Now showing items 21-40 of 1933

    • 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.
    • UMSOM Holds Virtual Commencement to Celebrate Class of 2020

      University of Maryland, Baltimore. School of Medicine (2020-05-15)
    • Commencement 2020

      Jarrell, Bruce E.; Hogan, Larry J., 1956-; Perman, Jay A.; Yang, Shi (Porter); Phelan, Mary T. (2020-05)
    • Targeting of Intracellular Survival Pathways in Human Prostate Cancer Cells

      Adediran, Samusi; Hussain, Arif, M.D. (2019)
      Although significant advances in the treatment of prostate cancer have been made, advanced prostate cancer is essentially incurable and remains the second leading cause of cancer-related deaths among men in the U.S. The current treatment landscape underscores the fact that additional strategies are needed to improve treatment outcomes of both castration sensitive and castration resistant advanced prostate cancer. Prostate cancer is characterized by a remarkable degree of heterogeneity in terms of its underlying biology and clinical behavior. Although pre-clinical models cannot adequately reflect the varied clinical behavior of prostate cancer, they are important for enhancing our understanding of prostate cancer biology and potentially identifying new therapeutic targets. In this thesis, we undertook the approach of studying several cell culture models of prostate cancer that in aggregate encompass many of the varied aspects of the prostate cancer disease spectrum seen clinically. Using these models, we focused on the erbB-PI3K-AKT axis as a paradigm of growth factor-receptor tyrosine kinase (RTK) signaling, and the androgen-AR axis, as well as the compensatory cross-talks within and across the pathways, to study the effects of combinatoric targeting of specific nodes in these signaling pathways. Chapter 1 provides background, pre-clinical and clinical context, and translational relevance of the studies presented in this thesis. Our research work is divided into two broad areas, namely the AR negative prostate cancer cells that include both drug-sensitive and drug-resistant models (Chapter 2), and the AR positive prostate cancer cells. These are characterized by a range of molecular signatures, including changes in AR/AR-V7 expression, functional PTEN status, wild-type (wt) or mutant p53 background, and altered underlying sensitivities to androgen receptor axis targeted (ARAT) agents (Chapter 3). The present work adds to the significant body of literature in this area of study and highlights the potential relevance of combinatoric targeting across a range of underlying molecular lesions that define the different phenotypes of prostate cancer.
    • What's the Buzz? 2020

      University of Maryland, Baltimore. School of Medicine, 2020
    • Respiratory protection safety culture beliefs among healthcare workers

      Hines, Stella E.; Oliver, Marc; Gucer, Patricia; Frisch, Melissa; Hogan, Regina; Roth, Tracy; Chang, James, C.I.H.; McDiarmid, Melissa (2019-10-30)
    • Effective Collaboration Towards Positive Perioperative Outcomes for the Cardiac Surgery Patient: A Perioperative Services - Cardiac Surgery Performance Innovation Project

      Perry, Mary; Fortuno, Maridulce Belen (2020)
      Nurses in the University of Maryland Medical Center (UMMC) Procedure Readiness Evaluation and Preparation (PREP) Center care for complex patients and support multiple surgical specialties, including cardiac surgery. Workflows related to cardiac surgery, specifically identification of blood type and antibodies (“blood banding”), presented a particular challenge for the clinic, and cardiac surgery patients were not always ready for the operating room (OR) on the day of surgery. PROJECT AIMS: To provide an efficient outpatient pre-operative 30-day blood band process in the PREP Center for the outpatient adult Cardiac Surgery patient. To establish a mutually beneficial internal work flow between UMMC Perioperative Services and Cardiac Surgery staff to minimize downstream OR delays.