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Diana Bautista, Department of Molecular and Cell Biology, University of California, Berkeley

"Examining Sex Differences in CIPN-Associated Neuropathic Pain"

Abstract:

Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating chronic pain condition that affects about 40% of cancer patients undergoing chemotherapy (1). Previous studies have reported sex differences in the development and prevalence of neuropathic pain (2). However the molecular mechanisms that drive pain hypersensitivity in some chemotherapy patients are unknown. I will test the hypothesis that there are sex differences in CIPN-associated pain by measuring mechanical pain behaviors in vivo. I am using a Paclitaxel model of CIPN and injecting mice on alternating successive days, over an 8 day period, triggering neuropathic pain in mice. I am measuring baseline mechanical sensitivity using calibrated force probes called Von Frey filaments to determine the pain thresholds in both sexes and then performing the same measurements after the Paclitaxel treatment on Day 12. Consistent with published literature, I’ve observed that both female and male mice develop mechanical hypersensitivity in this CIPN model. My preliminary data shows that there are differences in CIPN-associated pain between the sexes. Males developed more mechanical pain hypersensitivity than females (n= 6 males and 6 females; p=0.02). The goal of this project is to repeat this experiment with two additional cohorts of animals and compare the function of peripheral pain neurons isolated from the dorsal root ganglia of male and female Paclitaxel-treated mice. Examining sex differences of mechanical hypersensitivity can help us better understand why some individuals develop painful neuropathy and others do not, leading to the development of drugs and therapies to treat CIPN-evoked pain that are tailored specifically to the sexes.

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Andrew Dillin, Department of Molecular and Cell Biology, University of California, Berkeley

"4 Glial Cells Induce the Mitochondrial Unfolded Protein Response Cell Non-Autonomously in C. elegans"

Abstract:

Mitochondria have a specialized mechanism for maintaining protein homeostasis, the mitochondrial unfolded protein response (UPR­mt). It is known that its induction across the body can provide stress resistance and longevity benefits. Previous research has shown that it can be induced cell non-autonomously, that is activated by extracellular signaling, rather than a stress inside of the cell. Here, we attempt to answer whether UPRmt induction in the four cephalic sheath glia (CEPsh) is able to activate a non-autonomous response and increase lifespan and stress resistance.

We find that by expressing the UPRmt activating protein JMJD-1.2 under the control of the CEPsh specific promoter HLH-17p we are able to induce a stress response in distal tissues, which increases oxidative stress resistance and lifespan. We also test autonomous regulators of the UPRmt and demonstrate that this induction is dependent upon the transcription factor ATFS-1.

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Denis Titov, Department of Molecular and Cell Biology, University of California, Berkeley

"Optimizing and Improving the Inducible Expression of the Q System In Caenorhabditis elegans"

Abstract:

The ability to temporally control gene expression using inducible promoters is instrumental to studying biology. Currently, there are limited options in the toolkit of Caenorhabditis elegans allowing for this type of inducible expression, so we are attempting to improve the available tools in this area. We are working to optimize and improve previous research of implementing the Q system, an inducible expression system, into C. elegans. Currently, this system has not been extensively researched in C. elegans. The work that has been done focused primarily on neuronal promoters and we have found that using a ubiquitous promoter, QF appears to be toxic. Additionally, previous research only tested the QF activator, but since then new variations have been developed and tested in Drosophila. We have assembled a QF2 variant that is non-toxic and are working to optimize its induced expression levels. By changing the relative levels of QF2 to QS, we hope to minimize the bleed through in the absence of quinic acid while maximizing expression in its presence.

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David Schaffer, Department of Molecular and Cell Biology, University of California, Berkeley

"Minimizing off-target effects of CRISPR/Cas9 with an adeno-associated virus (AAV) delivered self-inactivation system"

Abstract:

CRISPR/Cas9 is a versatile genome-editing technology that has the potential to treat any disease-causing gene. This complex is comprised of a nucleolytic Cas9 protein and single guide RNA (sgRNA) that directs Cas9 to the gene of interest. More than one sgRNA can be co-delivered with the Cas9 protein, allowing for simultaneous editing at multiple genes, using adeno-associated virus (AAV) as a delivery mechanism. However, a major safety concern with AAV is the formation of stable episomal DNA that would cause persistent expression of Cas9 and increase the risk of creating cancerous cells. To address this problem, we have designed two self-inactivation strategies to reduce Cas9 activity while retaining editing at the gene of interest. The first strategy uses AAV to co-deliver two sgRNAs: one targeting the gene of interest and another targeting the Cas9 transgene. This method shows a significant decrease of Cas9 mRNA relative to a non-inactivation control. In addition, to achieve sufficient genomic editing and a therapeutic effect prior to inactivation, we demonstrate that a single nucleotide mismatch in the Cas9-targeting sgRNA could slow down the rate of Cas9 inactivation. Our second strategy utilizes a sgRNA to target the inverted terminal repeats (ITR) of the Cas9 transgene, causing its destabilization and degradation. This method successfully shows decreased Cas9 mRNA expression over time. For both strategies, on-target editing is maintained at the gene of interest. These self-inactivation strategies establish a critical proof-of-concept that Cas9 expression can be manipulated to reduce off-target activity while retaining on-target editing for future therapeutic applications.

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Polina Lishko, Department of Molecular and Cell Biology, University of California, Berkeley

"Unconventional endocannabinoid signaling in the mammalian ovary and its role in ovarian aging."

Abstract:

The α/β hydrolase domain-containing protein 2 (ABHD2) has been identified as a unconventional endocannabinoid signaling pathway that is initiated by a progesterone association with ABHD2 in the male sperm cells. The data shows that ABHD2 is also abundant in the ovarian stromal cells. Our goal is to assess ABHD2 expression as well as its interaction with progesterone depending on mouse estrous stage. To be able to observe the interaction we used the modified progesterone (P4*) which allows preservation and visualization of its interaction with ABHD2. P4* cross-links with adjacent proteins in a UV-dependent manner and contains biotin moiety which allows visualization by fluorescently labeled streptavidin. Our preliminary results confirm membrane localization of ABHD2. The use of the modified progesterone showed promise in detecting ABHD2-progesterone interaction. However, the lack of structural information about hABHD2 limits our understanding of the mechanism of progesterone activated regulation of its activity. Further study of the structure of ABHD2 protein by cloning extracellular domain of ABHD2 using pET28a plasmid for prokaryotic expression system, then expression in E.coli BL21(DE3) and protein purification, will show how the binding mechanism of progesterone triggers the signaling pathway through ABHD2.

