Pelagiarctos thomasi from the Sharktooth Hill Bonebed of California (16.1–14.5 Ma).A number of aberrant walruses (Odobenidae) have been described from the Neogene of the North Pacific, including specialized suction-feeding and generalist fish-eating taxa. At least one of these fossil walruses has been hypothesized to have been a specialized predator of other marine mammals, the middle Miocene walrus Pelagiarctos from the middle Miocene “Topanga” Formation of southern California (17.5–15 Ma) allows a reassessment of the morphology and feeding ecology of this extinct walrus. The mandibles of this new specimen are robust with large canines, bulbous premolars with prominent paraconid, metaconid, hypoconid cusps, crenulated lingual cingula with small talonid basins, M2 present, double-rooted P3–M1, single-rooted P1 and M2, and a P2 with a bilobate root. Because this specimen lacks a fused mandibular symphysis like Pelagiarctos thomasi, it is instead referred to Pelagiarctos sp. This specimen is more informative than the fragmentary holotype of Pelagiarctos thomasi, permitting Pelagiarctos to be included within a phylogenetic analysis for the first time. Analysis of a matrix composed of 90 cranial, dental, mandibular and postcranial characters indicates that Pelagiarctos is an early diverging walrus and sister to the late Miocene walrus Imagotaria downsi. We reevaluate the evidence for a macropredatory lifestyle for Pelagiarctos, and we find no evidence of specialization towards a macrophagous diet, suggesting that Pelagiarctos was a generalist feeder with the ability to feed on large prey.A new specimen of Pelagiarctos adds to the knowledge of this problematic taxon. The phylogenetic analysis conclusively demonstrates that Pelagiarctos is an early diverging walrus. Pelagiarctos does not show morphological specializations associated with macrophagy, and was likely a generalist predator, feeding on fish, invertebrates, and the occasional warm-blooded prey item.This new specimen of  Odobenus rosmarus represents one of the most morphologically aberrant members of the Pinnipedia, and is the sole extant member of the family Odobenidae. Despite the low taxonomic diversity of walruses today, the fossil record indicates that odobenids over the past 16 Ma showed a wider variety of morphological adaptations and body sizes than at present, exploited a greater number of resources based on inferred feeding ecology, and inhabited a wider variety of marine environments including subtropical shallow marine settings The extant walrus Despite the extensive fossil record of walruses during the Neogene  share numerous derived cranial features, such as a transversely arched palate, a reduced pseudosylvian sulcus, and less than three roots on the M1The “Imagotariinae” are a group of stem walruses considered by Barnes Pelagiarctos thomasi was described on the basis of an isolated and fragmentary pair of fused mandibles and several isolated teeth from the middle Miocene Sharktooth Hill Bonebed in Kern County, California Pelagiarctos, the morphology of its postcanine teeth, robust mandible, and rarity in the Sharktooth Hill Bonebed, Barnes Pelagiarctos was a marine apex predator. Pelagiarctos was hypothesized to be adapted toward macrophagy and fed upon warm blooded prey such as marine birds and small marine mammals, “in addition to (or instead of) the expected diet of fishes”  was compared with the Pelagiarctos thomasi holotype and referred specimens described by Barnes (Ontocetus emmonsi and Kamtschatarctos sinelnikovae (represented by casts), we examined the original fossil and modern specimens of these taxa (published and unpublished). Anatomical terminology used in this study follows Mead and Fordyce The new specimen of y Barnes , in addiPelagiarctos with other walruses, a phylogenetic analysis was performed using maximum parsimony in TNT Enaliarctos emlongiPteronarctos goedertaeDesmatophoca oregonensisAllodesmus gracilisCallorhinus ursinus), and two phocid taxa, representing Monachinae  and Phocinae (Erignathus barbatus). The otariid and phocid taxa selected were chosen as they represent the earliest diverging extant lineages within their respective families and subfamilies. A complete list of specimens examined is presented within Pelagiarctos. These characters are fully illustrated on Morphobank. These characters were then included within a larger dataset employing the cranial and postcranial characters of Kohno Ontocetus, in which neither author has been able to view mandibular and cranial material referable to the same species. In this case, O. emmonsi forms the basis of our coding for mandibular and lower dental characters, while coding of other features is largely based on that of Kohno http://morphobank.org. Characters used in this analysis were