Recent Submissions

  • Whole-genome analysis of Plasmodium falciparum isolates to understand allele-specific immunity to malaria

    Shah, Zalak; Takala-Harrison, Shannon (2020)
    After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.
  • Placebo Analgesia in Neuropathic Pain: A Translational Investigative Approach from Rodents to Humans

    Akintola, Titilola; Colloca, Luana (2020)
    Pain is a complex phenomenon which can be influenced by various factors. Placebo analgesia (PA) is the experience of pain relief after the administration of a physiologically inert intervention via the expectation of benefit. However, adequate animal models of PA in chronic neuropathic pain were unavailable to determine how PA occurs in neuropathic pain. Neuropathic pain (NP) is a chronic pain condition characterized by a dysfunction of the peripheral or central nervous system. There is still limited progress in translating the findings of preclinical studies to address the clinical burden of chronic pain. This is thought to partly reflect difficulties in reliably assessing pain in animals. Hence, we employ a translational approach in both rodents and humans to explore the occurrence of PA in chronic NP. First, I tested the hypothesis that the facial grimace scale is a useful metric of spontaneous pain in rodents. We performed a chronic constriction injury of the infraorbital nerve (CCI-ION) and tested for changes in mechanical hypersensitivity and grimace scores. Results showed rodents with CCI-ION had significantly higher grimace scores and lower mechanical withdrawal thresholds compared to controls. These changes were reversed by an opioid, indicating the grimace scale as a sensitive metric for assessing ongoing pain in CCI-ION. Secondly, I tested the hypothesis that pharmacological conditioning with fentanyl would produce PA in a rat model of CCI-ION. Rats were pharmacologically conditioned with or without contextual cues. We administered a placebo and found marginally significant PA effect via the grimace scale but not in mechanical sensitivity. These findings suggest that PA may be more challenging to induce in rodents. Finally, in humans, I investigated how NP-like symptoms in Temporomandibular Joint Disorder alter PA. The effect of NP on PA is yet to be fully understood. I tested the hypothesis that the presence of NP-like symptoms would decrease PA in TMD. NP assessment was carried out both in the orofacial region and across the whole body using validated screening tools. Our results showed that the presence of co-occurring NP-like symptoms increased PA in TMD. We also show that this effect is mediated by reinforced expectation.
  • Identifying pathogenic mechanisms and new therapeutic targets for Gaucher disease using induced pluripotent stem cells

    Srikanth, Manasa; Feldman, Ricardo A. (2020)
    Gaucher Disease (GD), the most common lysosomal storage disorder, is caused by mutations in the GBA1 gene, which codes for the lysosomal enzyme β-glucocerebrosidase (GCase). GCase breaks down sphingolipids but when it is mutated, it causes the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). The common manifestations of GD include hepatosplenomegaly, anemia, thrombocytopenia, skeletal disease, and in case of severe mutations, there are also fatal neurological manifestations. The conventional treatment is not effective in managing the skeletal or neurological manifestations. Hence, a better understanding of the underlying mechanisms that cause GD pathology is required for development of effective therapeutic strategies. The goal of this thesis was to identify the molecular mechanisms responsible for phenotypic alterations in osteoblasts and neuronal cells from GD patients, thereby pinpoint molecular targets for therapeutic intervention. Our laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) harboring GBA1 mutations to model GD. We have previously differentiated these iPSCs to various cell types and have shown that we can recapitulate the pathologic hallmarks of GD. Thus, in this study, we generated GD-iPSC derived osteoblasts and neuronal cells and found that mutations in GBA1 disrupt the canonical Wnt signaling and lysosomal compartment in these cell types. The phenotypic consequence of this was observed in the form of defective osteoblast differentiation and maturation as well as loss of midbrain/hindbrain neuronal progenitors in the respective cell types. Due to the known lysosomal dysregulation in GD, we then explored the mTOR pathway which is upstream of lysosomal biogenesis. We found hyperactivation of mTOR in GD neuronal cells was mediated by the significant accumulation of GlcSph, a lysolipid of GlcCer. In addition, when we blocked the conversion of GlcCer to GlcSph using acid ceramidase inhibitors, we were able to reverse mTOR hyperactivation and restore lysosomal expression, suggesting that GlcSph is partly, if not fully, responsible for the lysosomal abnormalities observed in GD. In conclusion, our study reveals that activation of canonical Wnt pathway or suppression of mTOR pathway ameliorates the phenotypic abnormalities in GD and identifies b-catenin, mTOR and acid ceramidase as potential therapeutic targets for GD.
  • Expression patterns of the metastasis suppressor NME1 in metastatic melanoma

    Snyder, Devin; Kaetzel, David M. (2020)
    Localized melanoma is a curable form of skin cancer, with a 98% chance for survival. However, for individuals diagnosed with metastatic melanoma, the five-year survival rate is reduced down to 23%. To enhance patient survival, it remains imperative to fully differentiate melanoma metastasis mechanisms. Our motivation lies on depicting underlying patterns accompanying metastatic inhibition. Here, we examine inhibition of melanoma metastasis through analysis of the metastasis suppressor protein, NME1. NME1 is a known inhibitor of melanoma metastasis. Overall loss of NME1 protein in melanoma tumors is associated with reduced survival. Yet, not all melanoma tumors are the same. Melanomas are heterogenous and exhibit distinct differences across patients and across individual primary tumor cells. The prognostic implications of heterogenous NME1 expression within melanomas has not previously been defined. Our focus lies in characterizing how expression of NME1 across patients and across primary tumor cells may impact melanoma prognosis and overall metastatic incidence. We utilized a NME1-based approach to identify a mRNA expression signature associated with metastasis suppression in melanoma. Bioinformatic analysis of our metastasis suppression signature in a melanoma patient dataset exposed a subset of melanoma patients with altered survival. We were also interested in analyzing the heterogenous expression of NME1 protein within melanoma cells. Flow cytometry analysis revealed that melanoma cell lines contain a rare population of cells with low levels of NME1 protein. We utilized multiple methods to characterize the impact of NME1 loss on cell behavior. Experiments designed to mimic loss of NME1 through shNME1 produced results which suggest that NME1 is associated with melanoma spheroid formation. We further utilized an innovative approach, which relied on CRISPR technology, to study endogenous NME1 expression. Characterization of endogenous NME1 expression patterns lead to the identification of a previously unidentified group of cells, which have a neural crest-like phenotype and contain enhanced metastatic capacity. Overall, the expression pattern of NME1 within tumor cells and across patients provides an advantageous tool for identifying metastatic susceptibility.
  • Development and Application of Vertebrate Models to Investigate the Risk of Defense-Relevant Chemicals

    Narizzano, Allison; Pereira, Edna F. R.; Quinn, Michael J., Jr. (2020)
    Adverse effects from unintended exposure to chemicals have been widely described and have provided the basis for many chemical and environmental management regulations and policies that are intended to protect humans, animals, and/or the environment. The present work focuses on two classes of chemicals that are tested and/or used in training activities at military sites: insensitive munitions (IMs) and per- and polyfluoroalkyl substances (PFAS). As groundwater is a common source of drinking water, accurate risk assessments are critically needed to ensure protection of the environment and public health. IMs are more resistant to accidental, unintentional or incidental detonation than traditional explosives. Incomplete detonation may deposit munition components and by-products on impacted installations and, importantly, mobilize those constituents into groundwater. One component of several IM mixtures is 3-nitro-1,2,4-triazol-5-one (NTO). NTO is especially worrisome because it is highly water soluble and, thus, mobile in the environment. Additionally, laboratory tests with rodents have identified the testes and epididymides as targets of NTO. PFAS are also prevalent and problematic, nearly universal contaminants. PFAS are manufactured for use in paints, cleaning agents, non-stick cookware and food containers, water-impermeable products, and Aqueous Film Forming Foams (AFFFs). PFAS have attracted increased regulatory scrutiny because of their resistance to degradation, ability to bioaccumulate, and growing evidence of toxicity in animals. A considerable body of work has examined the effects of NTO in laboratory rodents. However, there is still uncertainty in the derivation of a safe workplace environmental exposure level (WEEL) for NTO. The present studies evaluate the effects of NTO in the Japanese quail (Coturnix japonica) and in Sprague Dawley rats. In concert, these toxicity data on NTO will improve risk estimation efforts, provide data to explore taxa read-across, and fill a regulatory needed data-gap. The toxicity of PFAS will be evaluated in the native mammalian species, Peromyscus leucopus. Given that the serum elimination half-lives within this class of chemicals can vary greatly from hours to years based on species, sex, and dose, extrapolating between species is difficult and inaccurate. As such, wild mammalian-specific data is novel and valuable for risk estimation in taxa that are directly site-specific.
  • Inducing DNA Repair Deficiencies in Triple Negative Breast Cancers Through Pharmacologic Stimulation of Innate Immune Signaling

    McLaughlin, Lena; Rassool, Feyruz V. (2020)
    Poly (ADP-ribose) polymerase inhibitors (PARPi) are FDA approved in a subset of patients with ovarian cancer or metastatic breast cancers who harbor BRCA gene mutations. These mutations generate homologous recombination deficiencies (HRD) and are the main predictor to PARPi sensitivity. Unfortunately, responses to therapy have not been durable and have failed for the majority of sporadic triple negative breast cancers (TNBC). We previously reported that DNA methyltransferase inhibitor (DNMTi) azacytidine (Aza) improves the efficacy of a new generation of PARPi, Talazoparib (Tal), through increased trapping of cytotoxic PARP-DNA complexes in both BRCA-mutant and -proficient TNBC. These trapped complexes lead to increased and persistent levels of lethal double strand breaks (DSBs), suggesting that DSB repair may also be impaired with this treatment. In the present study, we show that Aza/Tal treatment in BRCA-proficient TNBC cell lines significantly downregulates expression of HR and Fanconi Anemia (FA) genes, notably FANCD2, and decreases HR activity, thus generating HRD. DNMTi have also been established to induce a viral mimicry response which upregulate Type I interferon (IFN) signaling and production of inflammatory cytokines. We now link Aza/Tal facilitated HRD and induction of innate immune and inflammatory related genes, mediated in part through a STING dependent mechanism. Gene set enrichment analysis of RNA-Seq data derived from mono- and combination-treatments, reveal enrichment of innate immune and cytosolic DNA sensing pathways with significant increases of TNFα/NF-κB and IFNαβ gene sets. Overlap between HRD and immune related signaling was evaluated using the STRING database, which reveals a significant interaction specifically between FA pathway and TNFα/NF-κB and IFNαβ pathway genes. This inverse relationship was also validated in both METABRIC TNBC dataset and other TCGA data sets suggesting broad applicability of this observed transcriptional program independent of pharmacologic intervention. Additionally, Tal driven cytosolic DNA as well as an Aza augmentation in STING protein expression, emerges as the key node in Aza/Tal induced innate immune signaling to drive HRD. Induction of what we define as a pathogen mimicry response to drive HRD mechanism suggests that DNMTi-PARPi therapy strategies can expand the therapeutic scope of PARPi to encompass treatment of BRCA-proficient cancers.
  • Elucidating the Localization of Estrogen and Estrogen-related Receptors in the Inner Ear

    Lipford, Erika; Hertzano, Ronna P. (2020)
    Hearing loss is the most common sensory impairment, affecting hundreds of millions of people worldwide. Although both men and women are impacted by hearing loss, the incidence rate differs between the two sexes, with the prevalence of bilateral high-frequency hearing loss reported to be 2.7 times higher in males. Recent studies have implicated estrogen as having a protective effect against hearing loss in females. While the localization of the estrogen receptors within the cochlea is known, the roles of estrogen-related receptors in auditory function require further investigation. The aim of this study is to elucidate the localization of estrogen receptors 1 and 2 and estrogen-related receptors α, β, and γ within the inner ear of adult mice using RNAscope. Our results give insight into the molecular mechanism through which estrogen receptors and estrogen-related receptors may grant protection against hearing loss and contribute to the functionality of the inner ear.
  • The Effect of ZSCAN4 on Telomere Chromatin Remodeling

    Lin, Phyo Nay; Zalzman, Michal (2020)
    Telomeres are repetitive sequences at the ends of chromosomes that protect the coding regions of DNA. Telomeres shorten with every cell division and therefore operate as a biological clock. Thus, factors regulating telomeric chromatin impact cell replicative lifespan, tumor formation and growth. The murine Zinc Finger and SCAN Domain Containing 4 (mZscan4) promotes telomere homeostasis and genomic stability in mouse embryonic stem cells (mESCs). A transient expression of mZscan4 was shown to correlate with chromatin de-repression in mESCs. However, the function of human ZSCAN4 in its contribution to the epigenetic landscape changes at telomeric chromatin remains to be determined. In this study, we defined the effect of ZSCAN4 on histone 3 and 4 hyperacetylation at the telomere region which is associated with telomere extension. Understanding the mechanism by which ZSCAN4 affects the telomeric chromatin is important for designing new therapeutic approaches to target cancer cell replicative lifespan.
  • Understanding the Role of Small Ankryin 1 in Calicum Regulation in Excitable Cells

    Labuza, Amanda; Bloch, Robert J. (2020)
    Small Ankryin 1 (sAnk1) is a 17kD transmembrane protein that plays a role in stabilizing the network sarcoplasmic reticulum in skeletal muscle (Ackermann et al., 2011). Recent studies have shown that sAnk1 can bind to and regulate sarco(endo)plasmic reticulum Ca2+-ATPase1 (SERCA1) activity (Desmond et al., 2015). SERCA1 transports Ca2+ against its gradient into the SR after muscle contraction. SERCA is inhibited by sarcolipin (SLN) in fast twitch skeletal muscle and atrial cardiac muscle and by phospholamban (PLN) in slow twitch muscle and ventricular cardiac muscle. Like SLN and PLN, sAnk1 also interacts with SERCA at least in part through its transmembrane domain (Asahi et al., 2003; Hutter et al., 2002; Desmond et al., 2015). The interaction of SERCA with SLN and PLN has been studied individually and together, but the effects of sAnk1 and its regulatory activity have only recently started to be addressed (Desmond et al., 2015, 2017). Here I show that sAnk1 can interact with PLN or SLN independently of SERCA1. sAnk1 forms a three-way complex with SLN and SERCA1 that ablates SLN inhibition (Desmond et al., 2017). sAnk1 can also form a three-way complex with PLN and SERCA1 that abolishes all inhibition. I show that the complexes that sAnk1 forms with SLN or PLN and SERCA1 are distinct, suggesting unique roles for each protein in SERCA regulation. I also examined sAnk1 and SERCA in several CNS tissues, and found that sAnk1 is not expressed in neurons, but that it is expressed in astrocytes, where it has the potential to bind and regulate SERCA2B. Studying the multi-protein complex of SERCA, sAnk1, SLN, and/or PLN can help us better understand physiological SERCA regulation. This knowledge can lead to better treatment for diseases related to misregulation of calcium, including muscular dystrophies and potentially some neuropathies.
  • Cryo-Electron Microscopy Structure Determination of the Anthrax Toxin Protective Antigen Bound to its Lethal and Edema Factors

    Hardenbrook, Nathan; Krantz, Bryan A. (2020)
    Protein translocation is an essential function within all living cells. Translocons are dedicated protein translocation machinery, responsible for the unfolding and translocation of proteins. Due to the thermostability of most proteins in their native states, these translocons utilize various different forms of energy to drive the translocation of their substrates. This process is mediated by polypeptide clamps responsible for catalyzing the unfolding and translocation of the protein. Using lipid nanodiscs and cryo-electron microscopy (cryoEM), we have determined structures of heptameric anthrax lethal toxin and edema toxin channels to 4.6 and 3.2-Å resolution, respectively. Additionally, using cryoEM we have determined the first atomic structures of PA8 prechannel bound to full-length EF and LF to 3.3 and 3.7-Å resolution, respectively. In this pre-translocation state, the first α helix and β strand of LF and EF unfold and the α clamp, which resides at the interface of two PA subunits. The α clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins, supporting previous work indicating that the α-clamp engages substrate α-helices repeatedly during translocation. A PA loop in the binding interface is displaced between the prechannel and channel. This results in the loss of a salt bridge and leading to the weakening of the binding interface prior to translocation in the PA7EF structure. Lastly, EF undergoes a largescale conformational rearrangement when forming the complex with PA, compared the solution structure of EF bound to calmodulin. Recruitment to the PA prechannel exposes an originally buried β strand and enables domain organization of EF. Many interactions are formed on domain interfaces in both PA prechannel-bound EF and LF, leading to toxin compaction prior to translocation. This work has resulted in the first structures of PA bound by edema factor, as well as the first structures of PA bound to a full-length substrate. These structures have provided insight into important biophysical steps occurring in preparation for translocation They reveal structural plasticity within the binding α-clamp binding site, allowing the translocating substrate to be engaged multiple times. This provides a greater understanding of how anthrax toxin can invade the host cytosol.
  • Quantitative Analysis of Compartments in the Leg and Implications for Trauma Surgery

    Agandi, Lorreen; Puche, Adam C. (2020)
    Compartment syndrome is characterized as an excess in swelling leading to an increase in pressure in a limited space and a lower extremity fasciotomy is performed to mitigate the effects. A two-incision fasciotomy is performed on the medial and lateral sides of the leg, accessing the posterior superficial, deep superficial, anterior and lateral compartments respectively. Ongoing studies have shown the lateral compartment is commonly decompressed incorrectly. This error has led to the hypothesis that there is variability in septum position and that using the fibula as a landmark can lead to erroneous incision placement in patients. CTA scans were analyzed to assess septum position. Findings indicate that the septum position shifts anteriorly progressing distally down the leg, indicating variability at different points in the leg. If surgeons do not take septum variability into consideration when decompressing the lateral compartment, this can lead to incorrect decompression of the lateral compartments.
  • Insertional Mutants of Chlamydia caviae Display Altered Virulence

    Filcek, Kimberly; Bavoil, Patrik M. (2020)
    The genus Chlamydia encompasses multiple bacterial species capable of colonizing and causing infection in a diverse range of host organisms. The type III secretion system and secreted effector proteins of this genus are important subjects for elucidating chlamydial biology. One important group of secreted proteins is the Inc family which are integral membrane proteins of the chlamydial inclusion found in all Chlamydia. Another novel protein, SinC, is a type III secreted effector initially described in Chlamydia psittaci, a zoonotic pathogen, but whose role in pathogenesis remains largely unknown. Characterization of specific genes in Chlamydia has been uniquely difficult due to its genetic intractability, but recent advances in chlamydial genetics have provided us the opportunity to generate targeted, stable mutants. The Chlamydia caviae GPIC strain has been used to model ocular and genital tract infection in guinea pigs. However recent C. caviae isolates were implicated in zoonotic respiratory infection in humans. The GPIC strain carries the two genes of interest, sinC and incA. Unlike for C. psittaci, we were able to perform mutagenesis in C. caviae without the constraints of BSL-3 containment, and with the benefit of well-characterized animal models. Here I engineered site-specific insertional mutations in sinC and incA, the first instances of targeted mutagenesis in the C. caviae lineage, with the aim of investigating the role of these genes in chlamydial pathogenesis.
  • Targeting the crosstalk between AR3 and E2F1 as a prospective therapy for drug-resistant prostate cancer

    Xu, Jin; Qiu, Yun (2020)
    Background: Drug resistance is one of the most prevalent causes of death in advanced prostate cancer patients. Combination therapies that target cancer cells via different mechanisms to overcome resistance have gained increased attention in recent years. However, the optimal drug combinations and the underlying mechanisms are yet to be fully explored. Aim and methods: The aim of this study is to investigate drugs that inhibit the growth of cells that are resistant to standard chemo and androgen deprivation therapy, and determine the underlying mechanisms of their action. To achieve this aim, we established cell lines that are resistant to this standard combination drug treatment and tested new compounds to overcome this “double drug” resistance. Results: Our results show that combination of enzalutamide (ENZ) and docetaxel (DTX) effectively inhibit the growth of prostate cancer cells that are resistant to either DTX or ENZ alone. The downregulation of transcription factor E2F1 plays a crucial role in cellular inhibition in response to the combined therapy. Notably, the androgen receptor (AR) variant AR3 (a.k.a. AR-V7), but not AR full length (AR-FL), positively regulates E2F1 expression in these cells. Specifically, E2F1 regulates AR3 and forms a positive regulatory feed-forward loop. Moreover, this drug combination treatment also results in DNA double strand break via the E2F1-AR3 signaling axis. Importantly, we established new drug-resistant cell lines that are resistant to ENZ+DTX combination therapy and found that the expression of both AR3 and E2F1 was restored in these double drug-resistant cells. Furthermore, we identified that auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, overcame the drug resistance and inhibited the growth of drug-resistant prostate cancer cells both in vitro and in vivo. Conclusion and significance: This proof-of-principle study demonstrates that targeting the E2F1/AR3 feedforward loop via a combination therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer.
  • Effects of Acute Stress on Discriminative Fear Conditioning: A Key Role of Kynurenic Acid in the Medial Prefrontal Cortex

    Klausing, Alex; Schwarcz, Robert
    Stressful events can profoundly impact physiology and are implicated in multiple brain disorders. Likewise, the kynurenine pathway (KP) of tryptophan degradation is associated with neurological diseases. Both stress and the KP, in particular the metabolite kynurenic acid (KYNA), can affect behavior, specifically learning and memory. Moreover, stress has been shown to manipulate the KP, but an investigation into the link between stress, KYNA, and cognition has yet to happen. To this end, we utilized three acute stressors (predator odor exposure, restraint, and inescapable shocks) of differing degrees of severity to investigate stress-related effects on KYNA levels in the medial prefrontal cortex (mPFC) and cognitive behavior. We focused on fear discrimination because it requires the mPFC, is easily testable in rodents, and is a hallmark of many stress-related disorders, in particular post-traumatic stress disorder. Furthermore, the adrenal gland, critical for an organism’s stress response, has been shown to have effects on the KP and cognition. Therefore, we also examined how adrenalectomy (ADX) affects a stress-induced change in KYNA and behavior. Inescapable shocks stress (ISS) was the most severe form of stress tested, defined by the highest increase in plasma corticosterone levels in naïve rats. ISS induced a significant, sustained elevation of extracellular KYNA in the mPFC and impairments in fear discrimination. A KYNA synthesis inhibitor administered before the initiation of ISS decreased the stress-induced KYNA increase and normalized the fear discrimination impairments, suggesting a causal linkage. Restraint and predator odor exposure did not affect KYNA levels or fear discrimination. However, in ADX rats, the threshold for the severity of stress required appears to be lower to elicit the effects described above. Restraint stress produced an increase in extracellular KYNA levels in the mPFC of ADX rats as well as impairments in fear discrimination, contrary to rats with intact adrenal glands. Additionally, a KYNA synthesis inhibitor attenuated these biochemical and behavioral effects. Together, these findings suggest a causal relationship between the stress-induced increase in KYNA and cognitive deficits. Therefore, targeting the KP by pharmacological or other means may alleviate some of the detrimental symptoms seen in stress-related psychiatric disorders.
  • Social Interaction and Pain

    Jenne, Carleigh; Keller, Asaf (2020)
    Chronic pain is the most common cause of disability. Progress in research to alleviate pain is hampered by the fact that metrics for studying pain in animal models are controversial. Rodents highly value social interactions, preferring them even over drugs of abuse or other hedonic rewards. Here, I tested the hypothesis that pain will reduce preference for social interaction, thereby offering a novel tool to quantify pain behaviors. After training rats to self-administer social interaction, I found that acute pain causes devaluation of social interaction. This devaluation was specific to social interaction, because after training rats to self-administer food, acute pain elicited no change in valuation for food self-administration. My findings display the importance of social interaction in pain behaviors, and suggest a novel metric for pain studies.
  • Investigating the Role of Metastasis Suppressor 1 (MTSS1/MIM) in Cancer Biology

    Baxter, Shaneen; Zhan, Steven (2020)
    Metastasis suppressor 1 (MTSS1/MIM) is a multi-domain, membrane-associated protein that has been linked to progression and poor prognosis of several types of cancer. It was initially thought to be metastasis suppressor, but it has been shown to be overexpressed as well as downregulated in both metastatic and non-metastatic cancer, making its role in tumorigenesis unclear. There are also questions about how MIM becomes deregulated in certain types of cancer. In this study, we hypothesized that factors within the microenvironment such as inflammation, nutrient availability, chemokine gradients and autophagy are major contributors to the role of MIM in cancer progression and metastasis. As a member of the BAR domain superfamily, a family of proteins that bind and deform membranes, MIM is thought to be involved in intracellular membrane trafficking pathways such as endocytosis and autophagy. This lab previously reported that MIM interacts with E3 ubiquitin ligase, AIP4, and endocytic Rabs to regulate cell surface expression of the CXCR4 receptor. In this study, we further investigated the role of MIM in CXCR4 endocytosis by examining the interaction of MIM with Rab7 and Rab11. Upon internalization, CXCR4 may be guided into the lysosomal degradation pathway or the recycling pathway. We found that an interaction with Rab7 (a marker for late endosomes of the lysosomal pathway) is necessary for MIM to function in CXCR4 endocytosis. We also found that MIM does not interact with Rab11 (a marker for recycling endosomes), instead, another I-BAR domain protein, IRTKS, seems to mediate CXCR4 recycling. We examined the role inflammation may play in aberrant MIM expression and function, and discovered that inflammatory cytokines downregulate MIM in macrophages, indirectly leading to CXCR4 cell surface overexpression and increased migration of these cells towards a SDF-1 gradient. This is of significance because SDF-1 is often secreted by stromal cells within the tumor microenvironment and at common metastatic sites. We also investigated the role of MIM in autophagy, and found that overexpression of MIM inhibits basal autophagy, which has the potential to promote tumorigenesis. Overall, our results provided further insights into how MIM deregulation can lead to cancer progression and metastasis.
  • Optical Recordings of Action Potentials and Voltage Sensing Domains

    Banks, Quinton; Schneider, Martin F. (2020)
    Excitation-contraction coupling (ECC) allows muscle to translate an action potential (AP) into muscle contraction. Optical methods for measuring membrane potential changes can expand our understanding of excitable cell function. Applying the potentiometric dye Di-8-ANEPPS and high-speed confocal microscopy to flexor digitorum brevis (FDB) muscle fibers from adult mice we non-invasively measure the electrical properties of these fibers. We determined action potential conduction velocity by comparing the time course of action potentials initiated at either end of muscle fibers by using alternate polarity electric field stimulation. Action potentials propagated longitudinally at a velocity of 0.39 ± 0.02 m/s. Conduction velocity of calcium transients, using mag-fluo-4, a low-affinity calcium indicator, was 0.37 ± 0.03 m/s, similar to Di-8-ANEPPS. We used mag-fluo-4 to examine whether our approach could capture conduction changes due to ionic concentrations, fiber length, and a lack of dystrophin, and found that we could. A lumped component equivalent electrical circuit model of the muscle fiber’s passive properties reproduced the observed passive responses of muscle fibers. This method using dyes allows the study the action potential propagation in a non-invasive manner in FDB fibers under differing physiological conditions and in various disease states. In skeletal muscle, AP sensing is governed by Cav1.1. Cav1.1 has 4 voltage sensor domains (VSDs) located in the membrane that that move in response to changes in membrane potential. During an AP these VSDs shift, initiating calcium release. We have not yet elucidated VSD movement in Cav1.1. Here we transfect mouse FDBs with a version of Cav1.1 that has a cysteine near the VSD. This allows us to attach a thiol-reactive fluorescent probe near the VSD and track its movement. This did not noticeably affect trafficking or function of Cav1.1. In resting conditions, our cysteine of interest should be embedded in the membrane. Upon depolarization, this cysteine shifts out of the membrane, allowing us to detect its movement as a change in fluorescence after changes in fluorophore environment. Using field stimulation fluorometry, we observed the shifts of Cav1.1 VSDs in response to field stimulation and analyzed how they correspond to Ca2+ release in native tissue.
  • Long term intraocular pressure (IOP) lowering effect of Femtosecond Laser Assisted Cataract Surgery (FLACS) versus conventional phacoemulsification (PE)

    Ataei, Yasaman; Saeedi, Osamah J. (2020)
    This is a retrospective cohort study of 244 otherwise healthy eyes (from 244 patients) undergoing cataract surgery. Patients were followed postoperatively at 1, 3, 6, 12, 24, 36 months for IOP measurement. Combination procedures and glaucomatous eyes were excluded. 147 eyes underwent conventional phacoemulsification and 97 underwent FLACS. Amongst eyes undergoing FLACS, we observed 1.42 mmHg crude decrease in postoperative IOP from baseline over 3 years of follow-up (p = 0.003). Amongst eyes undergoing conventional phacoemulsification, we observed 1.18 mmHg crude decrease in postoperative IOP from baseline over 3 years of follow-up (p = 0.003). Amongst non-Caucasian patients, eyes undergoing FLACS had 1.90-3.38 mmHg lower postoperative IOP compared to eyes undergoing conventional phacoemulsification (ps = 0.04-0.009). Our study showed that in non-Caucasian patients who were followed for an average of 22 months, the postoperative IOP was significantly lower in eyes that underwent FLACS compared to eyes that underwent conventional phacoemulsification.
  • Neuroimaging Insights Into Neuromodulation for Addiction: Effects of Transcranial Direct Current Stimulation on Cognitive Circuits Implicated in the Nicotine Withdrawal Syndrome

    Aronson Fischell, Sarah; Stein, Elliot; Keller, Asaf (2020)
    Cigarette smoking is the leading cause of preventable death in the United States. The nicotine withdrawal syndrome (NWS) remains a barrier to successful smoking cessation; however, current pharmacological treatments minimally impact sustained abstinence. An emerging class of non-invasive neuromodulation devices, such as transcranial direct current stimulation (tDCS), have been proposed as novel therapeutics for smoking cessation. tDCS has the potential to modulate brain circuits by application of weak currents through the scalp; its use builds upon recent advances in mapping the large-scale network organization of the brain. Functional magnetic resonance imaging (fMRI) functional connectivity (FC) studies have identified three networks as particularly vulnerable to disruption in psychopathology: the Executive Control Network (ECN), Salience Network (SN), and Default Mode Network (DMN). The NWS has been hypothesized to be mediated by reduced FC within the ECN, and between ECN–SN; and increased FC within the DMN, and between DMN–SN. It is hypothesized that tDCS, applied to cortical nodes of the ECN (e.g. dorsolateral prefrontal cortex) and DMN (e.g. ventromedial prefrontal cortex), may remediate NWS network dysregulation. Network effects of tDCS were assessed by simultaneous task-based fMRI. 15 smokers (in sated and withdrawal states) and 28 matched nonsmokers participated in a double-blind, randomized crossover design of three tDCS conditions: anodal left-dlPFC/cathodal right-vmPFC (“An-dlPFC”), polarity reversed (“An-vmPFC”), and Sham. Although single-session (25min, 2mA) tDCS did not evoke task behavior changes, An-dlPFC tDCS robustly suppressed DMN nodes during a working memory task, and enhanced anterior cingulate activity (SN node) during a conflict monitoring task. DMN suppression within smokers was more pronounced during the sated (vs. withdrawn) state. Given that DMN and SN are hypothesized to be dysregulated in nicotine and other addictions, these data quantitatively support the hypothesis that tDCS may modify large-scale circuits implicated in addictive disease. Additionally, the observation of state-dependent tDCS effects in smokers suggests that tDCS may be most efficacious when combined with standard smoking cessation therapies. This work contributes a translational approach to assessment of tDCS, an emerging intervention at the crossroads of basic neuroscience research and clinical therapeutics in addiction and psychiatric disease.

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