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Christine Wildsoet, School of Optometry, Unviersity of California, Berkeley

"Effect of Retinal Simultaneous Competing Defocus of Opposite Sign on BMP Gene Expression in Chick RPE"

Abstract:

Myopia or near-sightedness is a growing public health problem that is projected to affect 50% of the world’s population by 2050. This type of refractive error is caused by the eye growing abnormally long, resulting in distant objects being focused in front of the retina instead of on the retina, thus reducing visual acuity. Because myopia is associated with potentially blinding pathology, it is important that we understand myopia in terms of eye growth, to develop better myopia treatments in the future. Of relevance to this study, the expression of bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β (TGFβ) superfamily, in the chick retinal pigment epithelium (RPE) was previously found to be sensitive to the optical defocus experience of the retina. Specifically, BMP2, BMP4, and BMP7 showed defocus sign-dependent, bidirectional changes in RPE gene expression that were also associated with changes in ocular growth. In brief, myopic defocus, imposed by single vision (SV) positive (+10 Diopter (D)) lenses, slows down eye growth and up-regulates BMP gene expression, while hyperopic defocus, imposed by SV negative (-10D) lenses, speeds up ocular growth and causes BMP down-regulation. In other studies, dual-power Fresnel lenses (FL), which allowed myopic and hyperopic defocus to be simultaneously imposed on the retina, were found to slow ocular growth, and in some cases, decelerate myopia progression across a range of species, e.g., chickens, rhesus monkeys, marmosets, and guinea pigs. However, the effect of such complex retinal defocus stimuli on RPE BMP gene expression has not been examined. Here, I sought to extend these investigations by examining the effects on gene expression patterns of BMPs in chick RPE due to Fresnel lens treatments that impose competing retinal defocus of opposite sign simultaneously. 14-day old White-Leghorn chicks wore monocular +10D/-10D Fresnel lenses (FL) with concentric annular zones of alternating powers for three different treatment periods, i.e., 15 min, 2 h, and 48 h. At the end of the treatments, the RPE samples were collected for differential gene expression analysis by reverse transcription semi-quantitative polymerase chain reaction (RT- qPCR) for BMP2, BMP4, and BMP7. From our qPCR results, the +10D/-10D FL treatments induced significant up-regulation of RPE BMP2, BMP4, and BMP7 gene expression, although varying with the treatment duration, i.e., 15 min, 2 h and 48 h for BMP2; 2h and 48 h for BMP4; and 48 h for BMP7. Altogether, this result, showing a dominant effect of myopic defocus over hyperopic defocus when the two competing defocus stimuli are presented simultaneously, implies that the retina and/or RPE’s response to myopic defocus is relatively more robust than that to hyperopic defocus. Also, these findings provide further supporting evidence that BMPs produced by chick RPE might play important roles in the retino-scleral signalling pathway that regulates eye growth, and thus the development of myopia.

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Gloria Brar, Department of Molecular and Cell Biology, University of California, Berkeley

"Investigating the Role of Alternative mRNA Transcripts in SOD1 During Meiosis"

Abstract:

Superoxide dismutase 1 (Sod1) is a highly abundant and conserved enzyme that plays many important roles, including neutralizing harmful superoxide radicals and regulating cellular respiration. During mitotic growth, budding yeast express a canonical mRNA, SOD1canon., that is approximately 600 nucleotides long. During meiosis, cells produce a dramatically extended mRNA isoform, SOD1LUTI (for Long Undecoded Transcript Isoform), that is 2,200 nucleotides long, the expression of which coincides with a decrease in SOD1canon. mRNA and Sod1 protein. Meiosis specific mRNA isoforms have been shown to repress canonical mRNA and protein expression in other proteins, but it was previously unclear whether SOD1LUTI functioned similarly. By blocking SOD1LUTI in meiosis and forcing expression in mitosis, I found that the SOD1LUTI is both necessary and sufficient in decreasing SOD1canon. and Sod1 protein. With this information, I moved on to investigate the potential significance of meiotic downregulation of Sod1 by studying two of its substrates. Yck1 and Yck2, casein kinase paralogs, are stabilized by Sod1 in mitosis, but little is known about their meiotic expression and activity. I found that levels of Yck1 and Yck2 are dynamic in meiosis, which agrees with previously obtained meiotic mass spectrometry data. Preliminary results suggest that disruption of SOD1LUTI has a modest effect on Yck1 and Yck2 activity, but more analysis is required. If we do find a link between Sod1 and Yck1/Yck2 expression in meiosis, future studies will include directly examining Sod1-Yck1/Yck2 interactions and looking at localization of Sod1 in relation to its function. Sod1 is an essential enzyme, and a better understanding of its regulation in meiosis is important both for improving our knowledge of Sod1 protein and the larger function of alternative mRNA expression in gene regulation.