based on personal observation as well as prior phylogenetic studies Proneotherium repenningi (USNM 314628), which is referable based on size, similarity with Neotherium mirum , and lack of a carnassial-like lower molar that characterizes Astoria Formation enaliarctine pinnipeds.To understand the evolutionary relationships of All parsimony analyses were run using the new technology options with 10,000 random addition replicates and an implied weighting scheme of K = 2–5. Using implied weighting allowed us to downweight characters prone to homoplasy, reducing their impact on the phylogeny Mammalia Linnaeus, 1758.Carnivora Bowdich, 1821.Odobenidae Allen, 1880.Pelagiarctos Barnes, 1988.Pelagiarctos is distinguished from the early diverging odobenids Proneotherium, Neotherium, and Kamtschatarctos by the following derived characters: rugose, vascularized bone at mandibular terminus; mandible that is dorsoventrally much deeper anteriorly, C1 with lateral sulci, inflated postcanine crowns, less prominent metaconid cusps, and medially swollen and crenulated lingual cingulum. Pelagiarctos differs from the later diverging odobenids Imagotaria and Pontolis in retaining the following primitive characters: double-rooted P2–4; metaconid cusps; sinuous ventral margin of mandible. Pelagiarctos is distinguished from all dusignathine and odobenine walruses in the retention of the following primitive characters: well developed paraconid, metaconid, and hypoconid cusps on postcanine crowns; double-rooted lower postcanines; lower canine with posterior crista; retention of M2.Neotherium sp.), a desmatophocid seal (Allodesmus sp.), an allodelphinid odontocete (cf. Zarhinocetus errabundus), and a kentriodontid dolphin (Kentriodon sp.). Although this assemblage still awaits description, all of these taxa occur within the middle Miocene (16.1–14.5 Ma) Sharktooth Hill Bonebed, suggesting a similar age of the two units Metaxytherium arctodites; more complete material of this taxon was described from the Los Indios Member of the Rosarito Beach Formation at La Mision in Baja California. The Rosarito Beach Formation also shares a number of marine mammal taxa  with the Sharktooth Hill assemblage Aepycamelus sp.), and a horse (Merychippus sp.), suggesting correlation with the Barstovian North American Land Mammal Age , while the anterior cusp (paraconid) and posterior cusp (hypoconid) morphology is unknown because of two large wear facets on the anterior and posterior edges of the crown  to possess a single-rooted morphology  the first two molar alveoli are identified as the M1 alveoli, and the third alveolus is identified as a single alveolus for the M2. The M1 alveoli are deep and separated by a well-developed bony septum, indicating widely divergent root lobes. The M2 alveolus is shallow, and along with the relatively long root exposure above the alveolar margin in SDNHM 131041, suggests that the root of this tooth may have been primarily embedded in the gums.Both M missing , 3. Thresensu2, metaconid cusps on P2–4, double-rooted postcanine teeth, and two lower incisors  from the “Topanga” Formation . Due to the small sample size, it is unclear whether the symphyseal fusion of the holotype specimen of Pelagiarctos thomasi is characteristic of that species, is variable, or represents a pathologic condition; further study of symphyseal fusion variability within Odobenus and other pinnipeds is required for further evaluation. Pelagiarctos sp. shares with all fossil and modern odobenids a mandible that is dorsoventrally deepest anteriorly, and differs from “enaliarctines” which are characterized by having a mandible deepest near the M1. Rugose and vascularized bone at the mandibular terminus distinguishes Pelagiarctos from Imagotaria and all other earlier diverging odobenids, Dusignathus santacruzensis, Aivukus, and Protodobenus.A few minor differences exist between ormation . All posOdobenus . Because. Ray in reportedPelagiarctos from “enaliarctines”, early diverging odobenids , Gomphotaria, Protodobenus, and Odobenus. Pelagiarctos differs from dusignathines and odobenines in lacking an elongate mandibular symphysis and divergent mandibular rami. Pelagiarctos lacks an edentulous mandibular terminus, which is present in the Odobenini; furthermore, a dorsal longitudinal symphyseal furrow is also absent, unlike Valenictus and Odobenus. The horizontal symphyseal portion of the mandibular ramus in Pelagiarctos differs from the condition in Ontocetus and Valenictus, where it is upturned. Pelagiarctos exhibits a straight ventral margin of the mandible, unlike the sinuous condition in Dusignathus, Pontolis, and some specimens of Imagotaria.A well-developed and knob-like genial tuberosity further distinguishes Pelagiarctos is generally plesiomorphic and close to the morphology