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Mallar Bhattacharya, University of California, San Francisco

"Molecular Programs of the Aging Lung"

Abstract:

Recognizing the structural and genotypic heterogeneity in aging lung tissue is crucial for providing optimal and patient specific care using preventative and pharmacological intervention. Previous studies have either been limited to a small sample size, lack of human models of study, or retrospective studies. To address this gap in knowledge, we extracted RNA from 86 donor lung tissue samples between age 16 to 76 to analyze the heterogeneity in their molecular profile. Upon processing our RNA samples through cDNA library construction, quality control, and sample alignment to the reference genome, bulk RNA sequencing via the DESEQ2 package revealed a significant upregulation of senescence and profibrotic markers with age, and Ingenuity Pathway Analysis revealed of proliferation consistent with senescence. Corroborating the discovery of senescence markers through sequencing, analysis of telomere length demonstrated progressive telomere shortening across age associated with p53 and p16 activation. Finally, we used the R package MuSiC to analyze the heterogeneity in cellular composition between young and old samples, revealing an increase in the proportion of fibroblasts and a decrease in the proportion of Alveolar Type 2 epithelial cells. Put together, our findings present an age-related fibrotic and senescent mechanism that catalyzes cellular and genetic remodeling contributing to lung function deterioration.

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Dr. Hernan Garcia, Department of Molecular & Cell Biology, University of California, Berkeley

"Does ParB Oligomerization Interfere with Transcription?"

Abstract:

The recently developed MS2 system has enabled us to visualize and quantify how enhancers regulate the production of nascent mRNA transcripts in Drosophila melanogaster embryos. Transgenic repeats are inserted into a noncoding region of a gene and form a characteristic MS2 stem loop in the transcribed mRNA, which is then bound by a fluorescently-tagged coat protein, MCP, allowing for real-time visualization of transcription. However, until recently, we have been unable to visualize the location of an enhancer in the genome before transcriptional activation. To solve this, lab members inserted a DNA binding motif, IntB, at a genomic locus of interest, which is specifically recognized by a DNA binding protein, ParB. After an initial ParB dimer binds to the IntB motif, additional ParB dimers oligomerize with it and spread along the DNA. Since we have fused the ParB protein to a fluorophore, this oligomerized structure appears as a bright, detectable fluorescent spot on our confocal microscope. One outstanding concern about using the ParB system as a DNA-tagging tool is that the oligomerization of the dimers might expand to a range which would interfere with the transcriptional machinery. Using transgenic Drosophila lines previously created in the lab, I imaged embryos from three different lines, one expressing ParB-mCherry, one expressing MCP-mCherry, and one expressing both ParB-mCherry and MCP-mCherry, each of which contained a reporter construct with both IntB binding sites and MS2 loops. Using computational image analysis, I will quantify and compare transcription levels between MCP-mCherry only embryos and ParB-mCherry, MCP-mCherry embryos (after baseline subtraction of ParB-mCherry fluorescence). If ParB oligomerization does not interfere with transcription, then we expect no changes in transcriptional levels. However, a significant increase or decrease in transcriptional activity in the combined line would indicate that ParB does, in fact, interfere with transcription.

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Richard M. Harland, Department of Molecular & Cell Biology

"The Function of the Endosymbiotic Dinoflagellate Symbiodinium and Neural Circuitry in Cassiopea Jellyfish Behavior"

Abstract:

The upside-down jellyfish Cassiopea xamachana has been studied to understand its interaction with the endosymbiotic dinoflagellate alga Symbiodinium, as well as its sleep-like behavior. The role of the symbiote in Cassiopea is multifaceted, with previous research showing symbiosis-driven development in this jellyfish, and potential for unknown functions of this symbiotic relationship. Recent work has also shown that jellyfish exhibit sleep-like functions, despite having a decentralized nervous system. Pulsation rates (muscle contraction) and ganglia nerve cluster usage differ between day (light) and night (dark) periods. Symbiote-negative Cassiopea polyps were grown, and induced to form medusae, to understand the role of Symbiodinium in Cassiopea development and behavior. We found that Cassiopea develops without symbiote, though they pulse less frequently, and do not thrive. Future work will determine the relationship between pulsing behavior, and sleep/wake, in the presence or absence of symbiote.

Previously, pharmacology was used to study neural circuits and the regulation of behavior in many animals. In Cassiopea, the cholinergic and GABAergic systems were tested via inhibitory and excitatory drug conditions in pulse tracker and ganglia tracker recording setups to understand their relationship to wake and sleep states. Cassiopea showed a decrease in pulsation behavior when inhibiting nicotinic acetylcholine receptors (nAChRs) with DTC, and increased pulsation behavior when exogenous acetylcholine (ACh) agonist was introduced: exogenous ACh recovered function of nAChRs and normal pulsation behavior in DTC inhibited jellyfishes. By activating GABA receptors, using agonist gaboxadol, we also observed an induction of quiescence. Understanding these aspects of jellyfish behavior and neural circuitry paves the way for Cassiopea to be utilized as a model for symbiosis and sleep research.