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Matthew Springer, University of California, San Francisco

"The role of e-cigarette-derived glycotoxins on vascular impairment and atherogenesis"

Abstract:

The use of electronic cigarettes (e-cigs) has been increasing recently due to their advertisement as a healthier alternative to cigarettes and the introduction of various flavors of e-liquids. Although many health risks of cigarette usage have been studied for several decades, the health effects of e-cig usage are relatively unknown. E-cig usage is associated with a high risk of atherogenesis from increased platelet activation and aggregation, increased oxidative stress, endothelial dysfunction. One of the possible mechanisms that may explain the vascular impairment and pro-atherosclerotic properties from e-cig usage is the interaction between advanced glycation end products (AGEs) and their common receptor RAGE. AGEs are the modification in proteins, lipids, or nucleic acids that result from glycation or oxidation by aldose sugar and its byproducts, lipids, or nucleic acids. Elevated levels of serum AGEs are commonly seen in patients with diabetes primarily due to the high blood glucose level. Although e-cig usage is not known to be associated with an increase in blood glucose level, the generation of AGE precursors, such as methylglyoxal, glyoxal, and acetaldehyde, from e-cig aerosol suggests the potential synthesis of AGEs in human body e-cig usage. The direct tissue deposition of AGEs and its interaction with extracellular membrane can increase arterial stiffness, trapping of macromolecules in the vascular wall, the risk of plaque build-up, and the adhesion of circulating blood cells. AGE-RAGE interactions are known to increase intracellular ROS, increase expression of adhesion molecules, decreasing nitric oxide (NO) production, lowering eNOS protein expression, alter gene expression and activate downstream NF-kB that contributing to an increase in pro-inflammatory cytokines and vascular dysfunction. Treatment of HUVECs with serums from e-cigarette users significantly decreased nitric oxide production and significantly increased vascular permeability. Although there was no significant decrease in eNOS protein expression, there was a clear trend that indicate lower protein expression among e-cigarette users. NOS3 gene expression was not significant across the groups, indicating potential post-translation modification of eNOS protein. However, these changes clearly indicate that e-cigarette can cause endothelial dysfunction and may increase cardiovascular disease risk. Works that were postponed by COVID-19 will be done in the future to link the role of AGE-RAGE axis in e-cigarette-induced vascular impairment.

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David Bilder, Department of Molecular and Cell Biology, University of California, Berkeley

"Identification of a Drosophila complement protein regulating tumor immune surveillance and host survival"

Abstract:

Paraneoplastic syndromes have been found to be a major component of both human and fly cancer physiology, but the molecular mechanisms by which these syndromes kill their hosts remain unclear. Using Drosophila melanogaster cancer models, the Bilder lab has identified numerous secreted proteins that are upregulated in a malignant tumor, and we hypothesize that these secreted proteins are molecular effectors by which tumors impact host physiology. Through genetic screening of many of these secreted proteins, we have focused on Tep3, a homolog of vertebrate α-2-macroglobulins and a component of the Drosophila innate immune system. Knockdown of Tep3 within the tumor via either of two independent RNAi lines results in a significant increase in the lifespan of tumor-bearing flies. We tested multiple paraneoplastic effects through which tumors impact host physiology and found that Tep3 knockdown significantly increases hemocyte recruitment to the tumor. This finding suggests that Tep3 may play a role in the tumor’s ability to evade recognition by the host immune system and, more broadly, suggests a connection between mammalian α-2- macroglobulins and cancer progression.

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Kristin Scott, Department of Molecular and Cell Biology, University of California, Berkeley

"Exploration of the bitter gustatory circuitry involving the novel interneuron Eilish in Drosophila melanogaster"

Abstract:

To avoid the ingestion of potentially harmful substances, animals use their taste system to detect potential toxins in their food, which register as an aversive “bitter” taste. Understanding the bitter gustatory circuitry of Drosophila melanogaster provides valuable insight into how detection of bitter compounds can shape feeding behavior. Currently, little is known about the circuitry downstream of bitter sensory neurons. Using an electron microscopy data set of a whole Drosophila brain generated by the Bock group at Janelia Research Campus, I reconstructed multiple bitter sensory axonal projections at synaptic detail. I used these sensory projections to identify Eilish, a novel second-order neuron immediately downstream of bitter taste inputs. To comprehensively evaluate the gustatory inputs of Eilish, I am currently annotating all synapses of this interneuron, which will allow me to explore whether Eilish receives input exclusively from bitter sensory neurons or whether it integrates information of other taste modalities such as sugar as well. Furthermore, I am using light level circuit tracing tools in order to gain an overview of the neuronal ensemble immediately downstream of bitter sensory neurons. This will allow me to draw conclusions about the overall number of second-order bitter neurons in addition to Eilish. Ultimately, I am also working to generate tools to access Eilish genetically, which will allow for behavioral studies of this novel cell type. My studies will expand our knowledge of bitter taste processing in Drosophila and contribute to our understanding of how sensory information can shape feeding behaviors.

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William Weiss, University of California, San Francisco

"Targeting TGFβ pathway dependencies in group 3 medulloblastoma"

Abstract:

Medulloblastoma is the most common malignant brain tumor in children. While 75% of patients have favorable prognosis, long term survivors have significant neurocognitive and neuroendocrine deficits. Medulloblastoma is comprised of 4 distinct subgroups (WNT, SHH, Group 3 and 4). C-myc (MYC) amplified group 3 medulloblastoma (Grp3 MB) has the worst prognosis with poorly understood biology, insufficient representation in mouse models, and as a result few actionable targets for therapies. Amplification of a group of genes called TGFβ family genes commonly occurs in Grp3 MB, often in conjunction with amplification of the MYC oncogene, however functional roles have not been demonstrated. We have established the first humanized model for Grp3 MB from human pluripotent stem cells (hiPSC) differentiated to neuroepithelial stem (NES) cells. Using this model as a tool to discriminate drivers from passengers, we demonstrated that TGFβ effectors drive tumor formation in vivo alone, and in combination with MYC. I therefore hypothesize that the TGFβ pathway contributes to the intrinsic resistance of Grp3 MB through deregulation of key genes and pathways. To test this hypothesis, I propose to characterize the transcriptional signatures altered in TGFβ driven Grp3 MB, utilizing a panel of NES-derived cells and tumors transduced with MYC alone and in combination with TGFβ effectors. I will integrate my findings and evaluate clinical agents that target the TGFβ pathway, including agents in phase I/II clinical trials. Finally, I will test efficacy in NES-derived tumors and human PDX models to determine if these new agents can overcome therapy resistance. Promising results from cell culture will be validated using in-vivo models, and treating animals in-vivo.