of other early diverging odobenids  with moderate support, which in turn was sister to Otariidae; Pelagiarctos as the sister taxon to a clade comprised of Imagotaria, Pontolis, and all later diverging odobenids; and members of Dusignathus and Gomphotaria in an unresolved polytomy with a monophyletic Odobeninae.Phylogenetic analysis of the complete character set using implied weighting (K = 3) recovers one most parsimonious tree , which is shown in Enaliarctos and Pteronarctos are recovered as the earliest diverging taxa within this study, with Pteronarctos sister to crown Pinnipedia with strong support (BS = 100). However, resolution within crown Pinnipedia remains poor, although the monophyly of Desmatophocidae  and Phocidae  is recovered with strong support. Otariidae, Desmatophocidae, and Phocidae are recovered as unresolved within crown Pinnipedia.The “enaliarctines” Prototaria is recovered as the earliest diverging walrus, the sister taxon to a poorly supported  clade comprised of the remainder of Odobenidae. Odobenidae is characterized by five unequivocal synapomorphies: anterior narial opening large, thick-margined and dorsoventrally elliptical (character 2); a dorsoventrally thick and laterally broad pterygoid strut (character 13); possession of a large epitympanic recess (character 23); Bony tentorium appressed to petrosal (character 29); and a triple-rooted M1 (character 77); One equivocal synapomorphy was also suggested, presence of well developed cuspules on the P1–2 cingulum (character 71).Within Odobenidae , Proneotherium was recovered as the next diverging lineage of walrus, the sister taxa to a poorly resolved clade comprised of all later diverging walruses. This clade is supported by one unequivocal synapomorphy, a short and slender zygomatic process of the squamosal (character 19) and one equivocal synapomorphy, absence of the fossa muscularis on the orbital wall (character 14). Neotherium and Kamtschatarctos are found in an unresolved polytomy with a clade comprised of all later diverging walruses. This clade is supported by the following six synapomorphies: ascending process of maxilla along nasal short with no contact between ascending process and frontal (character 3); palatine long and posterolaterally expanded (character 11); a narrow and parallel-sided interorbital bar (character 17); a flattened and plate-like paroccipital process (character 34); presence of a talonid basin in the form of a slight concavity or shelf in the lower postcanine dentition (character 69); and a reduced metaconid (character 70). Five equivocal synapomorphies included absence of a supraorbital process (character 16); humerus with the medial lip of the distal trochlea greater in diameter than the distal capitulum (character 84); the distal end of the radius expanded with a large radial process (character 85); insertion of the pollicle extensor and metacarpal I forms a pit (character 86); and a scapholunar forms a well-formed pit for the magnum (character 87).Pseudotaria is found as the earliest diverging lineage of a clade comprised of Pelagiarctos, Imagotaria, Pontolis, Dusignathinae, and Odobeninae, with moderate to strong support . Characters that supported this topology include the following synapomorphies: a transversely arched palate (character 9); a reduced or absent pseudosylvian sulcus (character 28); and a double-rooted M1. The next diverging lineage identified as a strongly supported  clade comprised of Pelagiarctos sp. and Imagotaria downsi and all later diverging walruses. This clade is characterized by six unequivocal synapomorphies, including presence of a shallow glenoid fossa of the squamosal (character 26); a broad and pentagonal basioccipital (character 30); a large mastoid process of the squamosal (character 33); lateral lower incisors greater in size than medial incisors (character 54); bulbous postcanine crowns (character 63); and single and bilobed P3 roots (character 74).Pelagiarctos sp. and Imagotaria downsi are recovered as sister taxa with strong support (BS = 83). Unequivocal synapomorphies for this clade included lower canine roots bilobate in cross-section (character 59) and presence of rough or crenulated postcanine lingual cingulum (character 67).Within this clade, Pontolis, which is recovered with strong support  as the sister taxon to a Dusignathinae+Odobeninae clade. Unequivocal synapomorphies for this clade included termination of mandibular symphysis at the level of P2 (character 42); a sinuous ventral border of the mandible (character 45); an enlarged digastric insertion (character 46); mandibular condyle elevated above the tooth row (character 47); absence of posterior crista on C1 (character 57); presence of thin or patchy enamel (character 62); and absence of lower postcanine paraconid cusps (character 66); and single-rooted P4 roots (character 76).The next diverging lineage is 1–2 weak and bulbous (character 71); single-rooted P2 (character 72) and P3–4 (character 73); single and cylindrical P3 roots (character 74); reduced or absent P4 protocone shelf (character 75); single-rooted M1 (character 77) and M1 (character 79); and absence of M2 (character 81). Equivocal synapomorphies included a long mandibular symphysis (character 36) and a sharply divergent mandibular arch (character 44).Dusignathinae and Odobeninae are recovered as a clade, with strong support . Unequivocal synapomorphies for this clade included a very broad and short interorbital bar (character 17); presence of two upper incisors (character 51); absence of hypoconid cusps on lower premolars (character 68); cingulum on P1 (character 37). One equivocal synapomorphy is also found, a single-rooted M2 (character 80).Dusignathinae is also recovered as monophyletic, but with poor support (BS = 46). Unequivocal synapomorphies for Dusignathinae included tusk-like upper canines without globular osteodentine (character 55); procumbency of lower canines (character 60); and presence of a single-rooted M3 (character 52); absence of enamel in adults (character 62); and absence of M2 (character 80). Within Odobeninae, Aivukus is the sister taxon to a moderately supported  clade comprised of Protodobenus and Odobenini. Unequivocal synapomorphies that supported this topology included a palate that is arched transversely and longitudinally (character 9); tusk-like upper canines with globular osteodentine (character 55); C1 reduced in size and premolariform (character 56); and absence of M1 (character 78). Equivocal synapomorphies included a telescoped palatine that underlies the alisphenoid (character 11); a large and broad hamular process of the pterygoid (character 12); a sharply divergent mandibular arch (character 44); a mandibular condyle well elevated above the tooth row (character 47); root lobes of postcanines equal or narrower in width than the crowns (character 64); and absence of a talonid basin (character 69).Odobeninae is recovered as monophyletic with strong support . Unequivocal synapomorphies for Odobeninae included: a very short ascending process of the maxilla (character 3); a dorsally projected postorbital process of the jugal (character 18); a reduced squamosal fossa on the external auditory meatus (character 25); premolariform IValenictus and Odobenus are recovered as sister taxa, with moderate to strong support . Unequivocal synapomorphies for this clade included: fusion of the mandibular symphysis (character 35); genial tuberosity developed as a small tubercle or process (character 37); presence of a longitudinal furrow on the anterior portion of the mandible (character 40); loss of lower incisors (character 53); absence of P4 (character 76); and M1 (character 77); and deltoid tubercle of the humerus located off crest (character 83).Odobenini is recovered as monophyletic but poorly supported . Unequivocal synapomorphies for Odobenini included: a symphyseal region composed of rugose, vascular bone (character 38) and a mandibular terminus which possesses an edentulous pad (character 41). Equivocal synapomorphies included presence of a posteriorly positioned orbital vacuity (character 20); a funnel-shaped optic foramen and orbitosphenoid (character 21); a flattened anterodorsally projected lambdoidal crest (character 32); very large mastoid processes (character 33); presence of an upturned horizontal mandibular ramus (character 39) and presence of the deltoid tubercle of the humerus on the lateral edge of the crest (character 83). Within Odobenini, Pelagiarctos, and confirms that this taxon is a stem “imagotariine” walrus, in agreement with Barnes The phylogenetic analysis performed in this study is the first to include Imagotaria and Pelagiarctos is identified with excellent support on the basis of two synapomorphies. These synapomorphies included possession of lower canines bilobate in cross-section and presence of rough or crenulated postcanine lingual cingulum. Neither feature is found in other odobenids, although ontogenetic age and tooth wear may have impacted the coding of the latter character.A sister relationship between Pontolis as a stem walrus sister to a clade composed of Dusignathinae and Odobeninae. Identification of Pontolis as a stem “imagotariine” is consistent with phylogenetic analyses of Kohno Pontolis to be the sister taxon to Imagotaria. Characters in Kohno Pontolis, but this feature is also possessed by Dusignathus and Protodobenus. This character was originally found to unite Dusignathus and Pontolis within the Dusignathinae Imagotaria, with both character states represented in the taxon.Our analysis identified Pontolis as