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Jeff Belkora, University of California, San Francisco, Department of Surgery and Philip R. Lee Institute for Health Policy

"Consultation Audio Recordings via Smartphone Application and Summarization for Patients with Advanced Prostate Cancer"

Abstract:

Background: Patients diagnosed with a form of advanced prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), encounter situations where they must select a treatment from multiple options. Patient decision support-centered interventions such as making a list of questions to ask providers, audio recording appointments, and summarizing consultations have been demonstrated to improve patient knowledge and decision making quality.

Methods: We prospectively enrolled men with progressive, chemotherapy-naïve mCRPC with an upcoming genitourinary (GU) oncology appointment at UCSF and access to a mobile device. The feasibility, acceptability, and preliminary efficacy of patient-administered consultation recordings on personal smartphones was investigated through pre-consultation and post-consultation surveys administered via REDCap. The feasibility of audio recordings via the smartphone application Medcorder and changes in the Informed and Support subscale of the Decision Conflict Scale (DCS, Likert scale 1 to 3 and 7 to 9 items) were measured through surveys and post-consultation interviews. I summarized the GU oncology appointments from the patient-generated audio recordings and conducted semi-structured interviews with participants.

Results: In this pilot study, 15 patients were enrolled. Eighty percent of patients created an audio recording of their GU oncology appointment, seventy three percent of patients listened to the recording, and eighty seven percent of patients received a summary of their GU appointment. With this intervention, patients’ decisional conflict informed subscore and support subscore decreased from baseline to 2 weeks after consultation, corresponding to a decrease in decisional conflict and patients feeling more informed about their treatment options and supported in their decision making.

Conclusions: Patient-administered consultation audio recordings and consultation summaries were feasible for patients with mCRPC and improved patient decision-making quality.

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

"Examining the Evolution of cyp6g1 Gene Copies in Scaptomyza"

Abstract:

The Cytochrome P450 gene family (CYPs) is a conserved gene family that encodes Phase 1 enzymes critical for the detoxification of xenobiotics in most animals. CYP proteins are of particular importance to organisms that intricately interact with plants and their defensive chemicals. To understand the impacts of plant-produced xenobiotics on the evolution of plant-associated insects, I studied nine closely related fruit fly (Drosophilidae) species that differ in their dietary interactions with plant defensive chemicals. The flies Scaptomyza flava and S. montana attack the leaves of living plants in several different plant orders, S. hsui feeds on rotting plants, Drosophila mojavensis feeds on microbes in rotting cacti, and D. virilis, D. melanogaster, D. grimshawi, D. primaeva feed on microbes in rotting fruit. Here, I focused on the evolution of the cyp6g1 gene, which has been linked to contrasting detoxification abilities in species such as Drosophila melanogaster and has undergone multiple gene duplication events in S. montana and S. flava1 -- from two ancestral copies to six. Using comparative analysis, we found evidence of relaxed selection in herbivorous lineages as well as sites undergoing positive selection across the cyp6g1 phylogeny.

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Robert Dudley, Department of Integrative Biology, University of California, Berkeley

"Hummingbird Dietary Preference for Fermented Nectar"

Abstract:

Frugivores and nectarivores are naturally exposed via their diets to low levels of alcohol produced by fermentative yeasts. Previous studies have suggested a preference in many primates for fermented fruits and nectars; however, behavioral predilection for ethanol by nectar-feeding birds has not yet been studied. Given their high metabolic rates and concomitant chronic ingestion of floral nectar, hummingbirds represent an ideal taxon with which to investigate behavioral response to low-level alcohol concentrations within sugar solutions. In collaboration with members of the UC-Berkeley Animal Flight Laboratory, I investigated dietary preference for ethanol-enhanced sugar solutions by the hummingbird Calypte anna, a native to the western coast of North America. In binary-choice experiments, captive hummingbirds were exposed to sucrose solutions containing either 0%, 1%, or 2% ethanol concentration; rates of volitional nectar consumption were measured over a 3-hour interval. Nearly complete data sets with repeated measurements per treatment were obtained for two male individuals. Both birds demonstrated consistent ingestion of all three ethanol-containing solutions with no significant difference among concentrations, with the exception of one individual which showed a preference for 0%- relative to 2% ethanol concentration. These data, following ongoing replication with two more birds, will establish the behavioral threshold for ethanol preference by hummingbirds at naturally occurring concentrations within floral nectar.