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Arash Komeili, Department of Molecular and Cell Biology, University of California, Berkeley

"Investigating the Role of Ferrosomes in Iron Homeostasis in Shewanella putrefaciens"

Abstract:

In response to a universal need for iron, bacteria have developed several mechanisms to regulate iron import, efflux, and storage. Iron storage systems, such as the proteins ferritin (Ftn) and bacterioferritin (Bfr), function by mediating oxidative stress and preventing iron starvation. An additional iron storage organelle, the ferrosome, was recently discovered in phylogenetically diverse bacteria, including the metal-reducing microbe Shewanella putrefaciens. In past experiments, deletion of the “fez” genes required for ferrosome formation resulted in an increased lag time under iron starvation conditions. However, little is known as to how ferrosomes function in tandem with other systems to maintain iron homeostasis in S. putrefaciens. To this end, I have constructed mutant strains of S. putrefaciens with single, double, and triple deletions of the fez, ftn, and bfr genes. Iron starvation trials with these mutants suggest that ferrosomes are the primary iron storage compartment during anaerobic growth. These results prompted my interest in exploring the regulation of ferrosomes and other iron storage systems. In many bacteria, Fur, a global regulator of gene expression, is responsible for managing the overall influx and efflux of iron by controlling the transcription of ironsequestering genes. My deletion of the fur gene in S. putrefaciens resulted in cells that no longer produced ferrosomes visible by transmission electron microscopy, suggesting that Fur may positively regulate ferrosome production. Currently, I am probing the expression of the fez, ftn, and bfr genes in the ∆fur mutant and analyzing the broader Fur regulon with RNAseq to further develop these observed phenotypes. I am also purifying the FezB protein, a heavy metal cation transporter intrinsic to the fez operon, in order to understand how ferrosomes actively sequester iron. Ultimately, these studies will elucidate the mechanisms that regulate the expression and activity of a novel mode of iron stress response in bacteria.

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David Weisblat, Department of Molecular and Cell Biology, University of California, Berkeley

"Exploring the Function of lox5 in Helobdella Development: A Possible Role in Leech Nephridial Differentiation"

Abstract:

The hox gene lox5 in Helobdella austinensis, a glossiphoniid leech in Phylum Annelida, is expressed transiently in a prominent mesodermal cell during embryonic segment differentiation. To investigate the significance of this previously published expression pattern, my postdoctoral research mentor and I have perturbed the function of lox5 with CRISPR/Cas9-induced mutagenesis. Our experiments yield mutants characterized by substantial bloating during late embryogenesis. We have demonstrated the specificity of this CRISPR effect by: 1) observing the same phenotype for two different - and separately injected - gRNAs targeting lox5, 2) finding a substantial loss of wildtype alleles and the emergence of indel alleles in our lox5 CRISPR-injected embryos, and 3) finding a significantly higher presence of lox5 frameshift mutations in bloated vs unbloated CRISPR-injected embryos. We believe the bloated phenotype is caused by an inability to excrete fluids; therefore, the lox5-expressing mesodermal cells may either be nephridioblasts (the precursor cells of segmental kidney-like organs called metanephridia) or cells that indirectly regulate nephridial development. Work done by previous Weisblat lab members indicates that inx4 is a marker of nephridial differentiation. We find substantial reduction of inx4 expression in a high proportion of our lox5 CRISPR-injected embryos. This supports the hypothesis that lox5 functions to specify nephridioblasts. To help identify the lox5-positive cell type, we will conduct experiments to determine whether lox5 transcripts co-localize with those of pax3/7a, a transcription factor that was shown previously to be expressed in metanephridial precursors. Toward this end, we will combine fluorescent lineage tracing of mesoderm with fluorescent in situ hybridization (FISH) for pax3/7a to clarify the segmental position and cell morphology of the nephridioblasts. We will then combine the lineage analysis with FISH for lox5 to test whether it is expressed in nephridioblasts or adjacent cells. Lastly, to test whether lox5 mutation is affecting the development of non-nephridial mesodermal tissues, we will analyze other mesodermal elements in lox5 CRISPR-injected embryos. For example, we will use phalloidin staining to assess muscle development, and we will analyze the pattern of fluorescent lineage tracer and Hoechst nuclear label to assess visceral mesoderm development.

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Roberto Zoncu, Department of Molecular and Cell Biology, University of California, Berkeley

"Examining the Role of Phospholipase B Domain Containing 1 (PLBD1) in Pancreatic Cancer"

Abstract:

Pancreatic Ductal Adenocarcinoma (PDA) continues to have a poor prognosis and is currently projected to become the second leading cause of cancer-related deaths. PDA cells in culture exhibit significantly different cell metabolism from non-transformed cells, with a reliance on nutrient scavenging pathways like autophagy and macropinocytosis that converge on the lysosome. This reliance on the lysosome creates a liability that could be exploited therapeutically. PLBD1 (phospholipase B-like protein 1) is a protein shown to be highly upregulated in PDA cells but not in non-cancer pancreatic cell lines.

Determining what drives PLBD1 expression will help determine the role of PLBD1 in PDA, and perhaps why it is upregulated in exclusively PDA cells. Past research has shown that the MiT/TFE family transcription factors, which upregulate lysosome function and autophagy, are overexpressed in PDA cells. Our hypothesis is that PLBD1 expression is driven by TFE3. In collaboration with other lab members, we have shown that PLBD1 is upregulated in PDA lysosomes. I knocked down TFE3 using short hairpin RNAs in several pancreatic cancer cell lines. Knockdown cells showed decreased PLBD1 mRNA expression compared to a luciferase control by qPCR. This was a comparable decrease known TFE targets. An additional goal is determining the role of PLBD1 in signaling processes that promote PDA growth. I knocked down PLBD1 using shRNAs and showed an increase in ERK signaling and LC3 lipidation by Western. However, we did not see changes in other pathways such as mTOR signaling, which normally occurs at the lysosomal membrane. Current work includes confirming the role of TFE3 using a doxycycline inducible system, using CRISPR to knockout PLBD1 for metabolic studies, and further investigating the relationship between PLBD1 and ERK signaling.