a stem walrus is contrary to the phylogenetic analysis presented by Deméré Pontolis as the earliest diverging lineage of a dusignathine clade. The latter study recognizes the Pontolis as a dusignathine walrus on the basis of one character, development of tusk-like lower canines, and three potential equivocal cranial synapomorphies. We agree that Pontolis possesses enlarged lower canines, but can find no other characters of the mandible or dentition that support inclusion of this genus within or as sister taxon to the Dusignathinae. Given the poor support found for Dusignathinae as well as the possibly non-monophyly of Dusignathus, further research on odobenid phylogeny is needed.The recovery of Kamtschatarctos has previously only been included within a phylogenetic analysis by Kohno Kamtschatarctos was recovered as a stem “imagotariine” walrus . Examination of this character in a phylogenetic context suggested that symphyseal fusion evolved three times  are dorsoventrally deepest posteriorly near the molars, reflecting the primitive carnivoran condition where the mandible is thickest near the large carnassials, and becomes shallower nearer the anterior dentition  instead possess a mandible that is dorsoventrally deepest anteriorly near the canines and symphysis  and an otariid (Otaria). Odobenine mandibles show further modifications from the ancestral pinniped condition, including a mandibular symphysis that is greatly elongated and widely flaring mandibular rami, the latter relating to the extreme shortening of the rostrum Ontocetus and Valenictus  postcanine crown simplification, 2) tooth root fusion, 3) tooth reduction, 4) changes in tooth placement, and 5) tusk development. The postcanine dentition of extant walruses is remarkably simple in comparison to earlier diverging taxa like id cusps ; these ake crown .Imagotaria downsi was noted to have a stage of intermediate root fusion where the anterior postcanines were single-rooted or fused (but bilobate), and the posterior postcanines retained double-roots Pontolis exhibits a similar (but slightly more derived) pattern; dusignathines and all odobenines possess single-rooted postcanine teeth 1 in early pinnipedimorphs reflects an adaptation towards increasing functional gape. Because double-rooted teeth are anteroposteriorly more elongate than single-rooted teeth, Boessenecker Callorhinus, as well as the clade composed of “Otariinae”+southern fur seals), twice within Odobenidae, once within Desmatophocidae, and three times within Phocidae .Fusion of tooth roots was a major dental transition within the Odobenidae Imagotaria, with the incipient loss of the M2; although the M2 is present in PontolisImagotaria downsi and absent in others Dusignathus seftoni, and none in Gomphotaria; incisor loss independently occurred in the common ancestor of Odobenus and Valenictus. Incisor loss in odobenines may be related to allowing an oral pathway for suction feeding Gomphotaria, it is probably related to lower canine enlargement. Further postcanine reduction occurred in odobenines with the loss of the M1 in Ontocetus and Protodobenus, and further loss of the P4 in Odobenus. Also within odobenines, the C1 is reduced and premolariform. Extreme tooth reduction characterizes the most derived walrus, Valenictus chulavistensis, which lost all teeth with the exception of the C1Reduction of teeth began with geochronologically late “imagotariines” like 3 and I3) are enlarged in many pinnipeds, and the medial incisors are often smaller and within odobenids are the first to be lost 2) in early diverging odobenids is also associated with it being positioned posteromedial to the lateral incisor, so that the canines and incisors do not form a transverse arc, but instead are medially indented; this condition is present in Pelagiarctos, Neotherium, Imagotaria, and Ontocetus, and also many otariids and phocids Numerous changes occurred regarding the placement of anterior teeth, probably as a result of upper canine enlargement. The lateral upper and lower incisors  and the development of very elongate upper tusks (Odobenini). Within Dusignathinae, the tusks of ocumbent ; Fig. 9.Pelagiarctos  The robust morphology of the mandible; 2) the perceived similarity of the postcanine teeth with scavenging carnivorans (extant hyaenids and extinct borophagine canids); 3) The inferred large body size of Pelagiarctos; and 4) the apparent rarity of Pelagiarctos within the Sharktooth Hill Bonebed.Although known only from mandibles and isolated teeth, Barnes Pelagiarctos is far larger and more robust than that of Neotherium, and the holotype mandible possesses a large and fused bony symphysis, unlike other contemporary pinnipeds. Symphyseal fusion is not present in the “Topanga” Formation specimen, although the mandible is