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José Pablo Vázquez-Medina, Department of Integrative Biology

"Natural Tolerance to Endothelial Oxidative Stress in Elephant Seals"

Abstract:

Diving seals experience progressive hypoxemia and ischemia without suffering the reperfusion injuries characteristic of myocardial infarction or ischemic stroke in humans. Oxidative stress is a crucial driver of cytotoxicity during reperfusion injury via the formation of lipid hydroperoxides. Here, we show that primary endothelial cells derived from northern elephant seal (Mirounga angustirostris) placental arteries are more resistant to treatment with organic peroxides than equivalent human cells. We compared the expression and protein abundance of phospholipid hydroperoxidases, glutathione peroxidase 4 (GPx4) and peroxiredoxin 6 (Prdx6), between elephant seal and human endothelial cells treated with organic peroxides to investigate if these enzymes drive elephant seals’ extraordinary tolerance to oxidative stress. GPx4 and Prdx6 protein levels decreased in both species following a 2 h exposure to 100 µM tert-butyl hydroperoxide (t-BOOH). PRDX6 gene expression increased in human cells after t-BOOH exposure while GPX4 gene expression decreased in seal cells. Increasing t-BOOH concentrations increased cytotoxicity in both species; however, the magnitude of change was greater in human than in seal cells. Together, these results show that seal endothelial cells remain protected against oxidative stress despite decreases in GPx4 and Prdx6 gene and protein expression after treatment with organic peroxides.

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

"Exploration of a Feeding Circuit in Drosophila Melanogaster"

Abstract:

The sense of taste is pivotal to an animal’s responses to potential food sources: nutritious substances elicit an acceptance behavior while toxic substances elicit an avoidance behavior. In Drosophila melanogaster, these various feeding behaviors are controlled by dedicated gustatory circuits. However, our understanding of the neural circuitry between the Gustatory Receptor Neurons (GRNs), which sense chemical components of food, and the motor neurons which control muscles for feeding, is incomplete. To begin to identify these missing components, we mainly relied on a whole-brain Drosophila EM dataset, which allowed us to explore, with synaptic detail, how the gustatory circuit is organized in the Drosophila brain. Specifically, we examined potential circuits between sugar sensory neurons, the previously identified gustatory interneuron G2N-1, and the motor neurons innervating the muscles necessary for feeding. Previous light-level studies using GFP reconstitution across synaptic partners (GRASP) allowed us to select a specific target region of G2N-1, which we hypothesized to be directly connected to motor neurons, to search downstream of, which yielded information about both known and novel components of this circuit. Across 30 synapses, we identified 15 candidate downstream neurons, along with 94 unique fragments. These findings add to our understanding of the Drosophila gustatory circuit downstream of GRNs and may inform future studies that aim to uncover the full neural pathway from sensory taste intake to motor response.

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Diana Bautista, Department of Molecular & Cell Biology

"Functional Analysis of SARS-CoV-2 Ion Channel 3A"

Abstract:

SARS-CoV-2, the virus that causes the Coronavirus Disease 2019 (i.e. COVID-19), has rapidly overwhelmed global public health institutions from the winter of 2019 and onward. The present pandemic has heightened the ever-increasing need to study the fundamental basics of viral functioning, especially as new viral strains of SARS-CoV-2 evolve within different populations. Despite this rapid evolution, the ion channel 3A has been found to be highly conserved among families of Coronaviridae capable of infecting humans and shown to be expressed in infected cell membranes (1, 3). Ion channels in general are an understudied aspect of the viral genome that may play a crucial role in the virulence of viral infectious diseases. Be that as it may, there is still some conjecture concerning whether or not the 3A protein is a definitive ion channel, and if it is, which ions are capable of permeating through the channel. Previous studies within the field have demonstrated that SARS-CoV-1 3A is a non-selective ion channel with a slight preference for permeating Ca²⁺ and K⁺ over Na⁺ (2). Furthermore, 3A has also been suggested to play key roles in viral release and caspase 8-mediated apoptosis (1, 3-4). Based upon this knowledge, we hypothesize that SARS-CoV-2 3A is a Ca²⁺-permeable ion channel that promotes the inflammatory response and apoptosis triggered by SARS-CoV-2 infection. To test this, we performed Ca²⁺ imaging experiments using the calcium indicator FURA-2AM in order to quantify the consequences of 3A expression on Ca²⁺ stores and Ca²⁺ signaling pathways in heterologous cells. Our preliminary data show that there are no significant differences in the baseline Ca²⁺ levels in mock vs. 3A transfected cells (n= 1098 mock and 998 3A-transfected cells, p=0.31, two-tailed t test), but that Ca²⁺ influx and Ca²⁺ stores are increased (n= 1098 mock and 998 3A-transfected cells, p <0.0001, two-tailed t test). Further analyses have shown, however, that there is not a significant difference in baseline Ca²⁺ levels in mock vs. 3A-transfected cells (n= 5-6 wells of mock and 3A-transfected cells with 100-300 cells per well, p = 0.7211, two-tailed t test), Ca²⁺ stores (n = 5-6 wells of mock and 3A-transfected cells with 100-300 cells per well, p = 0.5508, two-tailed t test), nor Ca²⁺ influx (n = 5-6 wells of mock and 3A-transfected cells with 100-300 cells per well, p = 0.8361, two-tailed t test). These data suggest that 3A does not alter Ca²⁺ homeostasis in HEK293T cells. Future studies to investigate 3A channel gating and cellular localization will allow us to understand 3A’s role in the virulence and severity of COVID-19.