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Michael Rape, Department of Molecular and Cell Biology, University of California, Berkeley

"Investigating the domain architecture of an enzyme involved in the quality-control of myogenic regulators"

Abstract:

Metazoan development relies in the precise and spatiotemporal execution of a plethora of signaling pathways. To achieve this robust regulation, organisms rely on several regulatory mechanism that provide tight control of signaling cascades. Many of these signaling cascades rely on the formation of multimeric protein complexes, and recent work in our lab has shown that regulation of some dimeric complex is an ubiquitin-dependent process that is important for development. In order to identify regulators of development, we performed an in vitro myogenesis screen. This identified the ubiquitin ligase CUL3BTBD9 as an essential regulator of myogenesis. Intriguingly, immunoprecipitations coupled to mass spectrometry experiments identified robust binding to several multimeric proteins, including a tetrameric ubiquitin ligase the lab is currently studying: CUL3KCTD10. This prompted the lab to attempt to study the binding regions between KCTD10 and BTBD9 to better elucidate BTBD9 as a multimerization quality control enzyme.

The regulation by which ubiquitin ligases interact with substrates is not yet well understood, multimeric substrates in particular. Previous work in the Rape lab narrowed down the binding region of BTBD9 to KCD10 to its C-terminus. This region consists of two tandem F5/F8 domains, which we hypothesize are important for substrate binding. We used truncation analysis and mutational analysis of BTBD9 to further define the binding region that is required for KCTD10 binding, potentially identifying the substrate binding motif of BTBD9. This will provide a further understanding as to how developmental regulators are themselves regulated.

The data from the truncation analysis suggests that the F5/F8 domains within the C-terminus of BTBD9 are necessary and sufficient to bind KCTD10. Further mutation analysis will be performed in order to find the residues required for binding. Surprisingly, my data also shows evidence that the C-terminus of BTBD9 is able to interact with full length BTBD9, possibly in a KCTD10-dependent way. This provides a possible model of substrate regulation by BTBD9. We will also reconstitute these experiments in vitro. Understanding the architecture of BTBD9 and its interaction with KCTD10 will allow us to better understand the molecular mechanism by which BTBD9 functions as a quality control enzyme regulating multimeric protein complexes critical for metazoan development.

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Noah Whiteman, Department of Integrative Biology, University of California, Berkeley

"The Role of Gut Bacteria in the Evolution of Herbivory"

Abstract:

Plant-insect interactions compose a large fraction of the natural world’s predator-prey interactions. Many herbivorous insects feed on chemically-defended tissues. A recent shift to herbivory in the mustard-eating fly Scaptomyza flava, which must overcome chemical defenses called mustard oils in its host plants, makes this species an excellent model for studying adaptation to toxic environments. Here I begin to address the gut microbial facilitation hypothesis, which states that an organism’s ability to feed on plants which produce chemical toxins is partially due to their intestinal microbiome.1 Using strains of Enterobacter cloacae and Pseudomonas syringae, both of which degrade mustard oils with the enzyme SaxA, I tested if the bacteria colonized flies that ate them and if the bacteria persisted in fly digestive tracts. I found that only Enterobacter cloacae colonized and persisted in the fly digestive tracts. This work paves the way for future studies using genetically modified bacteria with a SaxA deletion to test if the ability to metabolize mustard oils enhances the fitness of host flies when consuming toxic mustard oils.

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Akiko Hata, University of California, San Francisco

"Mapping the E3 Ubiquitin Ligase Binding Site on the Human Drosha (hDrosha) Gene"

Abstract:

Background and Hypothesis: Drosha is a ribonuclease (RNAse) enzyme encoded by the Drosha gene that serves as a primary microRNA processor and can act as a principal regulator of microRNA processing. The microprocessor protein complex includes Drosha and the RNA-binding protein DGCR8. The Drosha protein forms pre-miRNA in the nucleus by cleaving the 3’ and 5’ strands of a primary miRNA stem loop structure. The hairpin shaped pre-miRNA is then exported from the nucleus to the cytoplasm to be further processed by the cytoplasmic Dicer protein, after which mature microRNAs are formed. E3 ubiquitin ligase is a protein that assists in the transfer of the small protein ubiquitin from an E2 ubiquitin-conjugating enzyme to a target protein substrate. Once ubiquitin is covalently attached to lysine residues of the protein substrate, the substrate is targeted for destruction by a proteasome. Point mutations in the Drosha gene have been identified in patients with diseases such as the Wilms Tumor and Hereditary Hemorrhagic Telangiectasia (HHT). Lapses in the normally tight regulation of miRNA processing are therefore linked to a variety of human diseases. Thus, mapping the E3 ubiquitin ligase binding site on the Drosha gene is an indispensable step to understanding the ubiquitination and proteasome degradation of the Drosha protein. Gao, Sheng, et al. identified NEDD4L as an E3 ubiquitin ligase for Smad2/3. More specifically, the WW domain of NEDD4L interacts with the PPGY motif in Smad 2/3. Since NEDD4 and NEDD4L belong to the same family, they are highly homologous. We therefore hypothesized that the WW domain in NEDD4 interacts with the PPGY motif in Drosha. Thus, if we mutate it into AAGY, it should abolish the interaction between NEDD4 and Drosha.

Methodology and Findings: The experimental design was based on designing primers (hDro-Bsu36I-REV, hDro- PPGY->AAGY-FOR, hDro-PPGY->AAGY-REV, hDro-PflMI-2-FOR) that would induce a mutation in the human Drosha (hDrosha) gene. Following PCR amplification of the hDrosha cDNA (cloned into pcDNA4/TO) with the designed primers, the amplified DNA was run by gel electrophoresis and isolated through gel purification. The plasmid pcDNA4-hDrosha was then cleaved by restriction enzyme (RE) digestion using the restriction enzymes PflMl and BSU36l in order to remove the original (unmutated) hDrosha cDNA. Using the NEBuilder HiFi DNA Assembly Reaction Protocol, the digested plasmid and the gel purified hDrosha PCR product were ligated. The ligation product containing the mutation was then transformed into competent cells and plated onto Luria Bertani (LB) agar plates with carbenicillin. The plasmids from the clones were then isolated through MiniPrep (plasmid preparation) and sent for sequencing. Following confirmation of successful mutation, resistance to E3 ubiquitin- proteasome degradation was confirmed by overexpression in HEK 293T cells.

