Phosphoinositide 3-kinase C2α (PI3K-C2α) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2α was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2α, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2α in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2α can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2α employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting. Phosphoinositide 3-kinase C2α (PI3K-C2α) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2α was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2α, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2α in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2α can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2α employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting. It is well established that both cargo and informational movement across the plasma membrane, as well as certain aspects of intercellular signaling, involve vesicular transport initiated by endocytosis from clathrin-coated pits. The machinery of endocytosis has been extensively studied, and its complexity would seem to reflect the challenge of providing a highly efficient yet controllable interface between the environment and the cell (1Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3056) Google Scholar). In addition to the structural protein clathrin, numerous other receptor binding adaptors are present, as are proteins involved in membrane curvature, detachment, coat dissociation, and recycling; these include AP-2 and other adaptors, epsin, dynamin, synaptojanin, and others (reviewed in Refs. 2Kirchhausen T. Annu. Rev. Biochem. 2000; 69: 699-727Crossref PubMed Scopus (500) Google Scholar, 3Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar, 4Robinson M.S. Trends Cell Biol. 2004; 14: 167-174Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 5Owen D.J. Collins B.M. Evans P.R. Annu. Rev. Cell Dev. Biol. 2004; 20: 153-191Crossref PubMed Scopus (357) Google Scholar). How the functions of these factors are controlled in space and time during the lifetime of the coated pit and the initial phase of the endocytic process remains a major challenge to our understanding. It has recently become apparent that membrane lipids, and specifically phosphoinositides, play major roles in the coordination of these processes. There is considerable evidence for an important regulatory role of phosphoinositide lipids in membrane traffic between multiple intracellular compartments. In the endocytic pathway, phosphoinositides were initially demonstrated to bind with high affinity to the plasma membrane adaptor AP-2 and to modulate its properties (6Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar, 7Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4437-4441Abstract Full Text PDF PubMed Google Scholar). Subsequent studies have confirmed these observations and demonstrated the interaction of phosphoinositides with other proteins involved in clathrin-mediated trafficking (reviewed in Ref. 8Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (240) Google Scholar). The actin cytoskeleton has also been strongly implicated in the function of the endocytic machinery (9Engqvist-Goldstein A.E. Drubin D.G. Annu. Rev. Cell Dev. Biol. 2003; 19: 287-332Crossref PubMed Scopus (493) Google Scholar, 10Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Merrifield C.J. Trends Cell Biol. 2004; 14: 352-358Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and phosphoinositide regulators (12Janmey P.A. Lindberg U. Nat. Rev. Mol. Cell. Biol. 2004; 5: 658-666Crossref PubMed Scopus (184) Google Scholar, 13Wenk M.R. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8262-8269Crossref PubMed Scopus (258) Google Scholar) tightly control its assembly and disassembly. Thus there is considerable in vitro evidence for roles of both PtdIns(4,5)P2 and 3-phosphorylated inositides in membrane trafficking, and the identity of many of the targets to which they bind has now been established. To dissect the spatiotemporal aspects of these interactions in the modulation of endocytic and cytoskeletal function, it is critical to identify and study the enzymes responsible for inositide metabolism at specific sites of action. We have recently shown that a specific phosphoinositide 3-kinase, phosphoinositide 3-kinase C2α (PI3K-C2α) 3The abbreviations used are: PI3K-C2αphosphoinositide 3-kinase C2αTGNtrans-Golgi networkCBDclathrin binding domainWTwild typeGSTglutathione S-transferaseMES4-morpholineethanesulfonic acid.3The abbreviations used are: PI3K-C2αphosphoinositide 3-kinase C2αTGNtrans-Golgi networkCBDclathrin binding domainWTwild typeGSTglutathione S-transferaseMES4-morpholineethanesulfonic acid. is a component of the clathrin-mediated transport machinery at the plasma membrane and in the trans-Golgi network (TGN) (14Domin J. Gaidarov I. Smith M.E. Keen J.H. Waterfield M.D. J. Biol. Chem. 2000; 275: 11943-11950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). PI3K-C2α is a member of the class II PI 3-kinases and is distinguished from other PI 3-kinases by its additional C-terminal C2 domain (15Virbasius J.V. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 13304-13307Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 16Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar). Three distinct mammalian members of the class II PI 3-kinase family have been characterized (17Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar), PI3K-C2α, PI3K-C2β, and PI3K-C2γ. Although all share the distinguishing C-terminal C2 domain as well as the characteristic 3-kinase motifs, the enzymes differ predominantly in their N-terminal regions. The cellular functions of class II enzymes are not well understood, though there is evidence that PI3K-C2β, and to a lesser extent PI3K-C2α, can be stimulated by activation of several receptor systems including epidermal growth factor (18Arcaro A. Zvelebil M.J. Wallasch C. Ullrich A. Waterfield M.D. Domin J. Mol. Cell. Biol. 2000; 20: 3817-3830Crossref PubMed Scopus (137) Google Scholar), insulin (19Brown R.A. Domin J. Arcaro A. Waterfield M.D. Shepherd P.R. J. Biol. Chem. 1999; 274: 14529-14532Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), chemokines (20Turner S.J. Domin J. Waterfield M.D. Ward S.G. Westwick J. J. Biol. Chem. 1998; 273: 25987-25995Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), and integrins (21Paulhe F. Perret B. Chap H. Iberg N. Morand O. Racaud-Sultan C. Biochem. Biophys. Res. Commun. 2002; 297: 261-266Crossref PubMed Scopus (16) Google Scholar); the former is also implicated in cell adhesion and movement (22Maffucci T. Cooke F.T. Foster F.M. Traer C.J. Fry M.J. Falasca M. J. Cell Biol. 2005; 169: 789-799Crossref PubMed Scopus (136) Google Scholar). By analogy with the Class I PI 3-kinase family, it has been suggested that activation of class II enzymes by signaling receptors may mediate specific aspects of downstream receptor activity. In vivo evidence for this possibility has recently been reported in studies of the single Class II PI 3-kinase in Drosophila melanogaster, PI3K-68D, which demonstrated its involvement in patterning processes involving the epidermal growth factor and Notch receptor pathways (23MacDougall L.K. Gagou M.E. Leevers S.J. Hafen E. Waterfield M.D. Mol. Cell. Biol. 2004; 24: 796-808Crossref PubMed Scopus (29) Google Scholar). Interestingly, the N-terminal region of PI3K-68D more closely resembles that of mammalian PI3K-C2β, consistent with the receptor-mediated signaling role for this family member implied by the studies in mammalian cells noted above. phosphoinositide 3-kinase C2α trans-Golgi network clathrin binding domain wild type glutathione S-transferase 4-morpholineethanesulfonic acid. phosphoinositide 3-kinase C2α trans-Golgi network clathrin binding domain wild type glutathione S-transferase 4-morpholineethanesulfonic acid. In addition to localizing PI3K-C2α to endocytic and TGN clathrin-coated membranes (14Domin J. Gaidarov I. Smith M.E. Keen J.H. Waterfield M.D. J. Biol. Chem. 2000; 275: 11943-11950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), we found that PI3K-C2α is enzymatically activated by clathrin through binding to a discrete clathrin binding domain (CBD) present in the unique N-terminal region of the protein (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Exogenous expression of PI3K-C2α in cells dramatically affected both clathrin distribution and function. Although plasma membrane clathrin-coated pits could still be observed, PI3K-C2α expression induced a striking accumulation of intracellular clathrin staining consistent with the presence of numerous cytoplasmic clathrin-coated structures. Such structures, distinct from those in the TGN, are virtually undetectable in control cells (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 25Santini F. Keen J.H. J. Cell Biol. 1996; 132: 1025-1036Crossref PubMed Scopus (56) Google Scholar). Inhibition of clathrin-mediated transport from both the plasma membrane and TGN was also observed under these conditions. It should be noted that the proliferation of uniform, punctate intracellular clathrin signal under these conditions was quite distinct from the aggregates of clathrin in granules that have been observed upon expression of dominant-negative forms of other clathrin-binding proteins such as auxilin or AP180 (26Zhao X. Greener T. Al-Hasani H. Cushman S.W. Eisenberg E. Greene L.E. J. Cell Sci. 2001; 114: 353-365Crossref PubMed Google Scholar). Given the ubiquitous roles played by many different phosphoinositides in membrane trafficking, clathrin binding and activation of PI3K-C2α suggests a possible mechanism for the localized and timed formation of 3-phosphoinositides at sites of membrane budding during endocytosis or Golgi transport. This notion is supported by the observation that both PI3K-C2α overexpression (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and the putatively reciprocal knockout of synaptojanin phosphatase activity (27Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar) result in the accumulation of unusual intracellular clathrin-coated structures, together suggesting defects in the coat recycling pathway. Elucidation of PI3K-C2α function under these conditions can be expected to provide insight into the mechanisms by which phosphoinositides regulate these aspects of membrane trafficking. To provide a basis for understanding PI3K-C2α function in cells, we report here an of the roles of the and of the lipid kinase activity in PI3K-C2α on clathrin in cells. Interestingly, we found that both the and the clathrin binding of PI3K-C2α play distinct and roles in clathrin distribution and function. and PI3K-C2α in have been (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The of PI3K-C2α to noted in were into sites of with a at the (14Domin J. Gaidarov I. Smith M.E. Keen J.H. Waterfield M.D. J. Biol. Chem. 2000; 275: 11943-11950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). PI3K-C2α and were in which and were to of cell and were into sites of of PI cells with or were as (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). cells were in were with a the and by of were for PI 3-kinase activity. was with protein for in The kinase was initiated upon addition of and at for lipids were with by and and a cells with and or were in with and PI3K-C2α and cells by and and cells were in and in this with and were for as L. J. Cell. Physiol. PubMed Scopus Google Scholar). of coated membrane was by at high for cell and were expressed in and as Keen J.