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Rodrigo Almeida, Department of Environmental Science, Policy, and Management

"Development of a Fiberoptic Array System to Test Insect Movement"

Abstract:

The bacterium Xylella fastidiosa is an insect transmitted plant pathogen that has various effects/interactions on insect vectors, such as blue-green sharpshooters (Graphocephala atropunctata) and meadow spittlebugs (Philaenus spumarius) as well as plant hosts like grape and almond. While there have been studies published regarding the effects of this bacterium on the biology and physiology of its insect-vectors, affected behavior is still a question to be answered. Previous assessments of vector insect movement behavior have focused on manual counts of insects that are feeding on infected vs. non infected plants. By developing a fiber optic array system that utilizes fiberoptics, a current amplifier, and an analog brainbox, I created a prototype system that measures changes in current that correspond to movement within the fiber optic system. By generating a python script that utilizes the coding language to transfer the analog signal of the amplifier into an ethernet signal that can be read and recorded on a laptop, I propose that this system can expand upon or replace previous methods of quantifying the movement behavior of insect vectors of X. fastidiosa like meadow spittlebug. Using an inanimate object comparable to X. fastidiosa insect vectors, I tested the accuracy and precision of the devices as well as potential detection limits. The next step will be test the device’s ability to track and record the movements of live insect vectors. Once this array is fully tested, the goal will be to use the system to assess potential differences in movement behavior among insects of varying infection statuses.

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Ting Wang, Pulmonary and Endothelial Lab, University of Arizona College of Medicine in Phoenix

"Particulate Matter Induces m6A RNA Methylation in Human Bronchial Epithelial Cells"

Abstract:

Particulate matter (PM) is a major part of air pollution and can enter and travel through human airways; thus, it poses as one of the highest risks to human health. Recently, many studies have been focused on the association between atmospheric PM and epigenetic alterations such as DNA methylation and histone modification. N6-methyladenosine (m6A) is the most common RNA modification on eukaryotic mRNA, and has numerous biological functions such as pre-mRNA splicing, mRNA stability, and translation, indicating its association with various human diseases. Key m6A enzymes include m6A methyltransferases, m6A demethylases and m6A RNA-binding proteins. The goal of this study was to investigate the potential role of PM in m6A methylation in epithelial cells. We treated human bronchial epithelial cells (A549 cells) with PM for 4 and 24 hours. Dot blot and anti-m6A ELISA were used to detect and quantify the m6A methylation in A549 cells. Protein expression of methyltransferases METTL3 and METTL14 and demethylases FTO and ALKBH5 were detected by western blot. Demethylase activity was measured by m6A Demethylase Assay Kit. METTL3-binding proteins were pulled-down by METTL3 antibody and detected by western blot. METTL3 siRNA was utilized to knockdown the gene expression of METTL3 in A549 cells. We showed that PM induced an increase in overall m6A methylation, METTL3 and METTL4 expression, and methyltransferase complex (METTL3 and METTL14) activity at 4 hours. However, there was no difference in FTO and ALKBH5 expression at 4 hours. Moreover, METTL3 does not move from the nucleus into the cytoplasm as a result of PM treatment. The demethylase activity didn't change significantly after PM treatment. PM-induced m6A methylation up-regulation was reduced after knockdown of METTL3. METTL3 silencing down-regulated the protein expression of ICAM1 and ERK, however it had a minor impact on NF-κB signaling pathway. Our research revealed that PM upregulated m6A methylation in epithelial cells at a short time by increasing the protein expression and methyltransferase activity. METTL3 plays a key role in PM-induced m6A methylation in A549 cells. This study provides an original mechanism for PM-induced epigenetic change in epithelial cells. Further studies are required to investigate further downstream effects of PM-induced m6A methylation in inflammation as well as apoptotic pathways.

