Sci. Sci. Sci. Free energy determinants of secondary structure formation: I. alpha-Helices. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon. doi: 10.1016/j.matbio.2009.10.003, Tu, Y., Wise, S. G., and Weiss, A. S. (2010). Biotechnol. Domain 26 of tropoelastin plays a dominant role in association by coacervation. Matrix Biol. Molecular dynamics modeling of aggregating tropoelastin-derived peptide chains points to the maintenance of a hydrated, disordered, liquid-like state due to the formation of short-lived inter-chain bonds (Rauscher and Pomes, 2017; Reichheld et al., 2020), mostly likely due to the inducement of random coils by PG repeats (vide supra). Pediatr. Dissection of human tropoelastin: solution structure, dynamics and self-assembly of the exon 5 peptide. doi: 10.1021/acs.biomac.0c00546, Bochicchio, B., Floquet, N., Pepe, A., Alix, A. J., and Tamburro, A. M. (2004). (1973). Elastagen was one of the inaugural recipients of MDF funding in 2013 and will be the first to successfully repay the funding amount. Engineering a highly elastic human protein-based sealant for surgical applications. Hailed as a giant leap forward in hair rejuvenation, NULASTIN transforms the look of eyelashes up to 120% thicker and 61% longer. J. Biol. (2014). doi: 10.1074/jbc.M104985200, Toonkool, P., Jensen, S. A., Maxwell, A. L., and Weiss, A. S. (2001). Tropoelastin is typically used as a thin coating on tissue culture surfaces. doi: 10.1046/j.1432-1327.1998.2580001.x, Wang, Z., Liu, L., Mithieux, S. M., and Weiss, A. S. (2020). doi: 10.1016/j.tibtech.2019.08.005, Wise, S. G., and Weiss, A. S. (2009). Mol. 432, 57365751. Int. 9:643110. doi: 10.3389/fbioe.2021.643110. The computational model uncovered the contributions of each molecular region to the flexibility of the molecule. Indeed, studies examining the consequences of domain insertions and deletions note changes in the intrinsic functionality of tropoelastin, corroborating the hypothesis that domain insertions and deletions result in altered tissue mechanics (Jensen et al., 2000; Kozel et al., 2003; Yeo et al., 2016; Miao et al., 2017). Binding of EDPs to the ERC induces a number of biological effects including migration, adhesion, proliferation, protease expression, and secretion. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). doi: 10.1016/j.febslet.2015.08.029, Rodgers, U. R., and Weiss, A. S. (2004). A., and Rucker, R. B. The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit. Thus, elastin is responsible, in great part, for the properties of elastic fibers. doi: 10.1007/978-1-4684-9093-0_25, Scandolera, A., Odoul, L., Salesse, S., Guillot, A., Blaise, S., Kawecki, C., et al. AW is the Scientific Founder of Elastagen Pty. Proc. Elastases can be in the form of serine- (e.g., Ela-2), cysteine- (e.g., cathepsin I), or matrix metalloproteinases (MMP-2, -7, -9, and -12), and bioactive peptides include the VGVAPG peptide. J. Biochem. Biochemistry 36, 84138421. Mater. February 14, 2018 Michael Patrini. (1989). Differential expression of two tropoelastin genes in zebrafish. Identification of an elastin cross-linking domain that joins three peptide chains. Assembly proceeds from a nucleation event and undergoes elongation in a step-wise manner to form a multimer which can occur in a head-to-tail fashion (Wise et al., 2014). U.S.A. 115, 73387343. A cell adhesive peptide from tropoelastin promotes sequential cell attachment and spreading via distinct receptors. Unlike hydrophobic domains, cross-linking domains in isolation are unable to self-assemble; thus, they have been studied in the context of the cross-linking of elastin-derived biomaterials (Annabi et al., 2017; Yue et al., 2017). doi: 10.1006/excr.1995.1321, Hinek, A., and Rabinovitch, M. (1994). Hogan Lovells represented Elastagen Pty Ltd. on the deal. 84, 6880. J. Exp. 293, 1510715119. Tropoelastin is a precursor protein of elastin, and is composed of hydrophilic (lysine, valine and proline) and hydrophobic (glycine, valine and proline) domains. Implications for tissue specificity and durability of elastic tissue. J. 26, 125135. Its hydrophobic (pink) and cross-linking (blue) domains consist of repetitive motifs that contribute uniquely to elastin assembly. Matrix Biol. However, there are aspects of this model that warrant further investigation. J. Pathol. Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin. Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins. (2017). Investig. Cellular expression of tropoelastin mRNA splice variants. Cellular activities are mediated through interactions between tropoelastin or EDPs and specific receptors on the cell surface. 13, 739743. 67-kD elastin-binding protein is a protective companion of extracellular insoluble elastin and intracellular tropoelastin. Biosci. Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice. doi: 10.1074/jbc.270.30.17778, Cain, S. A., Baldock, C., Gallagher, J., Morgan, A., Bax, D. V., Weiss, A. S., et al. 275, 2844928454. It has become increasingly evident that tropoelastins sequence, structure and subsequent functionality are in a delicate balance; thus, perturbations to tropoelastins sequence can have far-reaching consequences for self-assembly and the resultant architecture of elastic fibers. (2020). Most organisms contain one copy of ELN, other than teleosts and amphibians which notably possess two distinct types of ELN genes (He et al., 2007; Miao et al., 2007, 2009). (2006). Parameter, Testing, and Method. Unsubstituted models are also critical to understanding self-assembly. Due to the lack of a full-atomistic structure, a number of elastin derivatives, including isolated tropoelastin domains (Toonkool et al., 2001; Mackay et al., 2005; Dyksterhuis et al., 2007; Dyksterhuis and Weiss, 2010) and synthetic elastin-derived peptides (EDPs) (Luan et al., 1990; Tamburro et al., 1992; Kentsis and Sosnick, 1998; Kumashiro et al., 2006; Reichheld et al., 2014, 2017; Muiznieks et al., 2015; MacEwan et al., 2017; Tarakanova et al., 2017) have been studied to understand tropoelastins domain level properties. Acta Gen. Subj. The elasticity of organs and tissues in vertebrates is due to the presence of elastin, and an insoluble protein whose soluble monomer, tropoelastin, is enzymatically cross-linked through -amino groups of lysine residues. Developmental changes of tropoelastin synthesis by rat pulmonary fibroblasts and effects of dexamethasone. Tropoelastin #5052. Cell Biol. These interactions trigger a wide range of processes including wound healing, elastogenesis and maintenance of stemness (Yeo and Weiss, 2019). doi: 10.1016/j.matbio.2007.05.005, He, D., Miao, M., Sitarz, E. E., Muiznieks, L. D., Reichheld, S., Stahl, R. J., et al. Elastagen has received investment from an international syndicate of venture groups including AmorePacific Ventures, Brandon Capital, Cell Innovation Partners, Cicada Innovations, GBS Ventures, Korea Investment Partners and the Wellcome Trust. Sci. 28, 432441. Sci. Tropoelastins sequence and domain arrangement. Lyophilized Powder. Front. Chem. Similarly, a mutation model associated with cutis laxa, or loose skin disease (Hu et al., 2006), was found to exhibit higher stability compared with the wild-type molecule due to the increased longevity of its salt bridges (Hu et al., 2006; Tarakanova et al., 2018). 15, 786801. Biophys. doi: 10.1038/nature04085. The conversion of a single lysine to an allysine results in structural stabilization, which may serve as a checkpoint during self-assembly to ensure that molecules incapable of forming multiple cross-links are not incorporated into the growing chain. doi: 10.1203/00006450-199010000-00015, Nonaka, R., Sato, F., and Wachi, H. (2014). The ELN gene provides instructions for making a protein called tropoelastin. Natl. For many years it was difficult to study tropoelastin at an atomic scale, but the application of computational methods, such as full-atomistic molecular dynamics and elastic network models, in combination with powerful low-resolution structural studies, have expanded the field and delivered an enhanced understanding of the mechanisms that contribute to self-assembly. The Weiss Lab's research focuses on the use of human tropoelastin in tissue assembly and wound repair. Banded fibers in tropoelastin coacervates at physiological temperatures. The first successful experiments to define the 3D envelope of tropoelastin utilized small angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) (Baldock