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cells PI3K-C2α were in with and at for in a of glutathione S-transferase or were with and binding was by with studies and cells with wild or PI3K-C2α were as (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). of for and of clathrin-coated was by of and cells in the from multiple were for in the recycling was the of Ref. 25Santini F. Keen J.H. J. Cell Biol. 1996; 132: 1025-1036Crossref PubMed Scopus (56) Google a the of the cell were for of clathrin coat structures. was cells were in with were at for in a were in and with The were with or coat assembly was as J.H. J. Cell Biol. PubMed Scopus Google Scholar). of clathrin or with of AP-2 or was clathrin were by and of were by clathrin induced by were visualized by staining as J.H. J. Cell Biol. PubMed Scopus Google Scholar). PI3K-C2α is a protein protein to the activity of the and and C2 at its C and a distinct clathrin binding domain (CBD) in the N-terminal of the protein (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Expression of PI3K-C2α in cells to in clathrin distribution and function, as proliferation of intracellular clathrin-coated and inhibition of clathrin function in endocytosis and TGN (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). To the role of the kinase activity in these we a lipid In this in the region to in the protein kinase between all of PI 3-kinases as well as other lipid and protein kinases, was to In and involved in and to protein kinase and were with The and wild type proteins were from of cells and for kinase activity. of activity was in the protein in to the wild type protein that kinase activity been by the protein was expressed in cells and to it a punctate the with in the region characteristic of the TGN, a from that of the protein and Ref. I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). with the wild type in the M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) was results suggest as the protein was Further, the results that the kinase activity of the protein is not for its targeting. we to the of on the proliferation of intracellular clathrin-coated and inhibition of clathrin-mediated endocytosis that expression of the wild type expression of the protein also resulted in proliferation of intracellular clathrin-coated structures, virtually from the of wild type cells were also in endocytosis of of multiple confirmed these The on clathrin distribution and on were observed at of wild type and that other the lipid kinase activity of PI3K-C2α are to the on clathrin distribution and function. Cell upon PI3K-C2α more the of and expression upon clathrin we established cell of these proteins in cells. with the cells that both the and the proteins are localized to punctate the and are in the they an in intracellular clathrin and a inhibition of not of the cell important in coated membrane In cells, characteristic pits were observed on the plasma membrane and as discrete In the cells, was observed in the of plasma pits not a striking in the of intracellular coated was observed these the of coated on the of of the of these coated suggests that their is more in the cells coated in the coated of the cell also a in intracellular coated membrane coated results the observations of a proliferation of intracellular clathrin-coated reported here and (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Further, they that the lipid kinase activity of the PI3K-C2α is not to the In the ultrastructural suggest the presence of on membrane of these are in discrete coated coated on the of or other coated membrane more ultrastructural studies such as of protein in cells may provide an and results in our are consistent with the presence of clathrin-coated highly I. and J. in in that the lipid kinase activity of PI3K-C2α was not to the in clathrin distribution or inhibition of to which of the protein to these several different of PI3K-C2α were and expressed in cells. we expressed the N-terminal of PI3K-C2α the This N-terminal expressed well and the punctate of cytoplasmic with characteristic of both the and protein (14Domin J. Gaidarov I. Smith M.E. Keen J.H. Waterfield M.D. J. Biol. Chem. 2000; 275: 11943-11950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). it was to formation of intracellular clathrin-coated the it with these the it was to endocytosis of at expression with those of or proteins results in the that kinase activity the structural presence of the C-terminal of the protein is to the proliferation of intracellular clathrin and inhibition of the N-terminal