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Eva Harris, School of Public Health, University of California, Berkeley

"Investigating the SARS-CoV-2 Spike-Cell Surface Interactions That Contribute to Vascular Leak Using Site-Directed Mutagenesis"

Abstract:

Severe cases of COVID-19 are associated with cytokine storm-induced vascular leak in the lungs, and a deeper understanding of the mechanism behind this pathology is urgently needed. Preliminary data from the Harris lab has shown that the SARS-CoV-2 Spike glycoprotein, as well as its receptor binding domain (RBD), causes endothelial dysfunction in human pulmonary microvascular endothelial cells (HPMECs) independently from interaction with the ACE2 receptor or viral replication. Interestingly, we found that in contrast to CoV-2 Spike, SARS-CoV-1 Spike does not induce endothelial hyperpermeability in HPMECs. Together, these data suggest that interactions between the CoV-2 Spike RBD and glycosaminoglycans (GAGs) on the cell surface, such as heparan sulfate (HS), may play an important role in COVID-19 vasculopathy and viral dissemination. To further define this mechanism, I used site-directed mutagenesis and bacterial cloning to generate Spike point mutants incapable of binding to either ACE2, HS, or Furin. As CoV-2 Spike demonstrates enhanced interaction with HS proteoglycans to facilitate viral entry, likely due to the increase in positively charged residues found in CoV-2 Spike compared to CoV-1 Spike, one of the putative HS binding residues on CoV-2 Spike was mutated to that found on CoV-1 Spike (N354E), while the corresponding residue on CoV-1 Spike was mutated to create a mutant with HS binding capacity comparable to CoV-2 Spike (E341N). Double mutants for ACE2 and Furin were designed to attenuate ACE2 binding and Furin proteolytic cleavage. The mutants were then functionally tested for their ability to induce endothelial dysfunction via endothelial glycocalyx layer (EGL) disruption. Our results indicate that there may be a causative link between Spike-heparan sulfate binding and the EGL disruption pathways that lead to endothelial hyperpermeability, thus highlighting a critical stage of the viral life cycle to therapeutically target in order to prevent Spike-induced vascular leak in the lungs.

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Maria Pia Cosma and Gary Karpen, Guangdong Regenerative Medicine and Health Guangdong Laboratory

"In-situ Visualization of Imprinted Genes Through OligoDNA-PAINT and OligoSTORM"

Abstract:

Somatic cells of diploid organisms, like humans, contain two alleles of each autosomal genes – one maternal and one paternal copy. For genetically imprinted genes, only one of the two is expressed and its activation status is dependent on its parent of origin. In eukaryotes, the chromatin structure and methylation status of the genes regulate their expression. De-compacted or “open” chromatin state usually correlates with actively transcribed genes and accessibility to RNA Polymerase. The methylation status and gene expression have been previously employed to understand genomic imprinting, but there is no visualization to reveal the relationship between the spatial structure of the gene to its imprinting status. New developments on super-resolution microscopy (SR), specifically single molecule localization microscopy (SMLM), that can achieve higher resolution than light microscopy, allows us to study the chromatin alteration in a sequence-specific, single-cell manner. In this study, Stochastic Optical Reconstruction Microscopy(OligoSTORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography(OligoDNA-PAINT) will be used to observe specific DNA regions including Nanog, H19, IGF2 and Peg3 at nanoscale resolution.

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Sona Kang, Department of Nutritional Sciences and Toxicology, University of California, Berkeley

"Identifying JMJD8 as a Significant Regulator of Obesity-Associated Insulin Resistance"

Abstract:

Jumonji domain-containing protein 8 (JMJD8) is expressed in various tissues, but its role in adipose tissue remains largely unknown. After examining JMJD8 expression levels in mouse adipocytes using RNA extraction with qPCR followed by Western blotting, we found that high-fat-diet (HFD) and obese (ob/ob) mouse models displayed significantly increased JMJD8 expression compared to controls. Expanding upon this discovery, this study aims to investigate the role of JMJD8 in regulating obesity-associated insulin resistance. Previous glucose uptake assays depicted that JMJD8 knock-down improved insulin sensitivity in mouse 3T3-L1 and iWAT cells and human SGBS cells. Following cell culture and JMJD8 lentiviral overexpression in 3T3-L1, iWAT, and SGBS cells, other glucose uptake assays portrayed strong inhibition of insulin-stimulated glucose uptake. These results inspired us to generate CRISPR-mediated JMJD8 knockout (KO) mice to assess JMJD8 function in adipocytes in vivo. Collaborating with other lab members, I conducted a GTT and ITT on JMJD8-KO mice on Chow diet and HFD. Remarkably, the GTT and ITT for HFD signified significantly more improved glucose and insulin sensitivity than Chow. These results sparked our interest in insulin’s regulation of the IRS-PI3K-Akt pathway, so we conducted Western blotting and determined increased phosphorylation of IRS1, IR, and Akt in eWAT of HFD JMJD8-KO mice. In further gain-of-function experiments, I carried out qPCR followed by gel electrophoresis from JMJD8 transgenic (TG) mice to confirm significantly higher JMJD8 expression levels in Adi-JMJD8-TG mice compared to wild-type, and then collaborated with lab members to conduct a GTT and ITT that depicted reduced glucose and insulin sensitivity in Adi-JMJD8 TG mice. These findings support the notion that JMJD8 expression is involved in the pathogenesis of adipose obesity-associated insulin resistance. Moreover, they pave the way for further exploration of the molecular targets and mechanisms of JMJD8 that may confer potential clinical translation in patients with metabolic disease.

