Poster Session Abstracts
Neural Instruments of Prediction in Early Auditory Cortex (PRE-CANDIDACY)
Kiefer Forseth1; Gregory Hickok 2; Nitin Tandon1,3
1Vivian L. Smith Dept. of Neurosurgery, UT Health Science Center at Houston, Houston, TX, USA, 2Department of Cognitive Sciences,University of California, Irvine, California, USA, 3Memorial Hermann Hospital, Texas Medical Center, Houston, TX, USA
Neural computations in the brain are not merely a passive, stimulus-driven response – rather, cortical networks could be expected to anticipate patterns of sensory events. Acoustic rhythms contain the requisite information for such the prediction of future events. While the importance of rhythm in auditory perception seems intuitively clear, the neural mechanisms of auditory entrainment and the interactions of entrained cortex with incoming stimuli are not fully understood.
Intracranial electrodes (n = 3247, 15 patients) implanted for pre-surgical evaluation of epilepsy furnish the full spectrum of human neural oscillations at millimeter spatial and millisecond temporal resolution. We used an innovative stimulus with a period of amplitude modulated white noise followed by a period of constant amplitude white noise. In half of the trials and at variable delay, a pure tone was presented in the second period (coincident and partly masked by the white noise). Patients were asked to report the presence of the tone in each trial: a single-interval two-alternative forced-choice task.
Analysis of the behavioral data showed a modulation of perceptual accuracy by the phase of the entraining rhythmic stimulus. Data from depth electrodes placed along the dorsal superior temporal gyrus revealed two primary effects in early auditory cortex. First, we observed a posterior-anterior gradient of selectivity for acoustic onset, entrainment, and offset. Second, we observed a consistent alignment of gamma, beta, and low-frequency (1-15 Hz) power and phase relative to the stimulus.
Finally, these same patterns of activity were observed while the patients actively listened to short sentences. These neural representations of rhythm may constitute an adapted computational solution – cascaded neural oscillators – to enable predictive coding and timing.
Cholinergic control of cortical circuit dynamics (POST CANDIDACY)
Rajan Dasgupta1,2, Frederik M. Seibt1 and Michael Beierlein1,2
1Department of Neurobiology & Anatomy, McGovern Medical School at UTHealth and 2University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA
Cholinergic neurons of the basal forebrain (BF) form extensive projections to neocortex and are critically involved in mediating numerous cognitive processes. To understand how acetylcholine (ACh) shapes cortical computations, it is important to gain a better understanding of the underlying cellular and circuit mechanisms. However, how synaptically released ACh influences cellular targets and ultimately controls cortical circuit dynamics remains poorly understood. We addressed this question using a combination of in vitro electrophysiology and optogenetics in mouse somatosensory (barrel) cortex. We found that the activation of BF cholinergic afferents led to strong reduction of evoked cortical activity, with nicotinic receptors (nAChRs) mediating transient suppression, and muscarinic receptors (mAChRs) mediating prolonged suppression. Postsynaptic cholinergic responses were prominent in layer 4 and were primarily mediated by mAChRs, leading to long-lasting IPSCs in excitatory neurons and EPSCs in regular-spiking interneurons. In agreement, cholinergic suppression of cortical activity in the isolated layers 4-6 was entirely dependent on mAChR activation. In contrast, cholinergic responses in supragranular layers were predominantly mediated by nAChRs expressed by GABAergic interneurons. Taken together, our results indicate that cholinergic control of cortical network dynamics occurs over different time scales and is mediated by nAChRs and mAChRs-dependent mechanisms expressed in distinct cortical layers and cell types.
Evaluating the Role of Plasma Membrane Lipid Structure on KRAS Function in Cancer (PRE-CANDIDACY)
Walaa E Kattan1, Dharini van der Hoeven1,2, Tien-Hung Lan1, Wei Chen1, Yong Zhou1, John F Hancock1
1Department of Integrative Biology and Pharmacology, Graduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center UTHealth, Houston, Texas 77030, USA, 2 Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center, Houston, Texas 77030, USA
Ras proteins, including isoforms H-, N- and K-Ras, are small GTPases that switch between active GTP-bound and inactive GDP-bound states. Mutated K-, H- and N- Ras are found in ~22% of all human tumors, with K-Ras mutations comprising 86% of RAS-driven cancer. K-Ras signaling occurs mostly on the plasma membrane (PM) and is compartmentalized into nanoclusters of K-Ras protein aggregates that selectively sort PM lipids. Super-resolution quantitative imaging techniques, such as electron microscopy and fluorescence lifetime imaging, show extensive K-Ras co-localization with acidic lipid phosphatidylserine (PtdSer). Depleting PtdSer from cell PM, via pharmacological agent fendiline, mislocalizes K-Ras from the PM, disrupts nanoclustering of the remaining K-Ras on the PM, abolishes K-Ras-dependent MAPK and PI3K signaling and proliferation in cultured cancer cell lines, and decreases the size of K-Ras-dependent tumors in animal models. Supplementation of exogenous PtdSer, but not other lipid types tested, rescues this phenotype. PtdSer level in the PM is dynamically maintained via transport mechanisms between PM and endomembrane compartments including the endoplasmic reticulum and lysosomes. We hypothesize that perturbing PtdSer transport between PM and endomembranes directly attenuates K-Ras function. RNAi screening in C.elegans using let-60-activated multi-vulva phenotype as a read-out of K-Ras activity identified 5 candidate genes involved in PtdSer transport. We are establishing stable knockdown cell lines across multiple cancer types to study detailed molecular mechanisms underlying PtdSer-mediated K-Ras signaling. This effort will shed light on lipid-mediated K-Ras spatial segregation on the cell PM and signal transduction, and potentially inspire novel drug development strategies for K-RAS-driven cancer.
Elevated Cochlear Adenosine-mediated Metabolic Disturbance Underlies Hearing Loss (PRE-CANDIDACY)
Jeanne M. Manalo1,2, Hong Liu1,2, Dalian Ding4, Travis Nemkov5, Angelo D’Alessandro5, Hong Yue6, Youqiong Ye1, Leng Han1,2, Wa Xian6, Richard Salvi4, Frederick A Perreira3, Rodney Kellems1,2, Yang Xia1,2
1Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, TX, USA,2Graduate School of Biomedical Science, University of Texas Health Science Center at Houston, Houston, TX, USA,3Huffington Center on Aging, Baylor College of Medicine, Houston, TX, USA, 4Department of Communicative Disorders and Sciences, University at Buffalo, The State University of New York, Buffalo, New York, USA, 5Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA, 6University of Houston, Houston, TX, USA
Adenosine is a signaling nucleoside involved in regulating physiological and pathological systems. One of the ways adenosine is cleared is through adenosine deaminase (ADA) catalysis. Ada-deficient patients have high levels of adenosine with serious health problems, including hearing loss (HL). Additionally, previous studies have shown noise-exposed mice have elevated cochlear adenosine, but the pathogenic mechanisms behind these phenomena remain elusive. Our lab has found a HL phenotype in Ada-deficient mice (Ada-/-) that parallels Ada-deficient humans. Moreover, our high throughput metabolic profiling in these mice revealed an accumulation of acyl-carnitines, succinate, and glutamate, as well as increased hair and neuronal cell loss. To precisely define the natural progression of adenosine levels in our Ada-/- mice, we use an FDA-approved drug, polyethylene glycol modified ADA (PEG-ADA), that lowers adenosine levels. Interestingly, PEG-ADA treated mice displayed improved hearing, decreased levels of metabolites aforementioned, and reduced hair and neuronal cell loss. Mechanistically, we intend on defining the role of adenosine receptors in cochlear homeostasis. We found adenosine A2B receptor (ADORA2B) to be unusually highly expressed in our genetic and immunofluorescent studies of cochlea from Ada-/- mice without PEG-ADA. We also found Ada-/- mice treated with an ADORA2B antagonist have improved hearing. With these findings, we hypothesize that elevated adenosine-mediated hearing loss is dependent on ADORA2B signaling that leads to mitochondrial damage and excitotoxicity. Our use of a clinically relevant mouse model, metabolomics, and pharmacological tools to target metabolic disturbances allow a novel perspective in understanding and treating millions of individuals suffering from HL.