et al., 2011). Matrix Biol. Dissection of human tropoelastin: exon-by-exon chemical synthesis and related conformational studies. Cell Biol. Characterization of the complete human elastin gene. doi: 10.1172/jci115204, Sherratt, M., Wess, T. J., Baldock, C., Ashworth, J., Purslow, P., Shuttleworth, C., et al. Cell. Proc. Elastagens tropoelastin is identical to that present in human tissue, and has many potential clinical applications, including treatment for acne scars, stretch marks, aesthetic skin repair and surgical wound repair. It has recently been suggested that fibrillins are capable of stabilizing tropoelastin, possibly for the purpose of selecting conformations that are favorable for elastin assembly (Lockhart-Cairns et al., 2020). (2019). (1989). A., Gray, W. R., and Franzblau, C. (1973). (2009). doi: 10.1016/j.biomaterials.2017.04.050, Keywords: elastin, elastic fibers, tropoelastin, computational modeling, assembly, Citation: Ozsvar J, Yang C, Cain SA, Baldock C, Tarakanova A and Weiss AS (2021) Tropoelastin and Elastin Assembly. The isoform-function theory is further reinforced by the presence of two different ELN genes within teleosts that are differentially expressed both spatially and temporally during development (Miao et al., 2007). Int. The tropoelastin that Elastagen manufactures is an identical copy of what naturally occurs in human tissue, meaning the clinical application opportunities for this has tremendous potential . Circ. Biochemistry 8, 28332837. J. Biomol. Cooperativity between the hydrophobic and cross-linking domains of elastin. doi: 10.1006/jmbi.1995.0502, Yeo, G. C., Baldock, C., Tuukkanen, A., Roessle, M., Dyksterhuis, L. B., Wise, S. G., et al. For example, their inclusion in peptides derived from hydrophobic domains confers decreased times taken for self-assembly, most likely due to their favorable interaction with aqueous solvent (Miao et al., 2003). Natl. Remodelling the extracellular matrix in development and disease. Communication: coacervation of tropoelastin results in fiber formation. 29, 152159. J. Mol. Allergan acquired Elastagen primarily for the injectable product portfolio (acne scars/stretch marks aesthetics). Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. Thus, at lower temperatures, the water surrounding these domains forms ordered, clathrate-like shells that prevent aggregation until the appropriate temperature is reached (Wu and Weiss, 1999; Miao et al., 2003; Dandurand et al., 2015). Chem. Biochemistry 42, 1334713362. I thank the Elastagen team for their hard work and dedication in developing our innovative tropoelastin product pipeline and look forward to working with Allergan to take these products to market., Commenting on the transaction, Elastagens founding scientist Prof Anthony Weiss added:Our technology has come a long way from the lab bench at the University of Sydney towards developing products for patients around the world. Microfibrils comprise multiple proteins, of which fibrillin-1 is the most common. J. Biol. Foster, J. Elastin is extensively cross-linked and difficult to break apart into monomers even under harsh conditions, thus, initial strategies included feeding animals a copper deficient diet to prevent lysyl oxidase (LOX) cross-linking of tropoelastin; however, this proved inefficient and time consuming (Wise and Weiss, 2009; Tarakanova et al., 2018). All authors contributed to writing and editing the review. It was first hypothesized that tropoelastin assembles in a head-to-tail manner similar to that of other ECM proteins such as collagen, which assembles into fibrils that subsequently associate laterally to form sheets and thicker fibers (Kadler, 2017). Res. Thus, the current computational model unifies the global and regional characteristics of tropoelastin, inferring mechanisms that complement observed experimental phenomena. . Elastic fibers are eventually formed after extensive cross-linking through a process termed maturation (Yeo et al., 2016). doi: 10.1002/bip.1981.360200611. Dominant driving factors of assembly were examined, including water, temperature and domain pair orientations. Coacervation is an endothermic, entropically favorable process through which tropoelastin