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Linda Wilbrecht, Department of Psychology, University of California, Berkeley

"Does early life adversity (food-insecurity) have long-term effects on decision-making?"

Abstract:

Adversity during the childhood and adolescence period can have profound effects on the brain and behavior, including learning and decision making processes. In the field of public health, adverse childhood experience with food insecurity is associated with negative effects on mental and physical health (Ke & Ford-Jones, 2015), yet it is not known if food insecurity is the causal variable. To study this question we turned to mouse models. In my lab’s previous work it was found that groups of adult male mice that diverged in their juvenile and early adolescent (Postnatal day 21-40) feeding history, either experiencing food insecurity or ad libitum food, differed in their cognitive flexibility when tested in early adulthood (P60) after a 20 day period of ad libitum food for all mice. My project examined if differences in feeding history during late adolescence (P41-60) could drive differences in cognitive flexibility in adult mice (P80-90), again after a 20 day period of ad libitum food for all mice.This experiment was inspired by the idea that there may be a sensitive period in development in which feeding experience could influence the development of circuits that support learning and decision-making behavior.

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Polina Lishko, Department of Molecular and Cell Biology, University of California, Berkeley

"Investigating mitochondrial uncouplers and their effects on human sperm physiology as a means of a novel unisex contraception"

Abstract:

The need for unisex non-hormonal contraception is now greater than ever. With the world population projected to reach 10 billion in 2025 (United Nations Population Fund) and many women unable to take traditional hormonal methods of birth control, new strategies must be explored. To meet this need, we are investigating the use of chemical mitochondrial uncouplers as sperm-targeted contraceptives. Uncouplers are chemical agents that disturb the mitochondria’s electrochemical potential and ability to make ATP (Kadenbach, 2003). Recent work from our collaborators showed that the majority of endogenous uncoupling is mediated by the Adenosine Nucleotide Transferase (ANT) family of proteins, which were previously known only to be responsible for ADP/ATP exchange (Bertholet et al, 2019). While ANT proteins can be found throughout the body, the ANT4 isoform is expressed only in gametes (Lim et al, 2015 & Brower et. al 2009), and ANT4 KO male mice were shown to be infertile (Brower et. al, 2009). We hypothesize that it may be possible to specifically activate ANT4-mediated uncoupling, thereby preventing sperm from producing the ATP needed to power their vigorous motility and ascend the female reproductive tract. To investigate this hypothesis, we have treated human sperm with uncouplers and used flow cytometry and immunocytochemistry to assess the effects on sperm mitochondrial potential, respirometry to assess sperm oxygen consumption, In Vitro Fertilization (IVF) to assess sperm fertilization capacity and Computer-Aided Sperm Analysis (CASA) to gauge the effects of uncouplers on human sperm motility. We found treatment with mitochondrial uncouplers caused significant decreases in mitochondrial membrane potential and sperm motility, and significant increases in sperm oxygen consumption. Of the uncouplers tested, NEN and BAM15 were shown to be the most effective with the greatest impacts on, mitochondrial potential and sperm motility.

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Dirk Hockemeyer, Department of Molecular and Cell Biology, University of California, Berkeley

"Elucidating the Mechanism of TIN2 Mutations in Dyskeratosis Congenita"

Abstract:

Patients suffering from Dyskeratosis Congenita (DC) present with abnormally short telomeres. Short telomeres put patients at risk for many medical issues, including bone marrow failure, cancer, and pulmonary fibrosis. Mutations in TIN2, a component of the telomere binding shelterin complex, cause the short telomeres associated with DC. This study aims to characterize the effects of mutant TIN2 (TIN2-DC) on telomerase-mediated telomere elongation and telomeric protection, while exploring the possibility of wild-type rescue of the mutant phenotype. To this end we have engineered a cell line with a single amino acid change in the TIN2 protein in human embryonic stem cells (hESCs) in order to have a disease relevant model system to study the TIN2 mutations seen in DC. Cells with this patient derived mutation display the short telomere phenotype seen in DC. I have worked to assess effects of mutant TIN2 on telomerase mediated telomere elongation, characterize role of TIN2 in the protection of telomeres, and consider the possibility of wild-type rescue the mutant phenotype of short telomeres

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Iswar Hariharan, Department of Molecular and Cell Biology, University of California, Berkeley

"The Role of Stress-Response Transcription Factors in Epithelial Tissue Growth"

Abstract:

A fundamental process in tissue development is how cells perceive and react to their microenvironment to control cell growth and proliferation. This cell-cell coordination is likely achieved via many distinct signaling pathways and is integral to both normal development and tissue regeneration. There remain many unanswered questions about how these different signaling pathways interact to effectively control growth. Recent work has highlighted the importance of stress-responsive transcription factors like the AP-1 transcription factor in the coordination of a wide range of cellular events, from apoptosis to cell proliferation. To better understand the molecular mechanisms by which these stress-responsive pathways can coordinate such diverse responses, we use the Drosophila melanogaster imaginal discs as a model for studying development. Imaginal discs are epithelium tissues that develop into adult structures and are amenable to genetic manipulation. We are able to specifically activate or inhibit various stress-responsive signaling pathways to investigate how these manipulations alter target gene expression, cell viability and cell proliferation within a developing tissue. Preliminary results suggest that the overexpression of different stress-responsive transcription factors regulate similar downstream target genes and future work will elucidate how these pathways interact with each other and provide insight into the mechanisms of tissue growth and regeneration.

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Chunlei Liu, Department of Electrical Engineering and Computer Sciences and the Helen Wills Neuroscience Institute, University of California, Berkeley

"Activation of ferritin-tagged TRPA1 ion channels with radio-frequency (RF) magnetic fields and visible light."

Abstract:

We previously devised the magnetogenetic technique FeRIC (Ferritin-iron Redistribution to Ion Channels) to remotely activate transient receptor potential (TRP) channels with non-invasive radio-frequency (RF) magnetic fields. FeRIC technology couples TRP channels with ferritin by fusing their intracellular domain with the ferritin-binding region (domain 5) of Kininogen. The FeRIC channels redistribute endogenous ferritin to their proximity. In cells expressing FeRIC channels, RF interacts with ferritin producing an increase of the levels of the labile iron pool. Free iron participates in diverse chemical reactions producing reactive oxygen species and oxidized lipids that ultimately activate the FeRIC channels. Here we show that the TRP Ankyrin 1 channel tagged with ferritin (TRPA1FeRIC) is activated with RF and light (UV and blue light). In Neuro2a cells expressing the TRPA1FeRIC, RF and light increased the cytosolic Ca2+ levels. The TRPA1 antagonist AM-0902 inhibited the RF- and light-induced activation of TRPA1FeRIC. Moreover, the light-induced activation of TRPA1FeRIC is also inhibited with PIH, an iron chelator. To conclude, our results suggest that the TRPA1FeRIC channels are RF- and light-sensitive. This finding indicates that TRPA1FERIC channels could be used as a magnetogenetic or an optogenetic tool.

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Danica Galonic Fujimori, Department of Cellular & Molecular Pharmacology, University of California, San Francisco

"Combatting Antibiotic Resistance: Screening for Cfr Inhibitors"

Abstract:

Antibiotics are drugs used to treat bacterial infections. However, antibiotic usage is threatened by the spread and bacterial acquisition of resistance genes that result in robust antibiotic resistance. The chloramphenicol-florphenicol-resistance (cfr) gene is commonly found in bacteria and is a prominent cause of antibiotic resistance. The cfr gene encodes an enzyme in the radical SAM family that post-transcriptionally methylates the C8 position of A2503 within the 23S rRNA of the ribosome. This methylation causes bacteria containing the cfr gene to be cross resistant to 8 antibiotics targeting the peptidyl transferase center (PTC) of the bacterial ribosome. My project aims to identify antibiotic adjuvants, small compounds that are co-administered with antibiotics to increase the efficacy of current antibiotics. Specifically, I aim to identify molecules that would rescue the efficacy of the PTC antibiotics that are ineffective in bacteria containing Cfr. To address these questions, I’ve developed a cell-based assay in E. coli to screen for inhibitors of the Cfr enzyme from a precompiled library. To develop this assay, I constructed 3 E. coli strains that contained single-gene deletions to increase cell permeability or reduced cellular efflux, thereby increasing the likelihood of small molecules entering or staying in the cell. I then determined minimum inhibitory concentration values for PTC antibiotics to evaluate which E. coli strain would be optimal for the screening assay. Finally, using data from our inhibitor screen, I’ve generated a preliminary IC50 curve that captures the efficiency of a known competitive inhibitor of the radical SAM family, which is likely to also inhibit Cfr, during optimization assays. Ongoing work involves further assay optimization to determine antibiotic and inhibitor concentrations that allow for identification of Cfr inhibitors with high confidence.

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Damini Jawaheer, CHORI/UCSF Benioff Children's Hospital Oakland

"Differences in gene expression between men and women with rheumatoid arthritis"

Abstract:

Gene expression goes beyond the central dogma of biology. It is possible for gene expression to contribute to pathology of a disease. The goal of this project is to first analyze if there is an association between the expression of one or more genes and rheumatoid arthritis (RA), separately in men and women, and then to determine if the results differ between the 2 genders. I researched for data on Gene Expression Omnibus and obtained gene expression datasets generated through RNA sequencing. I processed the data to get gene expression levels using bioinformatics methods. Then, I conducted two comparisons using the EdgeR analysis software to identify differences in gene expression levels between: (1) healthy men and men with RA and (2) healthy women and women with RA. Using the two sets of results obtained from the gender-specific analyses, I analyzed the similarities and/or differences between men and women in terms of which specific genes have expression patterns associated with RA. There are 2,594 genes significantly differentially expressed in men with RA while 1,798 are significant in women. Of those, 1,517 are common to both men and women. Amongst the genes that were found to be differentially expressed only in men, 4 genes have been associated with RA and they all have immune-related functions. Amongst the genes that are only significant in women, only 1 gene has been associated with RA. There are 8 genes that are associated with RA that are differentially expressed in both genders. The different genes that are differentially expressed only in males or only in females demonstrates that there are gender-specific gene expression signatures associated with RA. The difference in functions of genes that are differentially expressed only in males or only in females also indicates that there is gender-bias due to biological pathways operating in a gender-specific manner.

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Steve Garan, Lawrence Berkeley National Laboratory

"A Systems Modeling Approach to Understanding Metabolic Regulation and Immune Response"

Abstract:

We propose a systems biology model that presents a novel approach to characterizing aging. Current biological research have yielded many medical advances that continue to raise median life expectancy, but the aging process itself is still not well understood. The fundamental physiological pathways and regulatory mechanisms behind aging are complex - current theories mainly focus on individual factors that inadequately capture aging's complexity; we, however, use mathematical modeling of a network of integrated and interacting systems to build a more comprehensive picture of human physiological and metabolic processes, with the hopes that we can identify and develop solutions to the decay factors associated with aging. My work builds on pre-existing simulations of systems done both at this lab and found in existing literature, which focuses on the interactions between a core group of systems impacted by aging - circadian rhythm, hunger response, stress response, insulin-glucose response, and immune response. Using existing clinical data, I generate differential equations to model the relationship between food intake and melatonin, as well as between cytokines and insulin response, reinforcing the links between the various biological systems in our existing model.