Defining the regulatory interaction of Ssa1/Hsp70 with Hsf1 in yeast (POST CANDIDACY)
Sara Peffer1, 2, Kevin A. Morano1, 2
1Department of Microbiology & Molecular Genetics, McGovern Medical School, Houston, TX, USA. 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.
Eukaryotic organisms respond to proteotoxic stress through the heat shock response (HSR), mediated primarily by the heat shock transcription factor, HSF1. The stress-induced HSF1 transcriptional program results in increased expression of protein chaperones to maintain cellular proteostasis. HSF1 consists of DNA-binding, trimerization and carboxyl-terminal transactivation domains. In Saccharomyces cerevisiae, Hsf1 has an extended amino terminus and is localized to the nucleus, bound to recognition sequences within target gene promoters, and maintained in a repressed state. Mechanisms for Hsf1 repression and activation are not completely understood. Yeast and human Hsp70 contain reactive cysteines that can be modified by thiol-reactive compounds. Replacement of these cysteines with aspartate renders Hsf1 constitutively activated; in contrast, substitution to serine abolishes response to oxidants, but maintains the response to heat. This suggests Ssa1 acts as a proximal sensor for multiple stressors to activate the HSR. Furthermore, Ssa1 and Hsf1 interact under optimal conditions and dissociate during thermal stress. We have localized this interaction within residues 50-100 of the amino terminus, and carboxy terminal residues 520-568. This association is mediated through the Ssa1 substrate binding domain, as identified through co-immunoprecipitation, with residues 50-100 and 520-568. Additionally, native Hsf1 repression is inhibited by over-expression of residues 50-100 in an Ssa1-depleted background, suggesting 50-100 competes for Hsp70 binding. We infer that Hsf1 transcription is regulated in part by predicted, but heretofore undescribed “amino-terminal repression domain (NRD)” and a carboxy-terminal repression domain through direct binding by Ssa1. This regulatory circuit likely plays a critical role in governing response to proteotoxic stress in S. cerevisiae and other organisms expressing HSF molecules with extended termini.
Deciphering impacts of thiol stress on cytosolic proteostasis in yeast (POST CANDIDACY)
Amy E. Ford1,2, Catherine Denicourt1,3, and Kevin A. Morano1,2
1The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA. 2Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, TX, USA. 3Department of Integrative Biology and Pharmacology, McGovern Medical School, Houston, TX, USA.
Protein homeostasis (“proteostasis”) is essential for cellular function. An inability to properly fold proteins into their native state results in misfolding and accumulation of protein aggregates. Eukaryotes maintain two distinct redox environments in which proteins are folded: the oxidizing endoplasmic reticulum (ER) and reducing cytosol. Thiol-containing proteins are vulnerable to disruptions in redox conditions as cysteine (Cys) residues are maintained in either an oxidized or reduced state. Within the ER, redox imbalance can prevent disulfide bond formation and result in protein misfolding which activates the unfolded protein response. In the cytosol, misfolded proteins activate the heat shock response. Although these protective strategies have been characterized independently, little is known about how cytosolic redox imbalance impacts proteostasis. Here, we demonstrate a model for thiol-dependent misfolding of an endogenous protein in yeast. Using green fluorescent protein (GFP) fusions to potentially redox-sensitive cytosolic proteins and two modes of redox disruption: cadmium (Cd) and glucose starvation, we discovered that triose phosphate isomerase (Tpi1) aggregates upon thiol-reactive stress. Tpi1 aggregation is restricted to newly synthesized protein and correlates in severity with Cd concentration. The chaperones Tsa1 and Hsp104 were found to co-localize with Tpi1 foci, supporting the contention that nascent Tpi1 accumulates in protein aggregates. Tpi1 contains two Cys residues, and simultaneous substitution of both with alanine prevents formation of protein aggregates, demonstrating thiol-dependent aggregation that may occur through direct interaction with protein thiols. Lastly, human Hsp70-YFP transfected cell lines formed foci following Cd exposure suggesting Hsp70 is localizing to aggregated proteins in response to Cd stress. Our data shows that thiol-reactive stress is proteotoxic and Tpi1 is a useful model for understanding the link between redox balance and proteostasis.
Depletion of Cleavage Factor 25kDa Subunit (CFIm25) and Resulting Alternative Polyadenylation (APA) in Vascular Remodeling and Right Ventricle Hypertrophy in Pulmonary Hypertension (POST CANDIDACY)
Scott D. Collum1 , Tingting Weng1 , Adriana Hernandez1 , Ning-Yuan Chen1 , Jose Molina1 , Javier Amione-Guerra2 , Odeea Al-Jabbari2 , Raquel R. Bunge2 , Neeraj Singha2 , Michael R. Blackburn1 , Leng Han1 , Harry Karmouty-Quintana1
1UTHealth, Houston, TX, 2Debakey Heart & Vascular Center, Houston, TX
Cleavage and polyadenylation is a key step in the maturation of mRNAs in which a poly(A) tail is added. A reduction in the level of the cleavage factor I 25kDa subunit (CFIm25) can induce Alternative Polyadenylation (APA) events characterized by a shortening of messages containing multiple poly(A) signals. This shortening often eliminates key regulatory elements from the mRNAs allowing for dysregulated expression. Depletion of CFIm25 has been found to contribute to the proliferative phenotype of some cancers, thus we hypothesize that reduced CFIm25 expression drives the remodeling of the pulmonary arteries that closely resembles the proliferative cancer phenotype.
Western blot for CFIm25 in the bleomycin and hypoxsia-sugen5416 mouse models of PH shows reduced levels in isolated pulmonary artery. RNA-seq analysis of transcripts upregulated in pulmonary artery smooth muscle cells depleted of CFIm25 shows enrichment for transcripts producing ECM components. The most exciting of the genes that undergoes APA on CFIm25 depletion in this experiment was Hyluronan Synthase 2(HAS2). My finding show that reduced HAS2 expression is protective in our mouse models and that increased levels are seen in patient samples. This is consistent with CFIm25 mediated APA causing shortening and dysregulation of the HAS2 mRNA in PH. These findings of APA and HAS2 in the development of vascular remodeling will provide future therapeutic targets.
Understanding the role of transcription in regulating uracil enrichment in genomic DNA (POST CANDIDACY)
Norah A. Owiti1,2 and Nayun Kim1,2
1The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA. 2Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, TX, USA.
Damage to DNA can be lethal to the cell and if left unrepaired, can lead to genomic instability and enhance cellular defects. The distribution of genome instability events is non-random with certain hotspots, including highly transcribed genomic loci, bearing a larger load of DNA damage. Using a mutation reporter assay modified with tetracycline-regulatable pTET promoter, previous studies from our lab demonstrated an increase in mutagenesis when transcription is activated in yeast strains deficient in the base excision repair (BER) and/or nucleotide excision repair (NER) pathways. The Transcription-Associated Mutagenesis (TAM) was abrogated when uracil-DNA glycosylase was disabled, indicating that the relevant mutagenic lesions in this assay are abasic sites derived from excision of uracil in DNA. We sought to elucidate how mutagenic imbalance in nucleotide composition is achieved in highly transcribed genomic regions. First, we developing a method to detect uracil in DNA at a specific genomic site to test our hypothesis that levels of uracil in DNA is enhanced in highly transcribed areas of the genome. We utilized enzymes to specifically create strand breaks at the sites of uracil and then quantified DNA damage using qPCR technique. In addition, we directly quantified uracil in DNA following DNA damage and finally, we modulated levels of the yeast dUTPase Dut1 to determine whether incorporation of uracil in DNA is cell cycle-regulated. Our results indicate a significant accumulation of uracil in DNA when transcription is elevated and following damage to DNA. Additionally, overexpression of Dut1 during G1 and G2 reduced the rate of uracil-dependent TAM suggesting that a significant amount of uracil-incorporation into DNA can occur outside S-phase in the form of DNA repair synthesis. Our results provide a novel mechanism of TAM.