monomers self-assemble into higher order n-mer structures. Vallet, S. D., Miele, A. E., Uciechowska-Kaczmarzyk, U., Liwo, A., Duclos, B., Samsonov, S. A., et al. For Elastagen contact: Robert Daniels, CEO +61 2 9209 4054. Baul, U., Bley, M., and Dzubiella, J. Codistribution analysis of elastin and related fibrillar proteins in early vertebrate development. (2008). Cross-linking domains are capable of modulating self-assembly when incorporated into peptides and biomaterials derived from tropoelastins hydrophobic domains. Elastogenesis is the term that collectively describes the hierarchical process of elastic fiber formation, and is comprised of distinct phases: tropoelastin synthesis, coacervation, cross-linking, and deposition. 87, 18281834. PLoS One 7:e46130. Trends towards maximal association are apparent when each of these parameters is varied. A., Starcher, B. C., and Urry, D. W. (1974). Tropoelastin (TE) is the main (>90%) component of elastin, wherein crosslinked assembled coacervates of TE are exposed to lysyl oxidases and interact with microfibrils to expand elastin fibers. FEBS J. Biol. Instead, tropoelastin domains 1418 and 36 (RKRK sequence) have been found to bind to both integrins. Isolation and characterization of crosslinked peptides from elastin. Tropoelastins C-terminal domain 36 (yellow) does not fall into either category as it contains a distinct sequence capped with a Gly-Arg-Lys-Arg-Lys (GRKRK) motif and is primarily involved in cell interactions. (2018). doi: 10.1016/j.matbio.2006.10.002, Dyksterhuis, L. B., and Weiss, A. S. (2010). The hydrophobic domain 26 of human tropoelastin is unstructured in solution. The Wellcome Trust Centre for Cell-Matrix Research is supported by funding from Wellcome Trust (203128/Z/16/Z). Biochemistry 45, 99899996. The self-assembly properties and nanostructure of these extra teleost isoforms are yet to be investigated. In order to eliminate this limitation, human tropoelastin has been expressed in a recombinant bacterial system and the protein has been highly purified. Simul. The full-atomistic model of tropoelastin was developed based on replica exchange molecular dynamics (REMD) simulations, an accelerated sampling method for molecular dynamics (Tarakanova et al., 2018). Tropoelastin, the raw material used to create 'MeTro' elastic surgical glue developed with the University of Sydney, has been found to encourage stem cell growth - with the potential to ultimately help the body repair itself. Localizing alpha-helices in human tropoelastin: assembly of the elastin puzzle. Tropoelastin was revealed to be an asymmetric molecule with distinct N- and C-terminal regions that are respectively referred to as the "head" and "foot" of the molecule. Moreover, fibulins are essential for elastic fiber directionality, as fibroblasts with fibulin-4 and -5 knockdowns generate poorly formed elastic fibers (Yamauchi et al., 2010), and fibulin-4 / mice display aberrant, poorly cross-linked, and non-fibrous elastin (McLaughlin et al., 2006). J. Biol. 220, 312324. The business could generate net sales of approximately $7 to $8 billion in 2025 from $3.8 billion in 2017. Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Transl. Invest. Characterization of an unusual tropoelastin with truncated C-terminus in the frog. Tropoelastin massively associates during coacervation to form quantized protein spheres. Biopolymers 29, 16991706. Acad. Cryo-electron microscopy is a rapidly developing method and likely to contribute to structural knowledge of tropoelastin. For example, the highly flexible domains 25 that were noted to generate a twisting motion in N-terminus, were positioned beside domain 6, a relatively immobile region that may assist in stabilizing the subsequent regions. The smallest amino acid, glycine is known to promote flexibility within local structures due to lack of steric hindrance, whereas prolines restrictive sidechain inhibits conformational sampling and disrupts the formation of prolonged secondary structures (Roberts et al., 2015). doi: 10.1016/j.jmb.2020.08.023, Luan, C. H., Harris, R., Prasad, K. U., and Urry, D. W. (1990). J. Biol. Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. In addition to its involvement in molecular elasticity, the flexibility of tropoelastin also a key requirement for self-assembly into elastin, with mutations that perturb this having detrimental effects on tissue (Yeo et al., 2016, 2017). 10, 441454. Lysinonorleucine. J. Biol. J. Biol. Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly. Chem. Nature 246, 461466. Lab. Indeed, cryo-EM was recently reported to have a lower molecular size limit of approximately 50 kDa, however, it has not yet been successfully applied to characterize tropoelastin (Murata and Wolf, 2018). Delineation of unusual features in the 5-flanking region. Adv. doi: 10.1016/j.biocel.2008.03.017, Wise, S. G., Yeo, G. C., Hiob, M. A., Rnjak-Kovacina, J., Kaplan, D. L., Ng, M. K., et al. doi: 10.1016/0378-1119(94)00848-m, McLaughlin, P. J., Chen, Q., Horiguchi, M., Starcher, B. C., Stanton, J. 1862, 324334. Natl. Implications for microfibril and elastic fiber assembly. Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin. The bifunctional cross-links can undergo further condensation to form tetrafunctional desmosine or isodesmosine (Partridge, 1966). AT acknowledges support from University of Connecticuts Institute for Collaboration on Health, Intervention, and Policy Faculty Research Seed Grant. *Correspondence: Anthony S. Weiss, tony.weiss@sydney.edu.au, These authors have contributed equally to this work, Fibrous Assemblies: From Synthesis and Nanostructure Characterization to Materials Development and Application, Front. Overview of the computational and experimental methodologies that have recently contributed to our understanding of elastic fiber assembly. Tropoelastin is frequently described in the literature as an unstructured protein, mainly because models of elasticity invoke an element of disorder within the structure (4, 9, 10).While this concept appears to be the case at the fine, more subtle intramolecular level, chaos alone is unlikely to explain the ordered assembly of tropoelastin which culminates in the formation of organized . doi: 10.1016/j.matbio.2019.06.010, Reichheld, S. E., Muiznieks, L. D., Stahl, R., Simonetti, K., Sharpe, S., and Keeley, F. W. (2014). Combinations of these two amino acids result in domains that undergo rapid conformational sampling (Rauscher and Pomes, 2017), which, if perturbed toward a conformation that gravitate toward stability, will assemble into highly ordered amyloid-like fibrils that are not elastomeric (Rauscher et al., 2006; Roberts et al., 2015). Tropoelastin: a novel marker for plaque progression and instability. Elastic fibers are an important component of the extracellular matrix, providing stretch, resilience, and cell interactivity to a broad range of elastic tissues. Cell Res. J. Hum. Further to this, the current model of elastin assembly does not account for how long strings or sheets of tropoelastin are capable of forming spherules on a macroscopic scale (Tu et al., 2010). J. doi: 10.1016/j.biomaterials.2017.01.041, Tarakanova, A., Ozsvar, J., Weiss, A. S., and Buehler, M. J. doi: 10.1111/febs.14114, Lent, R. W., Smith, B., Salcedo, L. L., Faris, B., and Franzblau, C. (1969). N. Y. Acad. doi: 10.1021/acsbiomaterials.6b00564, Yeo, G. C., Keeley, F. W., and Weiss, A. S. (2011). Visconti, R. P., Barth, J. L., Keeley, F. W., and Little, C. D. (2003). Acta Histochem. Molecular docking studies utilizing tropoelastin and the 3D structure of LOX1 (Vallet et al., 2018) may assist in our understanding as to whether the enzyme has a preference for modifying particular tropoelastin residues. Biochemistry 37, 1461314622. Allergans success is powered by our global colleagues commitment to being Bold for Life. Such models could be used in conjunction with the full molecular model of tropoelastin to examine the self-assembly of discrete regions to understand the impacts of amino acid substitutions in disease. Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains. (1987). J. Biol. doi: 10.1016/j.jsb.2005.02.005, Martin, S. L., Vrhovski, B., and Weiss, A. S. (1995). The smooth muscle cell. J. Biochem. The prevention of early self-aggregation in vitro is thought to be mediated by chaperone proteins (Hinek and Rabinovitch, 1994; Miao et al., 2013). The importance of tropoelastins flexibility during self-assembly has also been examined in the light of natural allysine modifications, which condense to give cross-links within mature elastin (discussed further in the following section) (Ozsvar et al., 2019). Acad. doi: 10.1038/246461a0, Haust, M. D., More, R. H., Bencosme, S. A., and Balis, J. U. Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin. Pathol. Tropoelastin is encoded by the ELN gene, which is present in all vertebrates except jawless fish (Chung et al., 2006). Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA. The next stage of elastin assembly involves the deposition of tropoelastin spherules onto the microfibril scaffold of elastic fibers. (2005). Biol. Received: 17 December 2020; Accepted: 08 February 2021;Published: 25 February 2021. Pharm. The spherules grow in size and are deposited onto the microfibril scaffold where they fuse into fibrillar structures (Sherratt et al., 2001). (1983). 10:034102. doi: 10.1088/1748-6041/10/3/034102, Hinek, A., Keeley, F. W., and Callahan, J. A., Wachi, H., Davis, E. C., and Mecham, R. P. (2003). This reflects the experimentally observed heterogeneity of cross-links in mature elastin, again giving weight to the hypothesis that the flexibility of tropoelastin is imperative for assembly (Schrader et al., 2018; Tarakanova et al., 2019a). Biopolymers 107:e23007. Further down the molecule, the cell-interactive C-terminus was noted to be highly flexible, indicating that high conformational sampling could be conducive to tropoelastins interactions between cell-surface receptors and elastic fiber-associated proteins. Matrix 12, 156162. The company is particularly pointing to usage in the treatment of acne scars, stretch marks, aesthetic skin repair and surgical wound repair. Solving tropoelastins global structure (Baldock et al., 2011) allowed for 3D mapping on tropoelastin of the approximate locations of domains 10, 19, and 25, which were the first unequivocally cross-linked domains to be discovered (Brown-Augsburger et al., 1995). The amino acid sequence of domain 36 is unique; furthermore, its sequence confers a positively charged C-terminus comprising lysines between positively charged arginines, forming a RKRK sequence (Vrhovski et al., 1997). Upon secretion tropoelastin has the propensity to self-aggregate through a process known as coacervation. doi: 10.1016/j.matbio.2009.07.003, Mithieux, S. M., Wise, S. G., Raftery, M. J., Starcher, B., and Weiss, A. S. (2005). It has been postulated that the interaction between tropoelastin and cell surface proteoglycans is part of the assembly process of elastin before it is deposited on microfibrils. Allergan is focused on developing, manufacturing and commercializing branded pharmaceutical, device, biologic, surgical and regenerative medicine products for patients around the world. 26, 17001709. Elastagens revolutionary technology is based on recombinant human tropoelastin, the precursor of elastin which is a key component of youthful skin. Interestingly, the location and orientation of different domains that contacted each other during self-assembly was noted to be heterogeneous, resulting in head-to-head, tail-to-tail, head-to-tail, and lateral interactions (Tarakanova et al., 2019a). 3, 28322844. Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: clues to the evolutionary history of elastins. doi: 10.1021/acs.biomac.6b01759, Mackay, J. P., Muiznieks, L. D., Toonkool, P., and Weiss, A. S. (2005). Tropoelastin has 2 alternating domains: (1) hydrophilic, cross-linked, domains rich in Lys and Ala; and (2) hydrophobic domains (responsible for elasticity) rich in Val, Pro, and Gly, which often occur in repeats of VPGVG or VGGVG. Chem. Transglutaminase-mediated cross-linking of tropoelastin to fibrillin stabilises the elastin precursor prior to elastic fibre assembly. Allergan announced that it would be accessing Elastagen's next generation Juvederm dermal filler injectable portfolio, which will now also boast its "key 'youth protein' tropoelastin". Annabi, N., Zhang, Y. N., Assmann, A., Sani, E. S., Cheng, G., Lassaletta, A. D., et al. Res. Tropoelastin is a spring-like molecule that is extremely extensible prior to cross-linking.
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