shapiro lab stanford

Dahlberg, P. D., Saurabh, S., Sartor, A. M., Wang, J., Mitchell, P. G., Chiu, W., Shapiro, L., Moerner, W. E. Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution. 1967 Brooklyn College Dynamic protein localization is an integral component of the regulatory circuit that drives the Caulobacter cell cycle. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. liqunyu2@illinois.edu Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The partial IS sequences may represent silent evolutionary remnants or they could modulate the expression of genes carrying these sequences. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter. Currently: Assistant Professor of Bioengineering We are interested in how Arctic species and populations responded to environmental and habitat change throughout the Pleistocene, and what role ecology, natural history, climate and community-level dynamics played in the Chromosomal loci and many protein complexes are positioned at particular subcellular sites. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. Can we use ultrasound to remote-control the location and motion of specific cells? As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. When these two parameters are calculated for genes from nonmammalian eukaryotic organisms, genes from the same organism again have similar values, and genome-wide codon bias may also be predicted from intergenic sequences. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. View details for Web of Science ID A1970I035400018, View details for Web of Science ID A1968D122300009, View details for Web of Science ID A1968B197000024, View details for Web of Science ID A19667876500003, View details for Web of Science ID A19656243300010, View details for Web of Science ID A19657086600018, View details for Web of Science ID A19656243300011, Director, Beckman Center for Molecular & Genetic Medicine (2004 - Present), Dickson Prize in Science, Carnegie Mellon University (2020), Chan/Zuckerberg Investigator, Chan/Zuckerberg Biohub (2017), ASCB Women in Cell Biology Lifetime Achievement Award, American Society for Cell Biology (2013), Pearl Meister Greengard Prize, Rockefeller University (2013), Dean's Medal, Stanford University School of Medicine (2012), Louisa Gross Horwitz Prize, Columbia University Medical Center (2012), National Medal of Science, National Science Foundation (2011), Abbott Lifetime Achievement Award, ASM (2010), Distinguished Alumna Award, Albert Einstein College of Medicine (2010), Canada Gairdner International Award, Gairdner Foundation (2009), John Scott Award, Philadelphia City Trust (2009), Address the Swedish Royal Academy of Sciences, Swedish (2008), Hitchcock Professorship, UC Berkeley (2008), Selman A. Waksman Award, National Academy of Sciences (2005), Elected to the American Philosophical Society, American Philosophical Society (2003), FASEB Excellence in Science Award, Federation of American Societies for Experimental Biology (1994), National Academy of Sciences, National Academy of Sciences (1994), American Academy of Microbiology, American Academy of Microbiology (1993), American Academy of Arts and Sciences, American Academy of Arts and Sciences (1992), Institute of Medicine of the National Academy of Sciences, National Academy of Sciences (1991), Ph.D., Albert Einstein College of Medicine, Molecular Biology (1966), Molecular and Genetic Medicine (Fellowship Program), Department: Department of Developmental Biology. Ph.D. Student, Bioengineering One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Pasadena, CA, USA 91125, Copyright 2023 Mikhail G. Shapiro | Powered by, Scientists, Postdoc Scholars & Graduate Students. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. Stanford Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Herrmann, J., Smit, J., Shapiro, L., Wakatsuki, S. Two-Color Sted Microscopy to Visualize S-Layer Biogenesis in Caulobacter Crescentus. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell. We demonstrate that successive cleavage events involving regulated intramembrane proteolysis (Rip) occur as a function of time during the Caulobacter cell cycle. The Caulobacter crescentus fliQ and fliR genes encode membrane proteins that have a role in an early step of flagellar biogenesis and belong to a family of proteins implicated in the export of virulence factors. In addition to topological constraints, the cellular position of the replication origin is strictly controlled during the cell cycle. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. View details for Web of Science ID A1980JF34600034. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. By deciphering the underlying design principles, we hope to generate pure populations of these cell-types from embryonic and induced pluripotent stem cells for regenerative medicine. Postdoc. M.S. The Bejerano Lab focuses on a fundamental question in Human Genomics: the relationship between geno(me)type and phenotype. B.S. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology. View details for Web of Science ID A1995RP99400003. The PleA protein contains a region that is similar to a peptidoglycan-hydrolytic active site, and a point mutation at this site in PleA results in the loss of flagellum and pili biogenesis. The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda. Western University of Health Sciences, Dr. Brittany Moser The mechanisms underlying this regulation include protein phosphorylation and proteolysis. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level. Shapiro completed postdoctoral research at Stanford University Medical School and was named a Guggenheim Fellow at MITs Center for Cancer Research. Shapiro (2018). We find that the actin-like MreB protein mediates global cell polarity in Caulobacter crescentus, although the intermediate filament-like CreS protein influences cell shape without affecting cell polarity. How toxin activation triggers persistence and induces a systemic stress response in the alphaproteobacteria remains unclear. Ph.D. Chemistry, Harvard University Critically, many of these functions occur at defined locations within the cell, and therefore regulators of each module must communicate to remain coordinated in space. They are using full genome sequence and microarray technology to identify the genetic circuitry that controls the cell cycle in a bacterial cell with 3767 genes. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. Our work is greatly enhanced by the ability to manipulate expression of exogenous genes, or to knock down endogenous ones, using transgenic mice. These results argue that PleA facilitates the assembly of envelope-spanning structures at the cell pole. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. A systematic search for motifs associated with CcrM DNA methylation sites revealed four long (>100-bp) motifs (CIR sequences) present in up to 21 copies in Caulobacter crescentus. Environmental Science, Johns Hopkins University The global regulatory architecture of transcription during the Caulobacter cell cycle. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Gonzalez, D., Kozdon, J. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. Interview With Dr. Jonathan Schapiro Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. Surprisingly, many signal transduction proteins are dynamically localized to specific subcellular addresses during the cell division cycle and sporulation, and proper localization is essential for their function. The K+ channels are M-type, originally called such from their inhibition by stimulation of muscarinic acetylcholine receptors in sympathetic neurons. View details for Web of Science ID 000430563200493, View details for Web of Science ID 000430450000249, View details for Web of Science ID 000430563200402, View details for Web of Science ID 000430450000508, View details for Web of Science ID 000430563300065, View details for Web of Science ID 000430439600509. View details for Web of Science ID A1986E866400004. UPR activation is protective and is a powerful new prognostic marker in ERa positive breast cancer. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. Protein localization, notably of signal transduction proteins, chromosome partition proteins, and proteases, serves to coordinate cell division with chromosome replication and cell differentiation. Attempts to encode large numbers of polymeric, metallic or glass beads in random arrays or in fluid suspension have used a variety of entities to provide coded elements (bits)--fluorescent molecules, molecules with specific vibrational signatures, quantum dots, or discrete metallic layers. 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. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. x@caltech.edu, x=yxyao, Sangjin Yoo, PhD Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. View details for DOI 10.1111/j.1365-2958.2005.04912.x, View details for Web of Science ID 000233170700012. Millions of possible codes can be prepared this way. Pharmacological Chemistry, UC San Diego The IHF and enhancer sites are 3' to the transcription start site in this promoter. Brett SHAPIRO The polar localization of ClpXP is dependent on the polar positioning of the CpdR single-domain response regulator. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. Expression of the latter two phenotypes required complex media and both were repressed by glucose. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. B.S. The Min proteins that govern division site selection in Escherichia coli may be the first example of a system that generates positional information de novo. The phospholipid composition of Caulobacter crescentus CB13 and CB15 was determined. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis. Duplication of the chromosome and partitioning of the newly generated daughter strands are interwoven processes driven by the dynamic interplay between the synthesis, segregation and condensation of DNA. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. B.S. In bacteria, cell polarity has been observed by using both morphological and molecular markers; however, no general regulators of bacterial cell polarity have been identified. View details for DOI 10.1016/j.cell.2008.07.015, View details for Web of Science ID 000259318100015, View details for PubMedCentralID PMC2745220. Ph.D. B.S. The initiation of replication depends on the proteolysis of CtrA. CtrA plays key roles in asymmetric cell division and in the timing of chromosome replication. View details for DOI 10.1073/pnas.202495099, View details for Web of Science ID 000178391700119, View details for PubMedCentralID PMC130603. Temporally controlled proteolysis of the essential response regulator, CtrA, is critical for cell cycle progression in Caulobacter crescentus. View details for DOI 10.1073/pnas.1405188111. View details for Web of Science ID A1995RM70200022. Mutants in the hook operon or in basal body genes caused overproduction of both hook operon and basal body gene chimeric mRNAs, suggesting that negative regulation is superimposed on the positive trans-acting control for these early events in the flagellar hierarchy. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Welcome to the Shapiro Lab at the California Institute of Technology. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA. Because CckA approximately P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. Dividing cells must coordinate cell cycle events to ensure genetic stability. 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. WebJonathan Schapiro, MD, Adjunct Clinical Professor, Stanford University School of Medicine, Stanford, California . Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35). Postdoctoral Scholar In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. x@caltech.edu, x=sdalfonz, Marama Diaz-Asper WebShapiroLab Going Deeper We develop technologies to image and control the function of cells deep inside the body. Ryan Rezvani, Amgen Scholar 2014 PhD at UC Irvine View details for Web of Science ID 000178123100022. View details for Web of Science ID A1981LG93700035. Overall, the algorithm is a major paradigm shift in the way we analyze experimental accelerator data at facilities today, Roussel said. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. Ph.D. Student, Bioengineering Biology, Dongguk University A., Deacon, A. M., Shapiro, L. Cell fate regulation governed by a repurposed bacterial histidine kinase. ZS Associates, Undergraduate and Visiting Student Alumni, Mei Yi You, SURF Scholar 2018-2022 PhD at MIT 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. Yes! We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. What is shapiro test in R? - Projectpro Albert Zhai, Research Assistant 2018 BS CS, Caltech 2021 Strains with mutations in one of these genes, flaS, cannot transcribe flagellar structural genes and divide abnormally. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the -proteobacteria. View details for DOI 10.1128/mBio.02238-16, View details for PubMedCentralID PMC5347347. The tests have not been cleared or approved by the US Food and Drug Administration (FDA). Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template. View details for DOI 10.1021/jacs.6b05943, View details for PubMedCentralID PMC4996739. The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. The bound ATP plays an important role in dimerization of ErTadZ. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. Chromosome replication initiates in the daughter stalked cell but is repressed in the daughter swarmer cell until later in the cell cycle. Postdoctoral Scholar, 2014-19 Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Thanbichler, M., Iniesta, A. Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. Stanford Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location. View details for DOI 10.1016/j.molcel.2010.08.027, View details for Web of Science ID 000282377200016, View details for PubMedCentralID PMC2945607. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. View details for Web of Science ID A1978FP55600049. View details for DOI 10.1111/j.1365-2958.2011.07836.x, View details for Web of Science ID 000298087300007, View details for PubMedCentralID PMC3273039. x@vis.caltech.edu, x=rosie, Ruby Zhang Ross, P. L., Chen, X., Toro, E., Britos, L., Shapiro, L., Pappin, D., Whitelegge, J. P. Caulobacter crescentus as a whole-cell uranium biosensor. SLAC is a vibrant multiprogram laboratory that GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In Caulobacter crescentus, the origin of DNA replication is located at the cell pole. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. The relative order of the cleavage fragments was determined by specific cleavage of isolated restriction fragments, terminal labeling of both the whole genome and isolated fragments, and hybridization of isolated fragments to restriction fragments generated by other restriction enzymes. B.S. NSERC Fellow Ph.D. Student, Neurobiology (co-advised with David Anderson) Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. article, Thank you to the Howard Hughes Medical Institute for welcoming our group and supporting our vision of biomolecular ultrasound as an emerging technology for basic biology and medicine. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. 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. Assays of the differential placement of the promoter-less drug resistance proteins (encoded within the interrupted fla genes) allow us to determine whether the positioning of the fla gene products is controlled by signal sequences in their proteins, by specific mRNA-targeting sequences in the 5'-regulatory regions of these genes, or by specific transcription from only one of the two newly replicated chromosomes in the predivisional cell. View details for Web of Science ID A1996UM40600009, View details for PubMedCentralID PMC450171. Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. Brett Shapiro | LIGO Group B.S. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Lab Phone: 626-395-8955, Division of Chemistry and A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. In the past several years, we have also focused on transcriptional regulation of expression of the channels, both in normal and pathophysiological states, such as during epilepsy, stroke and traumatic brain injury. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme.

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