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Jeremy Thorner, Department of Molecular and Cell Biology, University of California, Berkeley

"Visualizing the interaction between the protein Nap1 and the septin cytoskeleton during the budding phase in Saccharomyces cerevisiae"

Abstract:

Septins are a family of highly conserved, eukaryotic GTP-binding proteins that assemble into a linear hetero-octamer, which can polymerize end-to-end into filaments. In yeast, a collar of filaments composed of septins Cdc3, Cdc10, Cdc11 and Cdc12 is erected at the incipient site for cell division in the isthmus between a mother cell and Its bud. Nearly 200 proteins that control the cell cycle and/or cell morphogenesis are localized at the bud neck; but, whether such bud neck-localized proteins associate directly with the septins or are recruited to this location via other means has not been established for the majority of these molecules. Nap1 is a multi-functional protein that prior work from our laboratory has demonstrated associates dynamically and transiently with a set of interacting proteins (Aim44/Gps1, Nba1 and Nis1) that localize at or near the bud neck [Perez AM, Thorner J (2019) Cytoskeleton 76: 15-32]. Therefore, the goal of my work has been to determine whether Nap1 is able to physically bind to septins and, if so, whether Nap1 interacts specifically with a particular septin. To assess Nap1-septin interaction in vitro, I developed methods to purify it for use in EM studies. To assess Nap1-septin interaction in vivo, I am constructing strains using CRISPR-Cas9 gene editing to apply a tripartite split-GFP method developed in this lab to visualize direct protein-protein interactions in yeast [Finnigan GC et al. (2016) Mol. Biol. Cell 27: 2708-2725].

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Danica Chen, Department of Nutritional Sciences and Toxicology, University of California, Berkeley

"The Effect of SIRT3 Overexpression on HSC Aging Phenotype"

Abstract:

It has been shown in previous publications that sirtuins protein is associated with various aging cellular processes. Knock out of Sirt3, one of the protein from the sirtuins family, has been shown to accelerate aging phenotypes. Sirt3 knock out hematopoietic stem cell has shown exacerbated phenotypes such as increase myeloid biased lineage markers versus lymphoid biased lineage markers. Since we already know Sirt3 know out can lead to aging, I am interested to find out whether Sirt3 overexpression can rescue aging phenotypes. In my research, I generated HSC specific Sirt3 overexpression mice and performed different experiments to test their aging phenotypes.

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Lee Riley, School of Public Health, University of California, Berkeley

"Prevalence of antimicrobial resistance in patients with urinary tract infection caused by Klebsiella pneumoniae"

Abstract:

Urinary tract infections (UTI) are a leading cause of antibiotic prescription in women, and Klebsiella pneumoniae infection accounts for approximately 9% of UTIs. K. pneumoniae is a member of the Enterobacteriaceae opportunistic bacterium with growing antimicrobial resistance, including carbapenem-resistance. It is the most common cause of carbapenem-resistant Enterobacteriaceae (CRE), that is considered by CDC to be an urgent threat pathogen. While CREs remain relatively uncommon as a cause of community-acquired (CA) UTI, its resistance to other antimicrobial agents is increasing. This project aims to characterize the prevalence of drug-resistant CA-UTIs among patients attending a county general hospital in San Francisco. The BSI bacterial isolates from the same hospital were also characterized by multilocus sequence typing (MLST) to determine the frequency and distribution of K. pneumoniae sequence types (ST) in this population.

Between May 2019 and September 2019, we collected 1,007 confirmed UTI urine isolates linked with patient information from Zuckerberg San Francisco General Hospital (ZSFGH) in San Francisco. Bacterial colonies were isolated from patient urine, screened against 13 antimicrobial agents (nitrofurantoin, trimethoprim/sulfamethoxazole, cephazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, ampicillin, ampicillin-sulbactam, ertapenem, meropenem, piperacillin tazobactam) and will be characterized by MLST.

Among the 1,007 Gram-negative bacterial urine isolates collected, 46 (4.6%) were identified as K. pneumoniae. All K. pneumoniae isolates demonstrated resistance to ampicillin. Eighteen (39%) isolates were resistant to at least one antibiotic other than ampicillin. Nine (20%) K. pneumoniae isolates exhibited nitrofurantoin resistance and 12 (26%) were resistant to trimethoprim-sulfamethoxazole. Five (11%) isolates were identified as extended-spectrum beta-lactamase (ESBL) producing, as reported by ZSFGH clinical microbiology laboratory. Two (4%) isolates showed resistance to ciprofloxacin and no isolate demonstrated resistance to ceftriaxone. COVID-19 impacts prevented the sequence typing of the UTI isolates, so provided bloodstream infection (BSI) data was used to simulate MLST data generated by this project.

K. pneumoniae demonstrated resistance to two antibiotics commonly used in the empirical treatment of uncomplicated UTI – nitrofurantoin and trimethoprim-sulfamethoxazole. Prevalence of resistance beyond the threshold of 20% puts these two therapies at higher risk, as 20% and 26% of isolates were resistant to nitrofurantoin and trimethoprim, respectively. Understanding the prevalence of resistance of K. pneumoniae to the common therapies has the potential to improve clinical management of UTI.

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David Schaffer, Department of Molecular and Cell Biology, University of California, Berkeley

"Optimizing the Dissociation of Human Pluripotent Stem Cell Aggregates Through Enzymatic and Mechanical Methods"

Abstract:

The scale-up of the production of human pluripotent stem cells from a two-dimensional to a three-dimensional system has been successful in expanding the yield of hPSCs and poses many advantages over a two-dimensional system. However, when hPSCs are grown in a 3D system, they grow in spherical aggregates as opposed to a monolayer of cells. The formation of cellular aggregates is important for growth in 3D cultures but limits the scale-up of hESC production. My project studies the role of cell-cell interactions in differentiating 3D hPSC-derived neural aggregates in order to develop a strategy to dissociate neural aggregates and create a viable single cell suspension. I aim to investigate neural aggregate dissociation by controlled testing of enzymatic and mechanical methods to create single cell suspensions while maximizing viability and yield. These findings will be implemented in the development and optimization of the magnetic-activated cell sorting (MACS) purification process. For MACS, the inability to successfully dissociate cell aggregates has posed problems in the sorting procedure, with aggregates being too large to be correctly labeled and sorted. Ultimately, these processes will be applied to analyze differences in sorted and unsorted cell populations and their implications in animal models of demyelinating diseases.