of PI3K-C2α the clathrin binding domain that we have identified at the N terminus of the protein (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), we our on this expression of the N-terminal of the protein the with clathrin on its distribution suggesting that the activity of this of the protein can be to the In we found that expression of this was to the formation of intracellular clathrin-coated structures, though at of expression This may reflect or of this upon expression or that there are additional the C terminus of the In the results that the of the PI3K-C2α to in clathrin distribution is at in to the of the The the of PI3K-C2α that the to the proliferation of intracellular clathrin-coated structures, we that the clathrin assembly activity that could for the formation of these through clathrin demonstrated that this region of the protein could bind to clathrin in cell (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The clathrin binding we noted in PI3K-C2α has been shown to specifically with the domain of the clathrin Keen J.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). expected from these we found that clathrin domain to was to the PI3K-C2α of from cells we clathrin with expressed under conditions in which clathrin assembly a factor to into coat J.H. J. Cell Biol. PubMed Scopus Google Scholar). assembly was by by The N-terminal PI3K-C2α supported efficient of clathrin into a in extent to assembly used as a control The of the assembly were visualized by staining the formation of clathrin in the presence of the PI3K-C2α N-terminal that PI3K-C2α clathrin domain and can clathrin It that this activity is responsible for the formation of clathrin observed upon expression of PI3K-C2α PI3K-C2α and the results to this that the is to the proliferation of intracellular clathrin and that the lipid kinase activity of PI3K-C2α is not To the activity of PI3K-C2α can independently play a role in the modulation of clathrin distribution in cells it was to the clathrin binding domain from of PI3K-C2α N-terminal in or proteins were and expressed in cells. the were the proteins were from high and the presence of clathrin was by the presence of the the to bind clathrin, as the was present in of both and of the also the presence of clathrin, providing evidence of a our and and ultrastructural results that enzymatically PI3K-C2α as well as the are to mediate the formation of intracellular clathrin-coated vesicular and the that this activity is a of the presence of the clathrin binding and assembly activity of the To the of the kinase activity to the of PI3K-C2α on clathrin, cells PI3K-C2α the were Interestingly, clathrin was in of cells the at with the protein for expression it was or in from cells the of the protein that the and activity of PI3K-C2α can independently to clathrin We and for the to modulate clathrin distribution in cells that expression of the to the proliferation of intracellular clathrin-coated vesicular structures. In the enzymatically was of clathrin distribution upon roles for the and clathrin binding of PI3K-C2α in modulation of clathrin function in cells. Further, they suggest that the on clathrin-mediated processes observed upon expression of PI3K-C2α in cells is the result of both of these The observations reported here that the lipid kinase and clathrin binding of PI3K-C2α and can to affect clathrin distribution and function. This mechanism of though by expression is in aspects of the involvement of PI3K-C2α in membrane trafficking processes. the results provide a mechanism for of PI3K-C2α to sites of clathrin coat This as a of both PI3K-C2α affinity for clathrin and of membrane binding by its and C2 X. A. G. X. J.V. Czech M.P. 2001; PubMed Scopus Google Scholar). This of involving both protein-protein and is to mechanisms that have been suggested to in other systems M.R. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8262-8269Crossref PubMed Scopus (258) Google Scholar, S. M. K. B. A. M. C. D.J. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Keen J.H. Dev. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the other the assembly activity of the of PI3K-C2α that we report here may also to clathrin lattice assembly upon PI3K-C2α important of the formation of at sites of endocytosis is the localized activation of the lipid kinase activity of PI3K-C2α (24Gaidarov I. Smith M.E. Domin J. Keen J.H. Mol. Cell. 2001; 7: 443-449Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). This may have several production of may affect the membrane many of the proteins involved in endocytosis epsin, and inositide and a for more highly M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (240) Google Scholar, 2003; PubMed Scopus Google Scholar), may to and these it be important to the of these proteins in the coated and with cell systems should to these important of the activation of PI3K-C2α in the coated pit may be the production of in membrane to the endocytic pathway. in these may be the downstream binding targets of the many and domain proteins that have been identified and may be a of the 3-phosphoinositides that the on the in of the PI3K-C2α identified here be to our understanding of these We for and of the of for with ultrastructural
No takes yet. Share an insight, caveat, or question.
Gaidarov et al. (2005) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: