

What is the structure of RAMOSA3 and what are its implications outside the trehalose pathway?

In corn, RAMOSA3 (RA3), a metabolic enzyme, is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of the RA3 protein have shown phenotypic differences in the development of corn, creating long branches at the base when compared to the wild-type corn stem. However, it is still unknown how altering this particular protein ultimately creates these phenotype differences and what exactly are the moonlighting capabilities of RA3. We suspect that RA3 has transitional regulation capabilities, whether through the regulation of RAMOSA1 (RA1), a known transcription regulator, or by its own means due to its acting upstream of RA1. We attempted many different approaches to express the plant protein RA3 into different strains of Escherichia coli (E. coli), testing many strains to find one most optimized for creating such protein. Further, we tested different conditions, altering temperature and time, in which to grow the bacteria in order to optimize protein expression. We also created a custom purification process best suited to isolate the protein of interest to run assays to test its function. Lastly, X-ray crystallography will be utilized with successfully grown crystals with RA3 in order to solve a structure. Our findings suggest that the C43 (DE3) strain of E. Coli was best suited to express this protein after being grown for three hours after induction at thirty-seven degrees Celsius. A purification process focused on the histidine tag within the protein was successful, as a process of a nickel column combined with imidazole washes isolated the protein. *Future assays and advancements in the project will allow us to reach further conclusions. At the moment, our results allow us to suspect that it will be possible to solve a structure of such protein and possibly create crystals to perform X-ray crystallography. Our future conclusions will allow us to confirm the function of RA3 as well as further develop the possibility to solving a structure.

 

*Assay results will be ready by poster presentation, along with possible other advancements into the project

 

Revised Abstract:

 

Investigating the possibility of solving a crystal structure RAMOSA3 through analysis of expression and enzymatic activity.

RAMOSA3 (RA3) is a metabolic enzyme in maize that is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of RA3 have resulted in phenotypic differences in inflorescence branching at the base of the axillary meristem within maize. However, the transcription regulation functions of RA3 and its signaling interactions with other molecules are still unexplained, making a solved crystal structure vital for further knowledge. This study investigates the possibility of crystallization through expressing large amounts of RA3 in bacteria, purifying the protein, and confirming the function of the isolated protein. We tested expression of RA3 in multiple Escherichia coli (E. coli) through Isopropyl β-D-1-thiogalactopyranoside induction and variating temperatures and growth time. Then, we designed a purification process targeting the hexahistidine tag in RA3 to isolate the protein. Sentence about assay here. Our results show that RA3 was most optimally expressed in the C43(DE3)pLysS strain of E. Coli at 37 ℃with a 3 hour growth time and isolated with a NiNTA Agarose column followed with step elution of imidazole. Sentence about assay results here. The study suggest that crystallization is possiblewith the condition of (conclusion depends on assay results).

 

How does sensory experience mediate FruitlessM and DoublesexM expression in the fly brain?

In Drosophila melanogaster, the genes Fruitless (Fru) and Doublesex  (Dsx) control sex specific sexual behavior and experience-dependent sexual behavior, respectively. Additionally, mutants of the male form of Fruitless (FruM) and flies in isolation show a lapse in sexual behavior, indicating that experience has effects on courtship behavior. However, it is unknown how experience modulates the expression of these genes, and how this modulation effects future decision making and behavior in flies. Therefore, we asked how does sensory experience mediate FruitlessM and DoublesexM expression in the brain? To answer this, we placed flies in either a group housed environment or social isolation for five days and then dissected their heads. Chromatin immunoprecipitation (ChIP) and later ChIP-qPCR were performed to examine gene regulatory differences and endogenous enrichment, respectively, in RNA Polymerase II and H3K27ac. Our data suggests that with social isolation, the transcription of FruM and DsxM decreases, specifically in Fru and Dsx connected circuits. We believe this effect is circuit specific because enrichment in controls, Choline acetyl transferase (+) and Gustatory receptor 5a (-), are insignificant. Thus, our data suggests that sensory experience plays a role in future sexual behavior in flies and may contribute to neuroplasticity in the fly brain.

Are the subpopulations of PAG neurons responsible for mice vocalization context specific?

Mice are such wonderful creatures to work with. Those that I habituate every day   have become so nice to me. But there are also those who vocalize enough to make you think yes it’s gonna work and then stop vocalizing right away to remind you the unpredictability in life. Once again, mice have taught me that you cannot force behaviors to happen naturally. Things take time in science, and that is okay! I haven’t got much data yet, but it has been great learning and practicing various techniques, and of course, working with micey of all kinds of mouse-alities.

We have adjusted the project a little bit. Below is an updated abstract:

The midbrain periaqueductal gray (PAG), a highly conserved structure, is crucial for vocalization. Lesions of PAG leads to mutism and PAG stimulation elicits vocalizations across species. Mice produce ultrasonic vocalizations (USVs) in various social contexts. Previously, we used a viral genetic tagging method CANE (Capturing Activated Neuronal Ensembles) to identify a subpopulation of PAG neurons important for female-directed courtship USVs produced by male mice. However, it is unknown whether the subpopulations of PAG neurons responsible for USVs are context specific. Here we tested the hypothesis that the same PAG-USV neurons are active when a mouse produces USVs in different social contexts. We first used immunohistochemistry to stain for the immediate early gene c-fos to examine whether PAG neurons are active when a male mouse produces USVs directed to a juvenile male social partner. We then used CANE to tag PAG neurons activated when a male mouse produced female-directed USVs, and c-fos staining to identify active neurons when the same male mouse produced either female-directed or juvenile male-directed USVs. Using confocal microscopy, we examined the overlap between neurons active during the same or different social contexts. Findings from this study can better our understanding of mammalian vocal control at neuronal level.

 

Week 6 – Will the Data Conflict?

To start off this post, I just want to say how surprised I was yesterday when I thought about how there are only 2 weeks left until BSURF ends. On Friday I performed my last RIA for the summer, and this upcoming week my mentor and I will start analyzing the data we collected so far. Although I’m excited to learn about the statistical analyses used in the research my mentor does (while hanging out more with her dogs), I haven’t been away from the bench since the first week of BSURF. And while I performed 27 RIAs this summer, whether or not my mentor and I find meaningful results from our data is not 100% guaranteed. Regardless of what our results show, I’ve had an amazing time in the lab and feel great about the work I’ve done so far.

Abstract: Do Alpha and Low-ranking Male Yellow Baboons Experience Different Sources of Stress?

In yellow baboons, Papio cynocephalus, an individual’s dominance rank reflects their access to resources, which can result in varying levels of stress. A previous study found that the concentration of fecal glucocorticoid (fGC) increases as rank decreases in males with the exception of the highest-ranking alpha male, who possessed a significantly high concentration of fGC. While high levels of fGC were identified in alpha and low-ranking males, the major sources of stress between the two remains unknown. Triiodothyronine (T3) radioimmunoassays were conducted on fecal samples collected during a longitudinal study on a wild population of yellow baboons. We hypothesize that alpha males possess low concentrations of fecal T3 (fT) due to participation in mating activities and agonistic interactions that cause high energetic stress, which suppresses T3 secretion. Alternatively, low-ranking males will have high concentrations of fT since they primarily experience psychosocial stress caused by limited food access and harassment by higher-ranking males. Differences in stress between alpha and low-ranking males provides insight on the range of social challenges experienced within a natural population. Further research on other wild primate populations can deepen our knowledge of the similarities between how social status relates to stress among primates.

Studying essential genes involvement in Cryptococcus deneoformans sexual reproduction using regulated expression constructs.

Cryptococcus deneoformans is an opportunistic human fungal pathogen that mainly infects immunocompromised patients, including AIDs patients and patients with organ transplants. It infects approximately 280 thousand people and contributes to about 180 thousand deaths per year. Few strategies are available in treating Cryptococcosis. Dr. Heitman’s lab discovered two essential cell cycle regulating genes involved in the endoreplication pathway during C. deneoformans unisexual reproduction. This research focuses on developing strategies in studying these two essential genes. We generated expression constructs using the copper repressive promoter pCTR4-2 and the galactose-inducible promoter pUGE2 to modulate gene expression. We also used DAmP technique to reduce gene expression by disrupting the terminator sequence. By up- and down-regulating these genes, we would like to examine how perturbation of gene expression could impact C. deneoformans unisexual reproduction and other cellular processes. Study of essential genes also provides the opportunity for discovery of novel drug targets that are essential for cell growth. We are interested in testing how these genes impact the virulence of the pathogen by using DAmP alleles in different animal models. Based on this work, we could potentially broaden essential gene studies in both C. deneoformans and other fungal pathogens and provide hope in novel anti-fungal target discoveries.

Episode 6- Abstract and Possible Progress?

Progress on my project has unfortunately been quite slow, due mostly to unavoidable issues that cropped up earlier into the process. In particular, the change from using FL green sea urchins to NC green sea urchins. Given that the project started originally with using the genomic information gathered from FL urchins and the vast genetic variation between population of sea urchins, the guide RNA created for the FL urchins, while successful on those urchins, has proven to be little effective on the NC variety. The reason for the switch from FL to NC urchins is the season in which the animals are reproductively active and thus when their eggs are most healthy for injections.

However, with the lack of genomic sequencing of NC-originating Lytechinus urchins, it has been impossible to create gRNA specifically for the urchins we are currently using. Luckily, one of the members in the lab has recently been sequencing some sections of the NC sea urchin genome, so we may soon be able to design new guides that have a higher effectiveness, assuming that section of the genome has been sequenced and the gRNAs initially created are indeed different enough to warrant new guides.

On a brighter note, I’ve been practicing injections most days of the week and have been able to better hone my skills on the practice as it can often take months to learn how to inject efficiently. Early on, I would often take up to an hour trying to inject a single plate of embryos and awhile to setup my materials for the day. Now, I’ve managed to better refine the protocol and can have everything ready in an hour and do one plate every 20 minutes. Though that’s still a bit long (the optimum time being 15 minutes) and I still need to work on injecting more embryos in that time frame, I feel as though I’ve improved quite a lot and learned a number of things in the process.

Abstract- What Are the Functions of Genes in the GRN of Sea Urchins?

Sea urchins are one of the most useful model organisms for studying early development; however, much is still unknown about the specific functions of genes within the network that regulates urchin development. One gene that has been well-studied is Endo16 and its importance in gut formation. Using CRISPR/Cas9 technology, this project has focused on producing successful knockouts of Endo16 by targeting the gene’s enhancer region. Thus far, there has been moderate success with knocking out Endo16 in Lytechinus embryos, though further experiments are needed to improve the success of the Cas9 knockouts by taking into consideration the significant genetic variation between populations of Lytechinus. Once the percentage of embryos that are injected produce Endo16 knockouts equivalent to the percentages seen in similar studies using other gene silencing techniques, the project will move forward with knocking out other genes whose functions are less well-understood compared to Endo16. This will further our understanding of the gene functions and relationships within the gene regulatory network of sea urchins, and allow comparative studies of gene function in Lytechinus and the closely-related, yet developmentally very different, species, Heliocidaris erythrogramma.

Hsp Regulation by PI3P is important for Plasmodium Falciparum

Throughout the world millions of people are affected by Plasmodium Falciparum, a parasite responsible for the transmission of the deadliest form of malaria.  Several molecules in the parasite work to keep it alive during transmission and infection; PI3P is one such lipid necessary for parasite growth but with an unknown signaling pathway. Previous work by the Derbyshire lab indicated that PfHsp70– a parasite heat shock protein– was a potential candidate for binding to PI3P in the parasite life cycle. Within PfHsp70 is a LID domain, usually responsible for binding to anionic lipids like PI3P. We believe that deletion of the LID domain in PfHsp70 will decrease its binding affinity to PI3P. To test this, both the Wild Type and LID-deleted PfHsp70 proteins were expressed (in yeast) and purified. Isothermal Titration Calorimetry will be used to quantitatively assess changes in binding affinity. In the ITC both the purified Wild Type and LID-deleted proteins will react with PI3P to produce a binding curve with heat released/absorbed plotted against concentration of PI3P. If a lower binding affinity is measured in the LID-deleted interaction, we will potentially know more about PI3P signaling and ways to disrupt PI3P synthesis to cause parasite death.

That’s Life…science

A song made famous by Frank Sinatra, That’s Life, talks about some important facts/lessons about life that also connect to science.

The song talks says life is all about the highs and lows about life.  Science, as I found out, is also all about the highs… and the lows Things do not always go according to plan.  The deletion that you wanted to make, does not delete.  Sometimes, you may try a mutagenesis reaction multiple times with no luck, but that is okay.  Science takes time, and eventually, it will come back around and you may get a result, so don’t quit.

Over the summer, I attempted to delete Sec24D from SW1353 (a chrondrosarcoma cell line), and SAOS-2 (a osteosarcoma cell line) with no luck. Additionally, I’ve attempted several mutagenesis reactions with minimal successes.  These were the lows.  However, I did have some highs. Other projects and experiments went smoothly, and I am happy for all experiences.

I am also thankful for my successes, and “failures”.   There is more to say about what I did over the summer, but I would like to save it for my poster presentation that will be held on July 27, 2018, and future blogs.

As a teaser for the poster presentation, I will post a draft (science is all about sharing current knowledge about a specific topic, so please understand this is subject to change) of my project’s abstract.

 

What role does SEC24D O-GlcNAcylation play in collagen secretion?

The COPII complex mediates traffic from the endoplasmic reticulum (ER) with five major components: SAR1, SEC13, SEC23, SEC24, and SEC31. Posttranslational modification plays an important role in the regulation of the COPII complex. Our lab and others have found that O-linked β-N-acetylglucosamine (O-GlcNAc), a single sugar modification added to serine and threonine of intracellular proteins, decorates many COPII components. I mainly focused on O-GlcNAcylation of SEC24D, a subunit of the COPII complex, because in humans, SEC24D mutations cause a subtype of osteogenesis imperfecta, a collagen trafficking disorder. I wanted to answer the question, “What role does SEC24D O-GlcNAcylation play in collagen secretion?” I hypothesized that SEC 24D O-GlcNAcylation is necessary for normal collagen secretion. To test this hypothesis, I am deleting SEC24D in osteosarcoma cells using CRISPR-CAS9 and creating unglycosylatable SEC24D mutants to reintroduce into the SEC 24D deleted cell line.  After reintroducing the mutants, I will look at collagen secretion using immunofluorescence microscopy (IF) to determine the role of SEC24D O-GlcNAcylation in collagen secretion. We expect the IF to show more collagen in the ER of the mutants than the wild type, showing SEC24D O-GlcNAcylation is important in collagen secretion.

 

Ayana’s Abstract

Cryptococcus neoformans is a fungus that is most commonly responsible for the disease meningoencephalitis, an infection of the central nervous system, and is potentially fatal in immunocompromised individuals such as those with AIDS. In addition, research related to C. neoformans has concluded that melanin production allows the fungus to cause disease and that the transcription factor, BZP4, is associated with this phenotype. This data leads one to question whether BZP4 actually has an influence on the fungus’ ability to cause disease. In order to test this theory, a strain of C. neoformans H99 with BZP4 absent was restored with the BZP4 gene via biolistic transformation and used to inoculate a 30 mice along with the deletion strain and wild type and observed  for 42 days for virulence and fungal burden. By the end of the experiment, it is expected that the reconstituted strain and wild type will present the most cell counts in the mice on average compared to the deletion strain. These two strains may also be able to cause disease and kill the mice they infected. Based on previous data, one can predict that the transcription factor, BZP4, is crucial  for C. neoformans virulence.

 

*The data does not exist yet but will in a couple of weeks

How things are going so far

My activities have slowed down in the recent couple of days because the lab is starting its mice experiments. All that is required is to grow my cultures out and prepare them into an innoculum for the mice. After that I simply inoculate the mice and watch them for days and record what I see. To be honest, this week has mostly consisted of me trying to get building and ID clearance so I can actually check on the mice for days to come. I have been able to get ID clearance but I’m waiting on going through orientation for the animal facility next week in order to actually observe the mice on my own. I did however innonculate 3 mice on my own, so I am proud of that.

I am a little worried in terms of time because I don’t actaully harvest the mice until the week of the poster session. As a result, I will probably be scrambling to collect the data from the dissection. In fact, we dissect on the day I’m supposed to submit my final draft poster for printing. So in short, I’m a little stressed as to how this is going to play out. I’m prepared to at least explain what the results appear so far in the poster.

Other than this, my experiments last week were successful for the most part. Although, I did make a mistake last week while doing  a PCR.

I was so confused when they came out. I didn’t think it was physically possible, but I made it happen. Basically, I screwed the canister that holds the tubes in the machine too tight and they melted. I couldn’t even get the pipette inside some of the tubes. So I had to do some of them over.