Category Archives: Week 6

The prevalence of neurodevelopmental disorders (NDDs) has increased rapidly in recent decades. Genetic mechanisms of these disorders have been studied extensively; however, environmental factors contributing to NDD development remain less understood. Current evidence suggests that alterations in synaptogenesis and pruning underlie NDD pathology. Sleep is essential to this synaptic remodeling process. Disturbed sleep is a highly conserved trait affecting nearly 86% of NDD patients. Additionally, sleep appears to be particularly sensitive to environmental perturbations, as negative sleep outcomes have been strongly linked to exposure to air pollutants. The Diesel Exhaust Particle and Maternal Stress (DEP/MS) paradigm was used to co-expose pregnant mice to DEP and a maternal stressor, modeling epidemiological data suggesting that combined prenatal exposure to DEP is strongly linked to socioeconomic stressors. Previous studies have revealed that DEP/MS offspring show male-specific social and behavioral traits consistent with NDD pathology. Based on preliminary data demonstrating sleep deficits in DEP/MS adult offspring, this study aims to characterize NDD phenotype of DEP/MS offspring during adolescence, focusing on possible female-specific sleep and behavioral alterations. To analyze sleep patterns in DEP/MS and control offspring, we performed electroencephalography and electromyography (EEG/EMG) recordings from P37 to P40. Additionally, we isolated astrocyte and microglia populations from parietal and frontal cortex tissue. Lastly, we conducted the forced-swim test to analyze depressive-like behavior, a common comorbidity in female NDD patients. We hypothesize that DEP/MS offspring will exhibit sex-specific depressive-like behavior and sleep deficits driven by changes in astrocyte gene expression.

Caenorhabditis elegans that hatch into an environment without any nutrients are arrested in the first larval stage of development, L1. Larvae can remain in L1 arrest for weeks and resume normal development once they receive food. However, many C. elegans that experience extended L1 arrest develop abnormal gonads in adulthood, including germ-cell tumors and uterine masses. These growths suggest a misregulation of cell growth. Previous results indicate that genes involved in processing somatic RNA interference (RNAi) could be causing abnormality formation. RNAi refers to a cell’s response to exogenous or endogenous double-stranded RNA. It functions as an internal regulatory mechanism as well as an immune response, allowing sequence-specific suppression of corresponding genes. We hypothesize that endogenous RNAi pathways are involved in the occurrence of starvation-induced gonad abnormalities. It is possible that aberrant small RNAs produced by somatic RNAi machinery are transported to the germline, causing abnormalities to form. We examined rrf-1, ppw-1, rde-1, dcr-1, rde-4, sid-1, alg-1, and rrf-3, which correspond to components of various RNAi pathway branches. Knockout strains and RNAi were used to evaluate the impact of these genes on gonad abnormality frequency in adults following 8 days of L1 arrest. So far, results align with previous findings about somatic RNAi and our transportation hypothesis. We expect future results to further corroborate these findings and clarify the role of different RNAi pathway branches in gonad abnormality formation. Overall, these experiments provide greater insight into the little-known mechanisms regulating pathologies that result from early-life starvation.

Camila Rodriguez

Mentors: Anya Varanko, Ashutosh Chilkoti, PhD.

Department of Biomedical Engineering

Elastin-like polypeptides (ELPs) are proteins based off of tropoelastin, a subcomponent of elastin, that are able to change solubility under different temperatures. By combining these proteins with the affibodies ZEGFR and ZHER2, the proliferation of cancer can be limited much more effectively than bispecific antibodies. ELPs were combined with affibodies in E. coli plasmids to create different protein constructs, purifying the proteins using heat cycling. SK-BR-3 cancer cells positive for both EGFR and HER2 were then treated with the previous proteins to create a proliferation assay. Each construct maintained the same amount of proliferation as the control at all concentrations, resulting in no color change for the assay. This suggests that the constructs had no effect on the growth of SK-BR-3, and both affibodies were unable to block the receptors. The potential of ELPs and bispecifics still require further investigation, as it may be possible ELPs inhibit the binding of the affibodies, that ZEGFR and ZHER2 are incompatible with ELPs, or other outside factors. The use of other bispecifics, as well as maintaining temperature while performing the assay, will be explored as research in ELPs continue. 

The activation of type I interferons has long been reported to regulate pain, but the precise mechanism has remained unclear. We tested the hypothesis that type I interferons are critical pain regulators and function via a neuronal mechanism. Sensory, motor, and behavioral testing in addition to immunohistochemistry were performed on type I interferon receptor knockout (Ifnar1 KO) and wild type (WT) mice. Heightened sensitivity in KO mice suggests an importance of type I interferons in regulating pain under basal conditions. The lack of any difference in motor performance and quantity of nerve fibers in tissue of WT and Ifnar1 KO, along with RNA scope, support a neuronal-specific mechanism of interferons in regulating pain. These findings suggest that type I interferons are critical in regulating both pain and neuroinflammation by a neuronal mechanism, even under normal, healthy conditions. These findings redefine our understanding of the role of interferons in pain and could translate to improved treatment of a heterogeneous array of chronic pain.

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 Cell, 41(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

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.

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.

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

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.

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.

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).

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.

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.

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.

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.