Selective Lipid Sorting Regulates Rac1 Function on the Plasma Membrane (POST CANDIDACY)
Kelsey Maxwell1,2, Yong Zhou1,2, John F. Hancock1,2
1Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston and 2Graduate School of Biomedical Sciences, Houston, TX, USA
The small GTPase Rac1 is an important driver of cell migration, and in consequence has been implicated in many different types of cancer for its role in promoting metastasis. Using high-resolution spatial mapping, we have found that like Ras GTPases, Rac1 localizes to the PM to form spatially distinct microdomains called nanoclusters. For Ras, these nanoclusters are segregated by isoform-type and GDP/GTP loading to act as signaling sites for effector recruitment and downstream signal transmission. Ras nanoclusters have distinct lipid compositions as well. For example, phosphatidylserine (PS) is an essential structural and signaling component of oncogenic K-Ras but not H-Ras nanoclusters, and removal of PS results in abrogated K-Ras signaling. The spatial organization of these small GTPases is largely determined by differences in the membrane anchors of the protein. Ras membrane anchors comprise a C-terminal farnesyl-cysteine-methyl-ester, plus dual palmitoyl lipid chains in H-Ras, a single palmitoyl in N-Ras and a polybasic domain in K-Ras. Rac1 is likewise prenylated, with geranylgeranyl at the C-terminal cysteine residue of the CAAX motif and contains both polybasic sequence and a single palmitoyl. We show that like Ras, Rac1 nanoclusters have a distinct lipid composition, and that phosphatidic acid (PA) and PIP3 are important structural components for this nanocluster formation. Additionally, we have found that key residues in the membrane anchor of Rac1 are necessary for nanocluster formation and association between Rac1 and PA/PIP3. These findings may ultimately lead to novel and innovative strategies for targeting Rac-related cancer and metastases.
Investigating the Role of Transmembrane Potential on Lipid Bilayer (PRE-CANDIDACY)
Vinay Nair1, Xubo Lin1 & Alemayehu Gorfe1
1 Department of Integrative Biology & Pharmacology, McGovern Medical School, Houston, TX, USA
Biological cells actively maintain a net negative charge in their interior by exchanging ionic solutes with their surrounding environment, which results in an electrochemical potential difference generated across the plasma membrane, called the transmembrane potential (TM-potential). The TM-potential, in turn, is believed to affect the structure and dynamics of the components of the plasma membrane.
In this study, we investigated the effect of TM-potential on the structure and dynamics of lipids in a lipid bilayer using molecular dynamics simulations. Our model system was a double bilayer consisting of 140 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 50 1-Palmitoyl-2-oleoyl-glycero-3-phosphoserine (POPS) lipids per leaflet, with the two bilayers separated by two aqueous compartments. Three systems with different numbers of sodium ions in the aqueous compartments (100-100, 99-101 and 98-102) were set up to simulate TM-potentials of increasing intensity. Each system was simulated for 1000ns and the data analyzed.
The TM-potentials in the three systems correlated strongly with the difference in the concentration of sodium ion, with the contribution of each sodium ion determined to be ~90mV. The sodium ions interact with the oxygen atoms of POPC & POPS head-group and glycerol regions. Further, the TM-potential had a greater effect on the clustering and flexibility of POPS lipids, as compared to POPC. Thus, for our simple two-lipid system, TM-potential significantly affected the overall structure and dynamics of the component lipids, particularly the charged POPS. Further studies with systems of increased complexities need to be performed to better understand this process.
LBPI: A web interface for the identification of allosteric ligand binding sites (PRE-CANDIDACY)
Nabina Paudyal1, and Alemayehu Gorfe1
1Department of Integrative Biology & Pharmacology, McGovern Medical School, Houston, TX, USA
The development of efficient tools for allosteric ligand binding site identification in potential drug targets is an important step for computational drug design. Ligand binding specificity analysis (LIBSA) is one of the protocols that utilizes filtering algorithms to assess the propensity of a site on a target structure or structures to bind a ligand. However, LIBSA requires expert skills to be properly executed. Thus, a Web interface, LBPI (Ligand Binding Pocket Identification) has been developed using Django, a Python based web framework. A Python Wrapper has also been developed to streamline pre-existing algorithms of LIBSA. The Wrapper helps in the preparation of files, execution of individual programs and generation of appropriate results. LBPI provides an ideal platform for making complex binding site identification protocols readily available for non-expert users to submit jobs and monitor the results. The goal of LBPI is to integrate available algorithms in a systematic way and make it easily available for both experts and non-experts.
Downregulation of Cleavage Factor Im25 (CFIM25) in Senescent Fibroblasts Contributes to Pulmonary Fibrosis through Alternative Polyadenylation (PRE-CANDIDACY)
Jingjing Huang1,2, Junsuk Ko2, Ning-yuan Chen2, Jose Molina2, Scott D. Collum2, Harry Karmouty-Quintana2, Fayong Luo2, Kelly Volcik2, Jonathon Davies2, Kemly Philips2, Eric Wagner2, Tingting Mills2, and Michael R. Blackburn2
1The Second Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu,China,2Biochemistry&Molecular Biology, UT Health Science Center at Houston, Houston, TX, USA
Idiopathic Pulmonary fibrosis (IPF) is a prevalent and deadly disease that is also a disease of aging. Fibroblast senescence is widely observed in the lungs of IPF patients; however, whether fibroblast senescence to the pathogenesis of IPF is not known. Alternative polyadenylation generates mRNAs with different lengths of 3′-untranslated region (3′-UTR), thus changing the binding sites available for RNA binding proteins and microRNAs and influencing the stability of mRNAs. Down-regulation of Cleavage Factor Im 25 (CFIm25), a CFIm component that directly binds to RNA, has been shown to shorten the 3’-UTR of multiple mRNAs and enhance the level of translation. Here we demonstrate that CFIm25 is down-regulated in the lungs of patients and mice with pulmonary fibrosis, with its expression negatively correlated with levels of fibronectin, a marker for pulmonary fibrosis. Immunohistochemistry and in vitro cell culture experiments revealed that CFIm25 is selectively down-regulated in alpha-smooth muscle actin (α-SMA) positive fibroblasts in the lungs of IPF patients and bleomycin-treated mice. In line with the findings that IPF is an aging-related disease, we observed that CFIm25 is also down-regulated in aged lungs and its expression can be directly suppressed by senescence-inducing agents in normal lung fibroblasts. Interestingly, siRNA targeted to CFIm25 significantly induces fibronectin and collagen I expression in normal lung fibroblasts. Using RNA deep sequencing, we identified 808 genes with shortened 3’-UTRs after CFIm25 knockdown, including those involved in the transforming growth factor-beta signaling pathway, the Wnt signaling pathway, and cancer pathways. These results suggest that deregulation of CFIm25 in senescent fibroblasts may directly contribute to the amplification of key proteins that promote the pathogenesis of IPF. Finally, we demonstrated that deletion of CFIm25 in fibroblast using the Col1a1 promoter exaggerated bleomycin-induced pulmonary fibrosis. Taken together, our results identified CFIm25 and APA as a novel mechanism that could account for the excessive amplification of extracellular matrix proteins and exaggerate the pulmonary fibrosis in aged people. (Funding: NIH R01HL070952 and P01-HL114457)
Molecular mechanisms of microdomain-dependent protein trafficking (POST CANDIDACY)
Barbara Diaz-Rohrer1,2, Kandice Levental1, Ilya levental1,2
1Department of Integrative Biology and Pharmacology, Graduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center UTHealth, Houston, Texas 77030, USA. 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA
Eukaryotic cells are organized into spatially and functionally distinct membrane-bound organelles, whose functions are defined by their lipid and protein composition. Accurate and robust sorting of membrane components between these compartments is necessary for the maintenance of organelle identity. For most membrane proteins, the determinants of their steady-state subcellular localization remain unknown. Lateral membrane domains known as lipid rafts provide an ideal platform for membrane sorting processes, and have been widely implicated in post-Golgi sorting and endocytosis/recycling. However, the structural determinants of protein association with such domains are almost entirely unknown. We have developed and characterized a robust experimental system for direct, quantitative measurements of raft affinity in intact plasma membranes and used it to explore the determinants of transmembrane protein recruitment into raft domains and the consequences of this recruitment on subcellular traffic. We identified several structural features associated with raft affinity, and established a quantitative and functional relationship between raft association and subcellular protein localization. Specifically, we observed that raft association is fully sufficient for PM recycling of certain proteins, and that abrogation of raft partitioning for these proteins led to their degradation in the lysosomes. These findings identify structural determinants of raft affinity for transmembrane proteins and support the conclusion that ordered membrane domains mediate recycling of specific membrane components from the endosomal compartments to the PM. We have proceeded to define the molecular machinery that mediates raft lipid and protein sorting and recycling to the PM. Using a set of orthogonal transmembrane proteins as probes of raft and non-raft domains, we developed a high throughput siRNA screen to dissect the molecular machinery and dynamics for raft-mediated sorting. We identified a number of validated hits including known players of the early endocytic traffic (Rab5 and EEA1), but also novel players that appear to define a distinct class of trafficking mediators specific for raft-associated proteins. This pathway is not dependent on the classical recycling pathways defined by Rab4 and Rab11, rather defining a novel route for PM recycling of raft-preferring cargo.
High Translation Fidelity Induces Filamentation in Salmonella Leading to Potential Resistance against Antibiotics and Macrophage-killing (PRE-CANDIDACY)
Kalyn Weiss1,2, Yongqiang Fan1, Christopher R. Evans1,2, Jiqiang Ling1,2
1 Department of Microbiology and Molecular Genetics, Medical School, University of Texas Health Science Center, Houston, TX 77030, USA, 2 Graduate School of Biomedical Sciences, Houston, TX 77030, USA
Filamentation has been implicated to act as a survival mechanism against numerous environmental stresses. Induced inhibition of cell division within bacterial communities generates a sub-population of filamentous cells that can evade phagocytosis by innate immune effector cells and gain resistance against antibiotics. While it is evident that filamentation is regulated by multiple systems of cell division and inhibition in response to the environment, the molecular mechanisms that lead to filamentation are not fully understood. Here, we provide evidence that translation fidelity plays a role in the induction of filamentation in Salmonella. Translation fidelity has been shown to be regulated by environmental factors, making this connection between translation fidelity and induction of filamentation interesting. Images taken of a high translation fidelity Salmonella strain (S. rpsL*) showed filamentous cells making up a significant percent of the total population. Time-lapse microscopy of S. rpsL* shows filamentous cells have promising advantageous characteristics over normal cells of the population during antibiotic treatment and infection of macrophages. Regarding the mechanism of filamentation, we show that S. rpsL* expresses high levels of RpoS, and deleting RpoS in S. rpsL* eliminates filamentation suggesting that that the induction of filamentation by increased translation fidelity is mediated via RpoS and relative pathways. Our work to understand the mechanism behind filamentation and its heterogenous induction within a bacterial population will provide information on how sub-populations of pathogenic bacteria persist during infections and antibiotic treatment and how to better target these persister cells.
Voltage-sensitive dye (VSD) recordings provide insights into the differential modification of spike frequency and burst timing that underlies L-DOPA mediated motor pattern selection in the feeding circuit of Aplysia (PRE-CANDIDACY)
Renan M. Costa1, Curtis L. Neveu1, Ryota Homma1, Shin Nagayama1, Douglas A. Baxter1, and John H. Byrne1
1Department of Neurobiology & Anatomy, McGovern Medical School at UTHealth, Houston, TX, USA
A general model for behavioral selection considers neural circuits to be multifunctional and selection to be mediated via modulation of neuronal subgroups within the circuit. To gain insights into the ways in which motor patterns are selected, we monitored activity in the buccal ganglia of Aplysia using a voltage sensitive dye (VSD) while varying the concentration of L-DOPA to select from among different buccal motor patterns (BMPs). Preference for intermediate patterns was significantly greater in low (40 µM) L-DOPA than control or high (250 µM) L-DOPA. In contrast, the preference for bite-like patterns was significantly greater in high L-DOPA compared to control or low L-DOPA. The overall number of BMPs was also significantly increased in high L-DOPA compared to control, but the increase in low L-DOPA was not significant. Motor pattern selection was accompanied by differential modulation of neurons active during the protraction or retraction phases of a BMP. Selection of intermediate BMPs was associated with a decrease in burst duration but no change in spike frequency in neurons active during retraction, while no change occurred in spike frequency or burst duration in neurons active during protraction. In contrast, the selection of bite-like BMPs was associated with an increase in spike frequency but no change in burst duration in neurons active during protraction, and conversely a decrease in burst duration but no change in spike frequency in neurons active during retraction. These results suggest that individual groups of neurons are modified in characteristic ways to mediate a switch in behavior.
BMP4 Regulates Cell Cycle Kinetics in Aging Muscle-Derived Stem Cell-mediated Osteogenesis and Bone Regeneration (PRE-CANDIDACY)
Haizi Cheng1,3, Xueqin Gao1,2, Aiping Lu1,2, Johnny Huard1,2*Corresponding author
1The University of Texas Health Science Center at Houston, Houston, TX, 2Steadman Philippon Research Institute, Vail, CO, 3University of Pittsburgh, Pittsburgh, PA
As mammals develop from premature to mature organisms, effective cell cycle regulation occurs under different extrinsic and intrinsic cues. Physiologically, such regulation maintains homeostasis, resisting loss of function; as an organism is reaching its lifespan, it may meet more challenges. Cell cycle quiescence is supposed to prevent stem-cells exhaustion; however, relatively over-arrested cell cycle may mediate stem-cell senescence. To better elucidate cell cycle regulation during aging process, we separately isolated muscle-derived stem cells from 3-week-old and 2-year-old mice. In our previous study, we have shown that the expression of cell cycle regulators differs between young and old muscle-derived stem cells. Old muscle-derived stem cells appear to exhibit slower proliferation and lower potential for multilineage differentiation (Haizi Cheng, ORS 2017). To further investigate osteogenesis in muscle-derived aging stem cells in vivo, we recently transduced muscle-derived stem cells (MDSCs) with bone morphogenetic protein 4/green fluorescent protein (BMP4/GFP) retrovirus and explored the role of cell cycle regulators in osteogenesis and bone regeneration in aged MDSCs. Aged MDSCs regenerated almost as much new bone as did young MDSCs in a critical-sized skull defect in 6-week-old CD-1 nude mice, and followed cell cycle kinetics under the regulation of cyclin-dependent kinase inhibitors. We found that BMP4 as a positive signal provoked the release of the arrested cell cycle in aged MDSCs; therefore, releasing the cell cycle could be a potential target to rescue aging.
Downregulation of Cleavage Factor 25 by TGFb1 Contributes to Pulmonary Fibrosis
through Alternative Polyadenylation of mRNAs (POST CANDIDACY)
Junsuk Ko1, Tingting Mills1, Tinne Marten1, Ning-Yuan Chen1, Fayong Luo1, Jose Molina1, Kelly Volcik1, Yang Zhou2 and Michael Blackburn1.
1Pulmmonary Center of Excellence, Department of Biochemistry and Molecular Biology, Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas 77030, USA, 2Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912, USA.
Idiopathic pulmonary fibrosis (IPF) is the most severe and common type of lung fibrosis affecting 60,000 people in the U.S. alone. Despite the relatively high prevalence and incidence of IPF, currently there is no cure. One feature of IPF is enhanced protein expression of pro-fibrotic mediators and extracellular matrix components by fibroblasts in the lungs. However, the mechanisms governing the robust protein expression in the fibroblasts are still elusive. Alternative polyadenylation (APA) of mRNAs is a cellular process that can drive robust protein expression globally. Human mRNAs can contain more than one polyadenylation of site on their 3’UTR, and polyadenylation of different sites makes mRNAs with different 3’UTR lengths. Therefore, this process is called APA. In highly proliferative cells, the polyadenylation occurs at proximal polyadenylation sites making mRNAs with relatively shortened 3’UTRs. The mRNAs with a shortened 3’UTR tend to be more stable due to the deletion of the binding sites for mRNA degrading proteins and miRNAs are deleted, leading to enhanced protein expression and cell proliferation. Recently, our lab discovered that reduction in CFIm25, a protein in the polyadenylation complex, is associated with IPF. In CFIm25 KO mouse and in vitro models, we found that CFIm25 depletion contributes to fibrosis progression presumably via global mRNA shortening and robust protein expression for fibrotic mediators. It was also found that transforming growth factor beta 1(TGFb1), a potent fibrotic mediator, significantly downregulates CFIm25 in healthy human lung fibroblasts through miRNA regulation. This result may indicate that downregulation of CFIm25 by TGFb1 contribute to pulmonary fibrosis through APA of mRNAs.