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Qili Liu, Department of Anatomy, University of California, San Francisco

"Investigating the Circuit and Molecular Mechanisms Underlying Sugar Specific Hunger"

Abstract:

Our lab has been investigating the neurobiological basis of nutrient-specific appetite, which energizes behavior to search for and ingest particular types of food in response to nutritional deficiency. Previous research has suggested that organisms possess nutrient specific hunger for macronutrients and micronutrients sodium and calcium. Using Drosophila fruit flies as a model system, we recently identified a dopaminergic circuit that encodes protein specific hunger. This circuit simultaneously promotes protein intake and restricts sugar consumption in protein starved animals. We wondered whether equivalent neural circuits exist that encode specific hunger for another macronutrient, carbohydrates. To investigate this in flies, I fed flies for 1-7 days on yeast, which is enriched in protein but contains negligible amount of sugar. Following that, I assessed their food preference using a high throughput two-choice plate assay that our lab has recently developed, and which I performed various tests to validate. From the assay, I observed a robust dietary switch from preferring protein to sugar in these sugar-deprived animals, providing evidence for sugar-specific hunger. Recent studies in flies have made substantial progress in our understanding of feeding regulation. Through literature survey, we have gathered driver lines labeling neurons that potentially play a role in sugar intake and energy homeostasis. Through a search on FlyBase, a database of Drosophila genes and genomes, I identified a list of candidate genes predicted to encode transporters and receptors for carbohydrates, as well as G protein-coupled receptors. With all these works serving as a foundation, future experiments will use genetic screen to identify the neural circuits and molecular pathways that mediate the dietary switch in sugar-deprived animals.

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

"Investigating the Dynamic Between Telomere Length and Proliferative Capacity in Human Hematopoietic Stem Cells (hHSCs)"

Abstract:

A stem cell population’s telomere length has been linked with its ability to proliferate. This correlation is observed in telomerapathies like Dyskeratosis Congenita (DC), where abnormally short telomeres lead to clinical presentations such as bone marrow failure, skin abnormalities, and lung fibrosis. Mutations in the TINF2 gene, responsible for encoding the TIN2 protein in the telomere-binding complex shelterin, have been identified in DC patients. Upon endogenously introducing these TIN2-DC variants in hematopoietic stem cells (hHSCs) and transplanting them into immunocompromised mice, it was previously shown in the lab that while the telomeres shortened in the cells harboring DC alleles, those disease alleles were not depreciated in vivo. On the other hand, it has been observed that partially knocking out telomerase, a reverse transcriptase responsible for maintaining telomeres in adult stem cells, leads to depreciation of edited alleles, but the cause is unknown as the edited cells die out before they can be harvested from the mice. This study aims to elucidate the source of this proliferative disadvantage in vitro through single telomere length analysis (STELA) and exclude the possibility of off-target effects of the previously used editing strategy, further establishing the relationship between short telomeres and proliferative capacity. My findings will deepen our understanding of telomere biology in stem cell populations in addition to the molecular mechanisms of DC that can be harnessed for better therapeutic interventions.

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Diana Bautista, Department of Molecular & Cell Biology

"Investigating Acute and Chronic Itch Behaviors in Mice"

Abstract:

Acute itch sensations arise in response to a variety of external stimuli such as insects, parasites, allergens, mechanical stimuli, and chemical irritants. For itch associated with infection by parasites, scratching is likely induced as a tightly linked reflex-like response uniquely suited to remove harmful substances/organisms embedded in the skin. Itch-evoked scratching is also a symptom of chronic inflammatory skin conditions, such as atopic dermatitis and psoriasis, liver or kidney disease, and peripheral neuropathy. The consequences of scratching are likely distinct in these different settings, but no functional benefit has yet been uncovered. The standard in the field for studying itch and quantifying scratching behaviors is to score the total count of scratch bouts over a given time period. However, what constitutes a bout is poorly defined. Shimada and LaMotte state: “The number of scratches during a bout could be one or many. A bout of scratching could last several seconds and was initiated by lifting of the hind paw to the region of the body to be scratched” (Shimada and LaMotte, 2008), while others in the field have used the number of scratches and number of bouts interchangeably. For my thesis, we re-analyzed data from previously published experiments that examined scratching behaviors in acute and chronic models of itch. I compared the total scratching time, bout duration, and bout number, and found that different acute and chronic itch models induce unique itch-evoked scratching behaviors. For example, animals with eczema itch display significantly more bouts of a shorter duration, as compared to dry skin that induces fewer, but longer bouts (number of bouts: p=.00021; average bout duration:p=.000029; one-tailed t-test; n= 26-69 animals). Our data show that the current methods for assessing itch do not encompass the full complexity of itch behaviors. We argue that accurate, quantitative behavioral measurements are essential for pre-clinical development of therapeutic interventions for acute and chronic itch disorders.

