Presidents Council Faculty Scholar Award, UT Health SA (UTHSCSA), 2013-2015. View details for DOI 10.1073/pnas.1906119116, View details for DOI 10.1016/j.bpj.2018.11.881, View details for Web of Science ID 000460779800789, View details for DOI 10.1016/j.bpj.2018.11.2446, View details for Web of Science ID 000460779802279, View details for DOI 10.1016/j.bpj.2018.11.056, View details for Web of Science ID 000460779800028, View details for DOI 10.1016/j.bpj.2018.11.1077, View details for Web of Science ID 000460779800965, View details for DOI 10.1016/j.bpj.2018.11.2475, View details for Web of Science ID 000460779802305. Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controlled by a trans-acting regulatory hierarchy. The Caulobacter bacteria now present another system in which direct analysis of these control mechanisms is feasible. Postdoctoral Scholar, 2014-16 Michael Yao, SURF Scholar 2017-21 (Housner Prize) MD-PhD at University of Pennsylvania The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. M.D. To determine whether IS elements could exert control through specific RNA transcripts, we hybridised lambda NNC1857 r14 (carrying IS1) and pBR322 (carrying a portion of IS2) to Northern blots of E. coli RNA. P(xylX) promoter activity was determined as a function of the composition of the growth medium both in single copy and on a plasmid using different reporter genes. Postdoctoral Scholar (co-advised with Mory Gharib) The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. It will bring together the resources and expertise of the national lab, the university and Silicon Valley to accelerate the deployment of batteries and other energy storage A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. By examining depletion and overexpression strains, we demonstrate that MreB is required both for the polar localization of the chromosomal origin sequence and the dynamic localization of regulatory proteins to the correct cell pole. Paige, M. F., Judd, E., Shapiro, L., Moerner, W. E. Complete genome sequence of Caulobacter crescentus. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. To approach this question, they are studying a bacterial cell, whose simple life cycle is focused on the generation of asymmetry in the predivisional cell. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro. Research Technician Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. Dr. Howard Shapiro and The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. B., Dingwall, A., Bryan, R., CHAMPER, R., Shapiro, L. POSITIONING OF GENE-PRODUCTS DURING CAULOBACTER CELL-DIFFERENTIATION, RATE, ORIGIN, AND BIDIRECTIONALITY OF CAULOBACTER CHROMOSOME-REPLICATION AS DETERMINED BY PULSED-FIELD GEL-ELECTROPHORESIS, ORGANIZATION AND TEMPORAL EXPRESSION OF A FLAGELLAR BASAL BODY GENE IN CAULOBACTER-CRESCENTUS, CONTROL OF SYNTHESIS AND POSITIONING OF A CAULOBACTER-CRESCENTUS FLAGELLAR PROTEIN. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. Quon, K. C., Marczynski, G. T., Shapiro, L. USE OF FLOW-CYTOMETRY TO IDENTIFY A CAULOBACTER 4.5 S RNA TEMPERATURE-SENSITIVE MUTANT DEFECTIVE IN THE CELL-CYCLE, A DEVELOPMENTALLY-REGULATED CHROMOSOMAL ORIGIN OF REPLICATION USES ESSENTIAL TRANSCRIPTION ELEMENTS. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. We show here that two genes, gyrB (encoding the gyrase B subunit) and orf-1, are specifically transcribed from the chromosome in the portion of the predivisional cell destined for the progeny stalked cell. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. Follow @StanfordBioX, Stanford University, Stanford, California 94305, James H. Clark Center, Stanford University, Stanford Bio-X Frontiers in Interdisciplinary Biosciences: 2019/2020, Department of Developmental Biology Homepage, Stanford Interdisciplinary Life Sciences Council. Yeh, Y., Comolli, L. R., Downing, K. H., Shapiro, L., McAdams, H. H. Imaging-Based Identification of a Critical Regulator of FtsZ Protofilament Curvature in Caulobacter. Postdoctoral Scholar (co-advised with Richard Andersen) Maria Paulene Abundo Cell division then yields a new swarmer cell and a stem-cell-like stalked cell. CrfA functions to stabilize the CC3461 transcript. The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. For this study, the team tried a new approach: They built a machine learning model that uses our understanding of beam dynamics to predict the distribution of particles positions and speeds within the beam, collectively known as the beam's phase space distribution. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. After graduating he worked for the NASA Marshall Space Flight Center developing non-destructive evaluation techniques for applications related to the US space program. View details for DOI 10.1126/science.1095191, View details for Web of Science ID 000221383300040. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene. Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. View details for Web of Science ID A1990DB07400012. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. Gloria Ha, SURF Scholar 2015 PhD at Harvard These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. View details for Web of Science ID A1985ALK8400022. A major breakthrough in understanding the bacterial cell is the discovery that the cell is highly organized at the level of protein localization. WebBrett Shapiro Gravitational waves are predicted to exist by Einstein's Theory of General Relativity. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non-replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria. Here, we combine single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modelling and a spatially resolved promoter activation assay to study signal exchange in and out of the 200nm cytoplasmic pole-organizing protein popZ (PopZ) microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4-8. SLAC is a vibrant multiprogram laboratory that SURF Scholar 2022- Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. View details for Web of Science ID A1994NX67800011. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. x@caltech.edu, x=sshivaei, Cameron Ashley Brandon Smith, PhD We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Shapiro, L., Rosen, O. M., AGABIANK, N., Hirsch, A. BACTERIAL DIFFERENTIATION AND PHAGE INFECTION. We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. The production of these different transcripts by the E. coli enzyme was dependent on salt concentration and, in at least one case, appeared to be the result of differential termination. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. As a step toward understanding this process, we have defined cis-acting sequences necessary for expression of a Class II flagellar operon, fliLM. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. Research Technician, 2016-2019 This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. We demonstrate that another single domain response regulator, DivK, promotes the polar accumulation of unphosphorylated CpdR and that CpdR is subsequently degraded at the cell pole by the localized ClpXP protease. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression. Ph.D. Student, Chemical Engineering The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. Ph.D. Student, Neurobiology (co-advised with David Anderson) Ph.D. Acoustic Physics, Physics for Medicine Lab and Sorbonne University Research Scientist Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. Congratulations to Przemek and colleagues in the Shapiro and Jensen labs on figuring this out with a beautiful CryoET structure. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology. Structure (2023). B.E. The developmental program by which a single cell proceeds to a fully-developed organism involves cell divisions that yield dissimilar daughter cells. View details for Web of Science ID A1976CE95700078. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation. View details for Web of Science ID A1987G196300016. x@caltech.edu, x=mswift, Rosie Zedan View details for Web of Science ID A1976BU75500037. Currently: Clinical Research Assistant A fusion of the receiver domain and last 15 residues of CtrA to YFP is properly degraded in living cells. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. A., Deacon, A. M., Shapiro, L. Cell fate regulation governed by a repurposed bacterial histidine kinase. View details for Web of Science ID 000165066300004. Director, High-throughput Screening Facility Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. View details for Web of Science ID A1993KT81000027. DEOXYRIBONUCLEIC-ACID SEQUENCE HOMOLOGIES AMONG BACTERIAL INSERTION SEQUENCE ELEMENTS AND GENOMES OF VARIOUS ORGANISMS, CELL-CYCLE-ASSOCIATED REARRANGEMENT OF INVERTED REPEAT DNA-SEQUENCES. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. x@caltech.edu, x=spolidor, Andrea Torres Nathan, P., Gomes, S. L., Hahnenberger, K., Newton, A., Shapiro, L. FATTY-ACID DEGRADATION IN CAULOBACTER-CRESCENTUS, TRANSCRIPTION INITIATION INVITRO AND INVIVO AT A HIGHLY CONSERVED PROMOTER WITHIN A 16 S-RIBOSOMAL RNA GENE. Thanks to the researchers from 10 countries who joined us for this inaugural event and to all the Shapiro Lab members who participated, helped organize, and hosted live demos. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. Researchers develop clever algorithm to improve our understanding of particle beams in accelerators, Now, researchers at the Department of Energys SLAC National Accelerator Laboratory, the DOEs Argonne National Laboratory and the University of Chicago have developed an algorithm that more precisely predicts a beams distribution of particle positions and velocities as it zips through an accelerator. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Ph.D. Student, Bioengineering, Defended 2020 These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Candidate, David Geffen School of Medicine at UCLA Explore SLAC events and learn how to participate. The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. We study how the distribution of such signals is regulated in tissues, how cells perceive and respond to distinct concentrations of signals, and how such signaling pathways arose in evolution. Kim, S., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression. We have begun studies with RNA polymerase purified from Caulobacter crescentus to determine whether cell factors or alterations in the enzyme structure serve to change the specificity of transcription during the cell cycle. Shapiro (2018). A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. B.S. View details for DOI 10.1073/pnas.1220824110, View details for Web of Science ID 000314558100027, View details for PubMedCentralID PMC3562846. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. Blackburn, E., Firtel, R. A., Shapiro, L. GENETIC-ANALYSIS OF A TEMPORALLY TRANSCRIBED CHEMOTAXIS GENE-CLUSTER IN CAULOBACTER-CRESCENTUS. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. Shapiro's laboratory question in developmental biology involves the mechanisms used to generate the three-dimensional organization of a cell from a one-dimensional genetic Mathematical and Computational Biology, Harvey Mudd College The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S(20, w) of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. We use STORM super-resolution nanoscopy to probe the multi-protein complexes underlying modulation of ion channels. Marczynski, G. T., LENTINE, K., Shapiro, L. REGULATION OF THE CAULOBACTER-CRESCENTUS DNAKJ OPERON. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry. View details for Web of Science ID A1994PA42600022. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. View details for Web of Science ID A1997XT77200016, View details for PubMedCentralID PMC179404. Computation and Neural Systems, expected 2025 The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. Comerci, C. J., Herrmann, J., Shapiro, L., Wakatsuki, S., Moerner, W. E. Spatial Organization and Dynamics of RNA Processing in Caulobacter Crescentus. View details for Web of Science ID A1984SP90900006. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. Whenever SLAC National Accelerator Laboratorys linear accelerator is on, packs of around a billion electrons each travel together at nearly the speed of light through metal piping. Biochemistry, Jagiellonian University A representation of a particle beam traveling through an accelerator. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. The main task of a bacterial cell is to survive and duplicate itself. The specific features of the Caulobacter system which make it a system of choice for studies of the control of sequential events resulting in cellular differentiation can be summarized as follows. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. Rice University, Prof. Jerzy Szablowski View details for Web of Science ID 000177770100004. Research in the Villeneuve lab is aimed at understanding the molecular and cellular mechanisms underlying the faithful inheritance and function of eukaryotic chromosomes. The basal body consisted of five rings mounted on a rod. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. A series of Tn5 insertion mutations in the flaD BC region were mapped. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. AN UNUSUAL PROMOTER CONTROLS CELL-CYCLE REGULATION AND DEPENDENCE ON DNA-REPLICATION OF THE CAULOBACTER-FLILM EARLY FLAGELLAR OPERON, PROTEIN LOCALIZATION AND ASYMMETRY IN THE BACTERIAL-CELL, FLOW-CYTOMETRY OF CAULOBACTER-CRESCENTUS - IDENTIFICATION AND CHARACTERIZATION OF A CELL-CYCLE MUTANT. x@caltech.edu, x=rzhang5, Undergraduate Students The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression. WebWelcome! Childers, W. S., Xu, Q., Mann, T. H., Mathews, I. I., Blair, J. We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. View details for DOI 10.1016/j.jsb.2006.05.007, View details for Web of Science ID 000242008100007, View details for Web of Science ID 000207781609166. Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. How this is brought about remains one of the most fundamental questions of developmental biology. Cellular functions in Bacteria, such as chromosome segregation and cytokinesis, result from cascades of molecular events operating largely as self-contained modules. 1986 Fudan University The phenotype of pH-conditional mutants was defined on medium with lactose as the sole carbon source. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. It was observed that the final step in the swarmer cell-to-stalked cell transition, stalk elongation, was inhibited under these conditions. A., McGrath, P. T., Reisenauer, A., McAdams, H. H., Shapiro, L. Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. B.S. WebGraduate student, Sarah Cohen laboratory, Romberg Tiburon Center for Environmental Studies, 3152 Paradise Drive, Tiburon, CA 94920, (415) 338-3754 daniellendesmet gmail.com We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G(1)-S transition by signaling the temporally controlled proteolysis of CtrA. The genes involved in these processes are widely separated on the chromosome. A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity.
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