A Novel Mechanism of Resistance Against Antimicrobial Peptides Involves Extracellular Sensing Coupled with Membrane Remodeling Mediated by a Single Protein (POST CANDIDACY)
Ayesha Khan1,2,3, M. Davlieva4, D. Panesso1,3,5S. Rincon1,3,5, T. Tran1,3, K.V. Singh1, W.R. Miller1,3, Y. Shamoo4, CA. Arias1,2,3,5
1UT Health McGovern Medical School, Houston, TX, 2Department of Microbiology and Molecular Genetics, UT Health, Houston, TX, 3Center for Antimicrobial Resistance and Microbial Genomics, Houston, TX, 4Department of Biosciences, Rice Univ, Houston, TX, 5U. El. Bosque, Bogota, Colombia
Background: The LiaFSR stress response system regulates daptomycin (DAP) and antimicrobial peptide (AMP) resistance in enterococci. LiaX, an effector of LiaFSR, has distinct C- and N-terminal domains and is highly secreted in DAP-resistant (DAP-R) strains. Its role in resistance is unknown.
Methods: LiaX and the C-terminal alone were deleted in DAP-susceptible (DAP-S) E. faecalis (Efs). Mutants were assessed for DAP susceptibility and anionic phospholipids (APLs) were visualized with nonyl-acridine orange. To determine if extracellular LiaX protects DAP-S strains from the DAP “attack”, MICs were determined in the presence and absence of supernatants recovered from DAP-R strains. LiaFSR and LiaXYZ gene expression was assessed in the liaX mutants and upon exogenous addition of purified LiaX. The ability of LiaX to bind DAP and LL-37 (AMP) was tested.
Results: Deletion of liaX or its C-terminus resulted in DAP resistance (MIC 12 ug/ml), cell membrane (CM) remodeling and significant upregulation of liaFSR and liaYZ gene expression. The addition of DAP-R supernatants increased the MICs of DAP-S strains 3-5 fold, well above the clinical breakpoint. Upregulation of liaFSR and liaXYZ was observed when purified LiaX was added exogenously in the presence of DAP. LiaX readily bound DAP and the antimicrobial peptide LL-37 with KDs= 0.05 and 8.3uM, respectively.
Conclusions: Our results show that LiaX controls CM adaptation by modulating phospholipid remodeling through its C-terminal domain, binding antimicrobial peptides via its N-terminal domain, and activating the LiaFSR stress response system. This dual function of a single protein to protect against antibiotic attack has no precedent.
In a pro-inflammatory environment, mesenchymal stromal cells exert antiinflammatory and pro-angiogenic effects on endothelial cells (PRE-CANDIDACY)
Kaavya Giridhar1,2, Nikunj Satani1, Bing Yang1, Xiaopei Xi1, Songmi Lee1, Jaroslaw Aronowski1 and Sean I. Savitz1
1Stroke Team, McGovern Medical School at UTHealth, Houston, TX, 2Graduate School of Biomedical Sciences, UTHealth, Houston, TX
Background and Aims: Cell-based therapies such as mesenchymal stromal cells (MSCs) have increasingly shown great promise for ischemic stroke recovery. An understudied target of MSCs may be endothelial cells (ECs). Our study explores the interactions between MSCs and ECs and how this interaction alters EC functionality when exposed to an inflammatory cell stimulus.
Methods: Primary brain ECs were isolated from postnatal 1-4 day old C57BL/6 mice pups. Primary MSCs were isolated from the bone marrow of C57BL/6 mice. At passage 3, both the primary cells were individually seeded at 200,000 cells and 100,000 cells per well respectively. To simulate a stroke-like inflammatory environment, we exposed MSCs and ECs to LPS (doses ranging from 0.01ug/ml to 100ug/ml). 24 hours post-LPS exposure, conditioned media (CM) from MSCs was collected and added to the treated ECs. Following this fresh media was added to both MSCs and ECs. After 24 hours, media was collected from MSCs and ECs. IL-6 and VEGF concentrations were measured using ELISA from all the media samples. Viability and proliferation of MSCs and ECs following LPS exposure were measured using MTT and Crystal Violet assays.
Results: Treatment of the ECs using MSC conditioned media significantly reduced the release of IL-6, a pro-inflammatory cytokine at dose ranges of 1ug/ml to 100ug/ml of LPS. The release of VEGF, a pro-angiogenic cytokine, was significantly increased. 24 hours after the removal of an inflammatory stimulus the changes in the ECs go back to baseline whereas in the MSCs the effects were lasting. There was no significant change in viability or proliferation of MSCs or ECs following different LPS doses.
Conclusion: MSCs may release soluble factors that modulate the responses of endothelial cells. MSCs exert anti-inflammatory and pro-angiogenic effects on ECs in a pro-inflammatory setting. These results identify new targets to better understand how MSCs improve recovery in stroke animal models.
The molecular mechanism of nonstop decay in Saccharomyces cerevisiae (POST CANDIDACY)
Minseon Kim1, 2, Kim-Trang Ho1, Alexander Berroyer1, 2, and Ambro van Hoof1, 2
1Department of Microbiology & Molecular Genetics, McGovern Medical School, Houston, TX, USA. 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.
Mistakes made during mRNA processing harm cells by accumulating abnormal mRNAs and proteins. To prevent this, eukaryotes exploit several quality control systems.
mRNAs that lack stop codons, nonstop mRNAs, can be generated by premature polyadenylation. Nonstop mRNAs cause ribosome stalling during translation, and they are degraded by the RNA exosome and cofactors, the Ski complex and Ski7, in S. cerevisiae. While the same factors are involved in normal mRNA turnover, the C-terminus of Ski7 is only required for nonstop mRNA decay, suggesting that Ski7 plays a specific role in nonstop decay.
Nonstop proteins translated from nonstop mRNA are not released from the ribosome. Their degradation is mediated by the Ribosomal Quality Control complex (RQC) that binds disassembled 60S ribosomal subunits containing a nascent peptide.
Quality control systems for nonstop mRNA and nonstop proteins are thought to be triggered by the ribosome stalling during translation. While physical interactions between the RQC and the ribosome are well studied, the interaction between the nonstop mRNA decay machinery and the ribosome remains unclear. Additionally, how and when the nonstop mRNA decay machinery and RQC are recruited to the ribosome is still unknown.
Here, we investigate the interaction between the ribosome and nonstop mRNA decay machinery by using a promiscuous biotin ligase to purify interacting partners followed by mass spectrometry analysis to identify them. Also, we investigate the genetic relationship between nonstop mRNA decay machinery and RQC to understand the exact mechanism of nonstop decay.
Neuronal pexophagy: studying the effects of mutant huntingtin (POST CANDIDACY)
Ndidi Uzor1,2, Jose Felix Moruno Manchon2, and Andrey Tsvetkov1,2.
1The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA, 2Department of Neurobiology & Anatomy, McGovern Medical School at UTHealth, Houston, TX, USA.
Peroxisomes are small organelles with great physiological significance: they promote cellular redox balance, among other functions, by scavenging reactive oxygen species (ROS), metabolic by-products that damage cell structures. Aged and dysfunctional peroxisomes are regularly degraded by pexophagy, the selective autophagy of peroxisomes. Recent studies of some neurodegenerative diseases report peroxisomes accumulating in neurons, along with cellular oxidative stress. This evidence suggests that either peroxisomes are not breaking down ROS, or are not being degraded via pexophagy. Not much is known about pexophagy regulation in neurons, healthy or diseased. To analyze neuronal pexophagy, we combined time-lapse longitudinal imaging with a fluorescent protein targeted to peroxisomes, Keima-per. Peroxisomes expressing Keima-per emit green fluorescence in the cytoplasm, and red fluorescence in acidic lysosomes. We transfected neurons with mutant huntingtin possessing disease-associated glutamine repeats (mHtt-46Q) and Keima-per, and imaged them over a period of time. We observed a significant increase in Keima-per red fluorescence over time in cells expressing mHtt, compared to control. This finding suggests that in neurons, mHtt upregulates general autophagy, leading to non-specific degradation of all organelles, or that mHtt damages peroxisomes, consequently increasing pexophagy.
Oxidative folding mediated by the thiol-disulfide oxidoreductase MdbA is required for stability of penicillin binding proteins in the actinobacterium Corynebacterium diphtheriae (POST CANDIDACY)
Belkys C. Sánchez1 and Hung Ton-That1
1Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA
Accurate disulfide bond (DSB) formation is important for proper folding, stability, resistance to protease degradation, and function of exported proteins. The process of DSB formation, termed oxidative protein folding, is catalyzed by thiol-disulfide oxidoreductase enzymes. Recently, it has been shown that the DSB-forming machine is required for cell growth and division in Gram-positive actinobacteria. MdbA, the primary thiol-disulfide oxidoreductase of the Gram-positive actinobacterial pathogen Corynebacterium diphtheriae, is required for virulence in this organism. Additionally, deletion of mdbA causes a severe temperature-sensitive growth defect, accompanied with abnormal nascent peptidoglycan distribution and increased sensitivity to β-lactam antibiotics. These results suggest that MdbA is required for oxidative folding of actinobacterial factors involved in cell wall biogenesis. Consistent with this premise, our bioinformatic survey of the C. diphtheriae secretome revealed that most penicillin binding proteins (Pbps) contain two or more cysteine residues predicted to form DSBs. Electron microscopy analysis demonstrated that C. diphtheriae mutants lacking pbp1A or pbp1B lost rod-shape morphology, and formed chains and clumps; these phenotypes are similar to the ΔmdbA mutant grown at the nonpermissive temperature. Simultaneous overexpression of corynebacterial Pbp1A/1B/2B in the ΔmdbA strain significantly rescued the morphology defects of this mutant. Furthermore, Western blot analysis showed decreased levels of both Pbp1A and Pbp1B in the ΔmdbA mutant, suggesting that these proteins are unstable in absence of MdbA. We propose that the thiol-disulfide oxidoreductase enzyme MdbA catalyzes disulfide bond formation in Pbps, which confers stability to these key components of the cell wall biosynthesis machinery in C. diphtheriae.
The Influence of a Sugar Import System on a Virulence Regulator in Bacillus anthracis (POST CANDIDACY)
Naomi Bier1, Troy Hammerstrom1, Theresa Koehler1;
1Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas – Houston, Houston, Texas, USA
Bacillus anthracis AtxA is the archetype PRD-Containing Virulence Regulator (PCVR) based on its crystal structure, post-translational modifications, and activity. PCVRs are transcriptional regulators, named for the regulatory domains subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS), termed PTS Regulatory Domains (PRD), and for their impact on virulence gene expression. As is true for other PCVRs, AtxA activity is affected by phosphorylation at specific histidines in each of its two PRDs. We determined that a PTS mutant deleted for HPr and EI had greatly diminished production of AtxA target genes, suggesting the PTS is responsible for AtxA phosphorylation. Interestingly, our physiological, genetic, and biochemical experiments testing the relationship between the PTS and AtxA did not support the hypothesis that the PTS phosphorylates AtxA. Rather, evaluation of atxA transcript levels revealed a 4-fold decrease in atxA transcript in an EI/HPr – null mutant compared to the parent strain. In a model established in the nonpathogenic Bacillus subtilis, HPr can affect target gene transcription via three different mechanisms, each dependent on the phosphorylation state of two sites within HPr protein. We measured atxA transcript levels in HPr mutants altered for phosphorylation or phosphotransfer activity. Our data reveal that the phosphotransfer activity of EI and HPr is required for control of atxA gene expression. Thus, we hypothesize that HPr transfers a phosphate to a downstream transcriptional regulator to influence atxA gene transcription. Further studies aim to identify the transcriptional regulator involved in this process.
Molecular Characterization of a unique LCP enzyme that glycosylates surface proteins in the Gram-positive actinobacterium Actinomyces oris (POST CANDIDACY)
Sara D. Siegel,1 Brendan R. Amer,2 Chenggang Wu,1 Robert T. Clubb,2 and Hung Ton-That1
1Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA; 2Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA
LytR-CpsA-Psr-related enzymes, called LCPs, characterized to date catalyze the attachment of teichoic acid to the N-acetylmuramic acid component of Gram-positive peptidoglycan. In Actinomyces oris, an LCP protein has been shown to be coupled to sortase-catalyzed cell wall anchoring of a glycosylated surface protein named GspA. Here we report the structural and biochemical studies of A. oris LcpA, ascribing its new function in a novel glycosylation pathway of GspA. By X-ray crystallization, the structure of the extracellular LCP domain was determined at 2.5Å resolution revealing a single domain with an α–β–α architecture, similar to that of previously reported LCP enzymes. This structure is most closely related to the Bacillus subtilis TagT enzyme, although it harbors a unique disulfide bond formed by Cys179 and Cys365, functioning to link the C-terminus to α-helix H2. Like the LCP superfamily members, A. oris LcpA contains three conserved arginine residues (Arg128, Arg149, and Arg266), which cluster together within a surface exposed catalytic pocket. To determine how Arg and Cys residues affect A. oris LcpA activity, we generated alanine-substitution mutants of these residues and examined GspA glycosylation biochemically. Unlike the parental MG1, strains expressing stable LCP proteins with individual Arg mutations failed to produce surface GspA glycopolymers, the phenotype similar to that of the lcp deletion mutant. While the Cys mutants destabilize the protein, leading to an intermediate glycosylation defect. Finally, we determined the A. oris LcpA exhibits pyrophosphatase activity. We propose that the mechanism of LCP-mediated glycosylation of GspA occurs through a phosphotransfer reaction.
Evaluating the role of the methionine sulfoxide reductases MsrAB on the oxidative stress resistance and pathogenicity of Fusobacterium nucleatum in a murine model of preterm birth (PRE-CANDIDACY)
Matthew Scheible1, Cuong Nguyen1, Ju Huck Lee1, Luong Thanh Truc1, Luis Vega1, Chenggang Wu1, and Hung Ton-That1
1Department of Microbiology & Molecular Genetics, University of Texas McGovern Medical School, Houston, TX, USA
Fusobacterium nucleatum (FN), an obligate Gram-negative anaerobe, is a key colonizer in the development of oral biofilms. As a predominant member of the normal oral flora, the presence of FN at distant sites is linked to many negative patient outcomes including oral infections, pregnancy complications, and colorectal cancer. How FN promotes bacterial pathogenesis is not well understood. This is mainly due to the lack of genetic tools and efficient methodology. Here we report the development of a versatile reverse genetic system, permitting for the first time robust genetic manipulation of FN. Subsequently, genetic and biochemical dissection of seven response regulators of the corresponding two-component systems in FN revealed that deletion of modR caused a significant defect in H2O2 resistance. modR, together with the sensor kinase-encoding gene modS, resides within a five-gene locus, modS-modR-msrAB-trx-ccdA, coding for homologs of the methionine sulfoxide reductase system MsrAB known to repair damaged proteins by oxidative stress. Consistent with its involvement in oxidative stress resistance, this gene locus was highly responsive to H2O2 treatment, and deletion of modS caused up-regulation of trx and ccdA, whereas deletion of modR resulted in down-regulation of these genes. Significantly, compared to the parental strain, a mutant lacking msrAB was severely defective in survival in macrophage and adherence to and invasion of epithelial cells. In a murine model of infection, the msrAB mutant was attenuated in induction of preterm birth as compared to the parental strain. Together, our studies suggest that MsrAB is critical in the pathogenic capabilities of FN.
Bone Cancer in a Dish: A Study of Rothmund-Thomson Syndrome Associated Osteosarcoma
Brittany Ellis Jewell1,2, Dandan Zhu2, An Xu2, Ruoji Zhou2,3, Linchao Lu4,5, Lisa L. Wang4,5, Dung-Fang Lee1,2
1Biochemistry and Cell Biology, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA. 2Department of Integrative Biology and Pharmacology, McGovern Medical School, Houston, Texas, USA.3Genes and Development, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA.4Hematology and Oncology Division, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.5Cancer and Hematology Center, Texas Children’s Hospital, Houston, Texas, USA.