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

"Intracranial Implantation of Oligodendrocyte Progenitor Cells and Medium Spiny Neurons Correct Clinical Phenotypes of Huntington’s Disease in a Mouse Model"

Abstract:

Huntington’s Disease (HD) is a rare, but serious inherited neurodegenerative

disease characterized by a loss of myelination on neuronal axons, manifesting

itself in physical and cognitive dysfunction in patients. No treatment currently

exists for preventing the disease. But two cell types, oligodendrocyte progenitor

cells (OPCs) and medium spiny neurons (MSNs), have emerged as potential

candidates for cellular replacement therapies in neurodegenerative diseases,

specifically in HD. The current work presented here focuses on the evaluation of

implantation of OPCs and MSNs for the correction of phenotypes associated

with a murine model of HD, since these cell types have known involvement in

myelin generation and sustainment in the brain. In order to test this hypothesis,

OPCs and MSNs were generated de novo by the differentiation of human

pluripotent stem cells (hPSCs). Following surgical intracranial implantation of

these cells in the striatum, notable improvements in cognitive function, physical

coordination, as well as overall lifespan in the surgically-treated mice were

observed, as compared to controls. In particular, lifespans were improved and

weight loss prevented among male mice with MSN and OPC injections. In

addition, anxiety and stress measurements were significantly lowered with

MSNs and OPCs. Further analyses indicated that motor function was slightly

improved with OPCs. Lastly, histology staining showed that cell survival and

migration was observed weeks after injection, indicating that this effect may be

durable. In attempts to increase cellular reuptake and engraftment, both 2D cell

suspensions and 3D hydrogel formats have been tested as surgical injection

modalities. In the future, we hope to optimize the scaled-up production of our cell

types to enable these initial experiments to be performed in more mice and

produce larger data sets. These data sets provide support for the translation of

cell-based therapies in order to achieve myelin regeneration for therapeutic

application for not only Huntington’s Disease, but all neurodegenerative

diseases involved with myelin dysfunction or loss.

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John G. Flannery, Helen Wills Neuroscience Institute, University of California, Berkeley

"The Effects of Immunosuppression on AAV Vector-Delivered GFP Expression in Mouse Retinal Bipolar Cells"

Abstract:

Adeno-associated virus (AAV) vectors are valuable gene delivery tools increasingly being used in gene therapy and optogenetics. Currently, they are being used to treat various ocular diseases, such as Retinitis Pigmentosa (RP), where individuals lose eyesight due to an inherited mutation that causes degeneration of photoreceptors, the first order neurons of the retina. To optogenetically treat RP with the aid of AAV vectors, second and third order neurons in the retina are targeted to be granted the ability to perceive light through the expression of light sensitive proteins since photoreceptors are not viable targets anymore in this disease. However, a major issue with AAV vectors, when recognized as antigens in vivo, is that their injection induces immune responses, especially in non-human primates and human patients. This inflammation inhibits successful viral transduction and decreases transgene expression in targeted cells, reducing therapeutic effect. To study this problem, two FDA approved immunosuppressants, Cyclosporine A and Dexamethasone, were intraperitoneally injected into adult C57BL6 mice before and after intraocular injection with AAV to determine if green fluorescent protein (GFP) transgene expression would be 1. detectable in more target cells, or 2. detectable at enhanced levels when these drugs are suppressing intraocular inflammation. We found that immunosuppression does not affect the number of transfected GFP+ cells, but there are gender differences in GFP+ cell density observed due to females having stronger immune responses. In terms of GFP fluorescence intensity, we found that Dexamethasone drastically increased GFP production in transfected cells compared to Cyclosporine A and the control, PBS treated group. Our findings indicate that Dexamethasone has undeniable potential in enhancing the therapeutic effect of AAV vector based optogenetic vision restoration in human patients.

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Lin He, Department of Molecular & Cell Biology

"Role of Rpl41 in Oogenesis and Fertility"

Abstract:

Ribosomal protein L41 (Rpl41) is a highly conserved ribosomal protein of the large 60S ribosome. Given the universal and critical function of the ribosome, mutations to ribosomal genes result in drastic phenotypic differences and often lead to lethality. The Rpl41 knockout (KO) mouse models were generated by CRISPR-Cas9 gene editing. To our surprise, KO mice could be recovered; however, female Rpl41 KO mice are sterile. In collaboration with He Lab members, we had preliminary histological analysis of two-, six-, and nine-month ovary samples which revealed that Rpl41 KO ovaries display a progressive degenerative phenotype. In KO ovaries, we had seen large cysts form, incomplete germ cell nest breakdown, and a failure in support follicle cell formation. Through carefully timed matings and precise microdissection of fetal ovaries, followed by quantitative-Real-Time PCR (qRT-PCR) analysis, I have identified 18.5 days-post-coitum (dpc) as the peak expression of Rpl41 across WT fetal and adult ovaries. Intriguingly, this timepoints perfectly overlap with late meiosis in females, when oocytes are arrested and stored for fertility. Therefore, I hypothesize that Rpl41 is an essential component of the intense translational needs for oogenesis to form viable oocytes. To investigate this, I did phenotypic comparisons at these timepoints through Hematoxylin and Eosin (H&E) staining and Immunofluorescence (IF) to identify morphological and molecular differences between WT and KO fetal and adult ovaries. I confirmed the morphological abnormalities in 2 month ovaries which form cysts and have abnormal follicle development but still have oocyte and follicle structures present. In 18.5 dpc ovaries, I found no obvious morphological differences but some molecular differences that suggest a defect in translation and the meiotic pathway. Establishing these is a starting point for understanding the molecular mechanism behind infertility in Rpl41 KO female mice and explaining the ultra-conserved nature of RPL41.

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Richard Harland, Department of Molecular and Cell Biology

"Design of Loss- and Gain-of-Function Constructs to Disrupt CXCR4, CXCR7, and SDF1 Signaling during late Gastrulation in Xenopus"

Abstract:

During gastrulation in Xenopus, a particular group of cells located at the organizer, called Bottle Cells (BCs), undergo apical constriction and invagination to form the dorsal lip of the blastopore. Then, the surrounding tissue ingresses inwards towards the anterior end of the embryo to form the mesoderm and the archenteron. However, the exact mechanism behind the invagination process and role of the BCs is not well understood. Based on previous studies performed on Xenopus and Zebrafish, we hypothesize that the triad composed of CXCR4, CXCR7 and SDF1 (CXCL12) play a central role on the invagination and enlargement of the archenteron, likely mimicking the mechanisms and pathways observed for the lateral system of Zebrafish, a well-established model of collective migration, which requires this triad. In this work, we looked for already described loss- and gain-of function mutations in other species and carried out an orthology analysis to assign and validate equivalent positions in Xenopus laevis. This review and in silico work constitutes the first step to describe the role of this protein triad, as well as discovering the downstream signals and genes that could trigger Bottle Cell behavior and archenteron formation.

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Raul Andino, University of California, San Francisco

"Adaptive Suicide and Immunosenescence in Kin-Specific Infection"

Abstract:

According to the adaptive suicide theory, an organism may choose suicidal strategy when the population is infected with highly sterilizing pathogens. However, we seldom observe the practice of suicidal behavior as a disease control mechanism among higher animals, which also share the pressure of kin-selective epidemic infection. This study aims to show that the reason behind the absence in higher animals' suicidal behavior might lie in their immune-driven resistance and disease tolerance. By using computer simulation and agent-based modeling, we found that given a highly sterilizing pathogen, the chronic type of pathogen would drive the evolution of adaptive suicide, whereas the acute type would select against it. Because of the diverging selection forces of the two pathogens, adaptive suicide is no longer advantageous as the species would be imperiled by the danger of extinction. When both types of pathogens are present, immunosenescence would be a more favorable evolution strategy.

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Phillip B. Messersmith, Departments of Bioengineering and Materials Science and Engineering

"One-Pot in Situ Synthesis of Bioinspired Polydopamine/Bacterial Cellulose Composites"

Abstract:

In nature, bloodworm Glycera dibranchiate possess a set of four mechanically robust jaws that can repeatedly protrude and retract to capture prey. The critical constituents of the jaw are melanin and proteins in a highly cross-linked network, which forms an organic-inorganic biomaterial with remarkable mechanical properties such as abrasion resistance and hardness. Inspired by Glycera jaw, this research aims to design a composite platform material using two bioinspired components, bacterial cellulose (BC) as a protein-like network and polydopamine (pDA) as the melanin-like structure. BC is a natural scaffold material possessing intriguing properties such as high-water holding capacity (hydrogels), high crystallinity, and high tensile strength. pDA, made from the precursor dopamine, is a synthetic version of melanin, capable of a variety of chemical reactions afforded by the catechol and amine functional groups3. Previously, BC has been shown to conform to 3D projects that are prepared with a pDA treatment, indicating the compatibility of pDA and BC growth; in addition, ex situ chemistry has enabled the formation of BC-pDA composite material with the help of chelating chemistry. Further, the aim of this project was to evaluate an in situ approach to incorporate the pDA inside the BC network during growth, by a one-pot synthesis through adding dopamine into the medium, allowing and controlling the oxidative polymerization into pDA whilst growing a sufficient amount of BC. The BC-pDA composites may offer a powerful chemical platform for a myriad of chemical and biological modification that potentiates great mechanical properties as a composite material.