Our current study is focused on a relatively new way to model bone osteosarcoma, the most common type of bone malignancy in children and adolescents. Remarkably, osteo sarcoma is commonly found in Rothmund-Thomson Syndrome (RTS) patients. About two thirds of people with RTS also have a mutation in RECQL4. This gene is a DNA helicase, known to be important in unpacking the DNA within the cell. Because of the similarities between RTS-associated osteo sarcoma and sporadic osteo sarcoma, RTS patients make an ideal cohort to explore the molecular mechanisms underlying osteo sarcoma. In order to explore the role of RECQL4 in osteo sarcoma genesis, we use the induced pluripotent stem cell (iPSC) disease model approach to study why impaired RECQL4 function results in RTS patients-associated osteo sarcoma. I reprogrammed RTS fibroblasts using Yamanaka reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) to generate RTS iPSCs. Forthcoming, we will differentiate these iPSCs to osteoblasts and use RNA-seq to explore global RNA expression profiles and look for clues into mechanisms of osteo sarcoma genesis. Further research will correlate these findings with the finding of studies of another bone cancer prone diseases (Li-Fraumeni syndrome, hereditary Retinoblastoma) to identify the central mechanisms involved in osteo sarcoma genesis. Additionally, RTS iPSC-derived osteoblasts will be used to screen several thousand drug candidates, potentially leading to an effective treatment. The ultimate goal of this basic research is to follow through to make an impact on the lives of those with osteo sarcoma. In the future patient iPSCs could be useful in personalized medicine to develop an individualized strategy for therapy.
Pregnancy exerts beneficial effects on dystrophic mice (MEDICAL STUDENT)
Guillermo Pechero1, Haiying Pan1, Yan Cui1, Aiping Lu1,2, Johnny Huard1,2
1Department of Orthopedic Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77054, USA, 2Steadman Philippon Research Institute, Vail, CO 81657, USA
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that occurs due to the loss of dystrophin. Pregnancy has shown to improve myogenic differentiation of myogenic progenitor cells (MPCs) in vitro and muscle regeneration in wildtype (WT) mice in vitro with cardiotoxin injury. The therapeutic effects of pregnancy are not fully understood on the dystrophic muscle.
The gastrocnemius muscle, sectioned at 10 µm, of two different mouse models of DMD was used. Currently pregnant and non-pregnant MDX (dystrophin -/-) mice were used to compare the severity of the muscular dystrophy. Previously pregnant and virgin dKO-heterozygous (dystrophin -/-/utrophin+/-) mice were used to assess if upregulated hormones seen during pregnancy were the main reason for the increased therapeutic effects observed. Trichrome staining was done to assess collagen deposition due to fibrosis. Mouse IgG was used to quantify muscle fiber necrosis. Embryonic myosin heavy chain (e-MyHC) was used to quantify newly regenerated myofibers. F4/80, a macrophage marker, was used to assess macrophage infiltration.
Pregnant MDX mice showed comparatively less fibrosis, an increased number of e-MyHC positive fibers, and less macrophage infiltration; however, the amount of necrotic fibers was not decreased in pregnant mice. Previously pregnant dKO-heterozygous mice showed comparatively less fibrosis and fewer number of necrotic fibers.
Parabiosis has shown to increase muscle regeneration potential in older mice when they are paired with younger mice. Pregnancy, a form of natural parabiosis, has shown positive effects in WT mice with cardiotoxin injury. The present study demonstrates that pregnancy, or a history of pregnancy, provides beneficial therapeutic effects in the dystrophic muscle of mice. Furthermore, the results support the notion that the positive effects are not only due to the upregulation of hormones, but also to the upregulation of other circulating factors during pregnancy.
The Zebrafish Gut: A Living Microfluidic Environment to Study Bacterial Mechanosensing (PRE-CANDIDACY)
Laurel Thompson1,2, Daniel H. Stones3, Jacqueline Rocha1,2, Anne-Marie Krachler1
1 Department of Microbiology and Molecular Genetics, University of Texas McGovern Medical School at Houston, Houston, Texas, USA; 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA; 3University of Birmingham, School of Biosciences, Institute of Microbiology and Infection, Edgbaston, Birmingham, United Kingdom
Foodborne infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC) can result in outbreaks of bloody diarrhea and, in severe cases, hemolytic uremic syndrome. Cattle are the main reservoir of EHEC, and infection occurs following exposure to contaminated meat, dairy, or vegetables. We have established a vertebrate model for foodborne EHEC infection using larval zebrafish (Danio rerio) as a host and the protozoan prey Paramecium caudatum as a vehicle. This model allows us to study intestinal colonization, microbe-host interactions, and microbial gene induction within a live host. Foodborne EHEC colonizes the gastrointestinal tract faster and establishes a higher burden than waterborne infection. Colonization occurs in the foregut and midgut, with a preference for the midgut, and induces the expression of the locus of enterocyte effacement (LEE), a key EHEC virulence factor. We have previously shown that LEE is activated in vitro by fluid shear stress and are now developing assays to measure gut motility and fluid shear to determine how mechanosensation influences colonization site preference in vivo. These findings will provide information on how physical cues result in physiological changes in the bacteria during an infection.
Mapping lipid binding sites on K-Ras surface using probe-based MD simulations (PRE-CANDIDACY)
Zuhal Ozcan1,2, Abdallah Sayyed-Ahmad3, Priyanka Prakash1, Alemayehu A. Gorfe1,2
1Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA, 2Graduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center UTHealth, Houston, TX, USA, 3Department of Physics, Birzeit University, Birzeit, West Bank, Palestine
K-Ras is a membrane-associated GTPase that regulates signaling pathways involved in cell division and proliferation. K-Ras mutations account for 85% of all Ras-driven cancers, which constitute 15-20% of all human tumors. For its cellular activity, K-Ras requires high affinity binding to the plasma membrane through hydrophobic and electrostatic interactions. It has been shown that K-Ras interacts with the membrane in two distinct orientation states (OS); OS1 involves helices 3 and 4, whereas OS2 involves interaction of helix 2 and beta strands 1-3 with the bilayer. Although molecular dynamics (MD) simulation studies have already been conducted to identify K-Ras-membrane interaction surfaces, the high computational expense and sampling limitation still remain a challenge. We hypothesize that using lipid head groups as probes will yield a faster and computationally less expensive approach to map lipid binding sites on the K-Ras surface. The novelty of this research lies in using probes to assess protein surface for lipid binding potential, instead of to identify drug binding sites. In this study, acetate, choline, and phosphate probes were used to set up four systems mimicking the membrane through varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-Palmitoyl-2-oleoyl-glycero-3-phosphoserine (POPS) in a model bilayer. 200-ns long simulations were performed for the systems using NAMD software and the CHARMM force field. Data analysis has shown that our findings regarding the protein residues that were in the vicinity of the probes throughout the trajectory agree with the literature. We are currently working on refinements in the data analysis by developing better filters to reduce noise and upon success we will test our methodology on other membrane-binding proteins. We believe our study will lead to a faster, robust method to determine membrane-interacting surfaces on any surface-bound protein molecule.
Morphologically different β-Amyloid fibrils induce distinct patterns of brain amyloidosis in animal models of Alzheimer’s disease (OTHER)
Ruben Gomez-Gutierrez1,2; Carlos Kramm1,3; Claudio Soto1, Rodrigo Morales1
1 Department of Neurology, UT Health Science Center at Houston, Houston, TX, USA, 2Department of Cell Biology, University of Malaga, Malaga, SPAIN, 3Universidad de Los Andes, Santiago, CHILE.
The accumulation of the β-Amyloid peptide as amyloid fibrils in the brain is associated with Alzheimer’s disease (AD) progression. Amyloid fibrils and deposits commonly exhibit different morphologies as a result of variations in the molecular structure of their forming protofilaments. In the case of Aβ1-40, two different types of fibrils, 2F and 3F, have been produced in vitro. Both the morphology of these fibrils and the molecular structure of their protofilaments self-propagate in a prion-like fashion. For this reason, we hypothesized that these differences could be responsible for different outcomes in vivo. In the present study, we injected 2F or 3F fibrils intracerebrally into Tg2576 AD transgenic mice, as well as control animals with an equal volume of aged wild-type or aged Tg2576 brain homogenate. After measuring PBS-insoluble Aβ40 and Aβ42 levels at 100 and 250 days post injection (dpi), we discovered that the animals receiving 2F fibrils exhibited significantly higher accumulation of Aβ40 than all other groups. Moreover, 2F injected animals also presented a significant increase in aggregated Aβ42 levels with respect to 3F injected animals, albeit similar to the animals inoculated with aged Tg2576 brain homogenate. We will extend these studies by performing histological characterizations in the brains of experimental and control subjects. Overall, we found that morphologically distinct β-Amyloid fibrils differentially induce β-Amyloid accumulation in vivo, thus raising the possibility that different amyloid morphologies have different pathogenicity affecting the outcome of AD patients.
Imaging and Pharmacokinetic Analysis of a 68Ga/NIR labeled peptide for somatostatin receptor targeting (PRE-CANDIDACY)
Servando Hernandez Vargas1, Sukhen C. Ghosh1, Julie Voss1, Kendra S. Carmon1, Melissa Rodriguez2, Agnes Schonbrunn2, Susanne Kossatz3, Thomas Reiner3, Ali Azhdarinia1
1Institute of Molecular Medicine, McGovern Medical School, Houston, TX, USA;2Department of Integrative Biology and Pharmacology, McGovern Medical School, Houston, TX, USA
3Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Objectives: Growing clinical evidence has shown that intraoperative imaging with fluorescent contrast agents can improve tumor identification. Fluorescently labeled probes have been dual labeled with a radionuclide to enable cross-validation with nuclear imaging. Here, we synthesized a dual-labeled octreotide analog using a multimodality chelator (MMC) and evaluated agent properties in somatostatin receptor-2 (SSTR2) expressing cells and tumor-bearing mice.
Methods: Synthesis of the MMC was performed by selectively attaching an azide-containing pendant arm and an acetate pendant arm to the tetraza chelator, DO2A. Tyr3-octreotide (TOC) was conjugated to the MMC on solid-phase, and the resulting peptide conjugate was fluorescently labeled with IRDye800. Potency of the peptide conjugate for cAMP inhibition and receptor internalization in HEK293-SSTR2 expressing cells was determined. Confocal microscopy along uptake and competition studies were carried out in SSTR2-transfected human colorectal carcinoma (HCT116(SSTR2)). PET/CT and near-infrared fluorescence (NIRF) imaging of 68Ga-MMC(IR800)-TOC was conducted in HCT116(SSTR2) tumor-bearing mice at 1, 3 and 24 h.
Results: MMC-TOC was synthesized using standard solid-phase techniques and dual labeling was confirmed by HPLC. 68Ga-MMC(IR800)-TOC effectively inhibited cAMP formation (EC50 = 0.066±0.012nM) and stimulated receptor internalization (EC50 = 48.7±9.9nM). 68Ga-MMC(IR800)-TOC uptake was 25±1.7% (% of total radioactivity added) in HCT116(SSTR2) cells and the findings were in agreement with 68Ga-DOTA-TOC. PET/CT showed 68Ga-MMC(IR800)-TOC localization in tumors with a tumor-to-muscle ratio of 3.8 (n=3), and correlated with NIRF images both In Vivo and Ex Vivo.
Conclusions: The MMC scaffold is effective for developing a dual-labeled octreotide analog. Further optimization of agent design could improve image contrast and utility of 68Ga-MMC(IR800)-TOC for multimodal imaging.
Functional characterization of peroxidases in C. elegans innate immunity (POST CANDIDACY)
Yi Liu1, George R. Tiller1 and Danielle A. Garsin1
1Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Texas 77030
Studying the role of reactive oxygen species (ROS) in innate immunity is crucial to our understanding of both its protective role and pathological effects. Dual oxidases (DUOX) produce H2O2 upon microbial infection. In some cases, H2O2 is converted into more potent antimicrobial agents by heme-containing peroxidases. Although there is recognition of the synergistic relationships between DUOX and peroxidases in recent years, the mechanisms by which immune associated peroxidases use H2O2 to contribute to the response are incompletely understood.
Previous studies in our lab have demonstrated that the DUOX, BLI-3, is responsible for the production of H2O2 and pathogen resistance upon E. faecalis infection. We hypothesize that BLI-3 may partner with other peroxidases for innate immune function. Through RNAi screen and CRISPR-Cas9 mediated knockout, we found two putative peroxidases, SKPO-1 and HPX-2, which are important for pathogen resistance. Localization study showed that SKPO-1 localizes to the hypodermis whereas HPX-2 expresses in pharynx. Functional analysis of skpo-1 and hpx-2 mutants revealed that they have different brood size, cuticle integrity, and peroxidase activity, suggesting differential roles between the two peroxidases in innate immunity. Our next efforts are to determine the difference in gene expression profile of skpo-1 and hpx-2 mutants by RNA-seq and to test any potential direct interactions between BLI-3 and the two peroxidases.
Together, our finding will provide us insight of the mechanism by which peroxidases utilize BLI-3 generated H2O2 to contribute to innate immunity. Since the peroxidases and NADPH oxidases are conserved from nematodes to insects to humans, our insights will be widely applicable to other organisms.
Takayasu Arteritis and Aortic Graft Infection: A Dangerous Duo (MEDICAL STUDENT)
McGovern Medical School at UTHealth, Houston
Objective: Takayasu’s arteritis (TA) is an inflammatory disease that often involves the aorta and its branches. Autopsy studies demonstrate 5.3% of TA mortality is attributable to aortic graft infection (AGI). We describe two cases with AGI that presented as aortocutaneous fistula (ACF).
Case-1: A 62-year-old woman with TA and occlusive aortic-arch disease s/p aorto-innominate, aorto-carotid, aorto-left-subclavian, and axillo-axillary bypasses presented with non-healing chest-wall sinus tract with purulent-discharge. Patient underwent aortic arch graft excision, ACF repair, ascending aorta (AAo) with proximal-arch replacement, redo innominate and left common carotid bypasses using Dacron grafts with omental flap, without any complications. Pre-and intraoperative cultures were negative. Postoperatively, she developed respiratory failure but recovered uneventfully following extubation on POD-4 and was discharged home on lifelong doxycycline suppressive therapy after 12 days. At follow-up, patient reported no complications, reinterventions, or reinfection.
Case-2: 31-year-old woman with TA, Crohn’s disease, CAB, aortic valve and composite-root replacement, composite-root AGI repair, and repair of ruptured MRSA+ sternal-wound seroma, developed ACF and AAo pseudoaneurysm (PSA). Patient underwent redo-sternotomy, ACF-repair and AAo PSA replacement using Dacron patch-graft and omental/muscle flap. Multiorgan failure complicated her postoperative recovery requiring month-long hospitalization following discharge. 4-months later, she was readmitted for acute right heart failure following complete aortic root dehiscence requiring emergent redo aortic valve, root, AAo and arch replacement. She eventually died of refractory heart failure.
Conclusions: Graft infection following aortic repair coupled with TA can escalate a dangerous complication to a fatal outcome.
Detection of Prions in Blood of Cervids at the Pre-symptomatic Stage of Chronic Wasting Disease (OTHER)
Carlos Kramm1,2,, Adam Lyon1, Tracy Nichols3, Sandra Pritzkow1, Claudio Soto1,2 and Rodrigo Morales1,
1Mitchell Center for Alzheimer’s disease and Related Brain Disorders, Dept. of Neurology, University of Texas Houston Medical School, Houston, TX 77030, USA.,2Universidad de los Andes, Facultad de Medicina, Av. San Carlos de Apoquindo 2200, Las Condes, Santiago, Chile., 3National Wildlife Research Center, United States Department of Agriculture, Fort Collins, CO 80521, USA
Chronic wasting disease (CWD) is a highly infectious and fatal illness affecting captive and free-ranging cervids, which is decimating the population of deer and elk in North America and has been recently detected also in Europe. A top priority to minimize the currently uncontrollable spreading of this disease is the development of a sensitive, specific and non-invasive ante-mortem diagnostic test that can identify CWD infected animals during the long and silent period between infection and the onset of clinical signs. Here, we optimized the protein misfolding cyclic amplification (PMCA) assay for highly efficient detection of CWD prions in blood samples. Studies were done using a panel of 98 field-collected samples of whole blood from white-tail deer that were analyzed for prion infection by post-mortem histological studies. The results showed a sensitivity of 100% in symptomatic animals, 96% in late pre-symptomatic animals and 53% in animals at early stages of infection. The overall diagnostic sensitivity was 79.3% with 100% specificity. These findings demonstrate a higher efficiency for CWD detection compared to previous studies utilizing immune-histochemical analysis of rectal biopsy tissue, which is currently approved for ante-mortem detection of CWD infected animals. The fact that this data was obtained with less invasive blood samples, offers a great promise for a routine, live animal laboratory diagnosis of CWD. Further studies are needed to confirm these results using larger number of samples.