similarly robust. Fusion and ankylosis of the mandibular symphysis, among pinnipeds, is only typically found in Pelagiarctos thomasi, Dusignathus seftoni, and the odobenines Odobenus and Valenictus; in odobenines it is hypothesized to reduce torsion during suction feeding Panthera), aardwolves (Proteles), bears (Ursidae), kinkajou (Potos) and a variety of mustelid taxa Pelagiarctos had a durophagous diet, something not observed in the new specimen or the previously described material. Given the poor correlation between diet and symphyseal fusion between caniforms, use of this character to determine the ecology of Pelagiarctos is probably premature, and cannot be used as evidence for hypercarnivory.The mandible of Pelagiarctos is similar to that of Neotherium and other earlier diverging “imagotariines”, with a prominent protoconid and less prominent metaconid and paraconid cusps , they are small relative to the size of the mandible. Pelagiarctos postcanine teeth are superficially similar to anterior premolars of borophagines and hyaenids, and although Barnes Pelagiarctos thomasi by Barnes Pelagiarctos generally lacks sharp cusps or cristae of any sort, unlike the aforementioned borophagous fissipeds. In general, the dentition of Pelagiarctos is similar to other early odobenids which have been interpreted as generalist piscivores.Overall, the morphology of ilities” :9, basedPelagiarctos may also provide useful information on its ecology. Based on length of the lower tooth row of SDNHM 131041 and linear regression equations based on measurements of modern taxa (Churchill unpublished data), we estimate that Pelagiarctos weighed ∼350 kg, about the size of an adult male South American Sea Lion (Otaria). Pelagiarctos was the first truly large odobenid, and started a trend towards ever larger body sizes within later diverging odobenids. The contemporary pinnipeds Neotherium and Allodesmus would have had weighed ∼280 and up to 1400 kg respectively, making Pelagiarctos intermediate in body size between the two taxa. Large body size has been indicated to be correlated with trophic level 2 = −0.01211; GLM analysis performed in R 2.15; Mirounga) and the lowest trophic levels  nor within the range of body size reported for the high trophic level Mirounga.The body size of Pelagiarctos within the fossil record has also been cited as a possible indicator of its ecology Pelagiarctos, preserves one of the largest and most diverse marine vertebrate assemblages of the Neogene and has been prospected for decades, remains of Pelagiarctos from this locality remain limited to six isolated teeth and a partial mandible. Given the abundance of fossil material referable to Neotherium and Allodesmus, this rarity appears to be a real phenomenon. Barnes Pelagiarctos may correlate with a fairly high trophic level, and that a significant portion of its diet may have consisted of other pinnipeds, limiting the population to a level far below that of contemporary pinnipeds. He identified the holotype as a male individual, based on the presence of large broken canines and a fairly rugose mandible; because some isolated cheek teeth fit into alveoli of the holotype mandible, Barnes Pelagiarctos possessed a partially allopatric distribution of genders, with females not ranging into the Temblor Sea. Given the limited amount of material of Pelagiarctos available for study and scarcity of studies quantifying sexual dimorphism in modern and fossil pinnipeds, evaluation of sexual dimorphism is probably premature and we question gender identification of isolated teeth. Other explanations for the rarity of Pelagiarctos within the Round Mountain Silt also exist. Many seals are prone to vagrancy Pelagiarctos within this fossil locality may indicate the existence of brief periods of unusual climate, which in turn could have led to shifts in the species composition of the local fauna and preservation of taxa not normally present along this section of coastline.The rarity of Pelagiarctos includes a pair of well preserved mandibles from the lower-middle Miocene “Topanga” Formation of Orange County, California. This fossil is more complete than the fragmentary holotype of Pelagiarctos thomasi, and confirms the referral of isolated teeth from the Sharktooth Hill Bonebed to this taxon. Because of minor differences including the lack of a fused mandibular symphysis, this new specimen is not referable to P. thomasi and is instead identified as Pelagiarctos sp. Owing to the more complete preservation of this new specimen, inclusion of Pelagiarctos within a phylogenetic analysis was possible for the first time, clearly establishing it as an early diverging odobenid, and as a sister taxon of Imagotaria. Although previously hypothesized to be a macrophagous predator, reevaluation of the evidence failed to support such an interpretation. Previously identified lines of evidence may have other explanations (rarity of Pelagiarctos within the Sharktooth Hill Bonebed), do not apply to pinnipeds (correlation of body size to trophic level), or were not verified in this study (borophagine/hyaenid like postcanine dentition with sharp cristae). The robust and fused mandibular symphysis may genuinely support the macrophagy hypothesis, but other evidence is instead suggestive of a generalist piscivore diet for Pelagiarctos.New fossil material of the enigmatic early walrus Text S1Includes a list of specimens examined during this study and a list of museum abbreviations.(DOC)Click here for additional data file.Text S2Includes a list of detailed descriptions for cladistic characters used in the phylogenetic analysis.(DOC)Click here for additional data file.


Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis.Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The  The Fox family of winged helix DNA binding transcription factors is quite large, with more than 40 known family members in mammals and 18 in flies foxA1 and foxA2 genes appear to have largely overlapping functions. The late endodermal loss of each gene alone has very little affect, whereas the simultaneous loss of both genes results in a complete failure of internal organs, such as the liver, to form foxA1 in the definitive endoderm and multiple endodermal derivatives, foxA2 is expressed earlier in the node and anterior primitive streak foxA2 results in the absence of the notochord, failure to form a gut tube and defects in derivatives from multiple germ layers The FoxA1-3 proteins, originally known as hepatic nuclear factors , are among the best-studied members of the Fox protein family C. elegans and the fruitfly D. melanogaster each encode only a single FoxA homologue, PHA-4 and Fork head (Fkh) Insight into the function and activities of the FoxA genes has also come from studies of model organisms in which redundancy is less of an issue; the worm reaper and hidPH4αSG1 and PH4αSG2, which are required to maintain uniform patent SG lumens In flies, studies have focused on the role of the FoxA protein Fork head (Fkh) in the embryonic and larval salivary gland (SG), although as with the worm PHA-4 and mammalian FoxA genes, Fkh is expressed in multiple embryonic cell types . In SGs,Drosophila Fkh have been analyzed to date, there have been no genome-wide surveys to identify the range of Fkh targets. Here, we use two approaches to obtain a more global view of Drosophila Fkh targets in both the SG and the whole embryo. Our studies demonstrate that Fkh plays a major role in SG maintenance and function. These studies also reveal unexpected roles for Fkh in endoreduplication and in blocking terminal gene expression in early embryos.Although several key SG targets of fkh loss-of-function mutant embryos. SG genes were chosen from the expression pattern database Release 2 of the Berkeley Drosophila Genome Project (BDGP [http://www.fruitfly.org/cgi-bin/ex/insitu.pl]), which is in the process of determining the embryonic expression patterns of all Drosophila genes. Whole mount in situ hybridization analysis of 190 different genes in WT embryos revealed 127 with reliable SG expression. Of the 127 genes, 59% (75 genes) had altered expression patterns in fkh mutants  genes depend on Fkh for their expression, we performed a large-scale in situ hybridization analysis of SG gene expression in WT and  mutants . Approxi mutants . Other gfkh mutants revealed that Fkh affects late gene expression far more than it does early gene expression. SG genes were grouped into three classes based on their WT expression pattern  showed no change in expression in SGs between WT and fkh mutants at stage 11; however, expression persisted much longer in fkh mutants than in WT , which encodes a bHLH-PAS transcription factor required for the development of the salivary gland ducts trh is expressed initially in both the duct and secretory gland primordia until stage 12 when Fkh downregulates its expression in the secretory cells nyobe (nyo) trh, although its expression persisted in the proximal secretory cells in WT SGs to stage 13 (nyo by Fkh: (1) like trh, nyo could be repressed by Fkh in late secretory cells or (2) nyo expression in duct cells could be activated by Trh and only indirectly repressed by Fkh. The remaining genes in the ‘upregulated later’ group are not expressed in the duct or duct primordia, suggesting that Fkh also represses late expression of a subset of early secretory cell specific genes, including other transcription factor genes. Expression of the ‘decreased and upregulated later’ group of early SG genes (two genes) was decreased or diminished at stage 11 but was also detected in SGs of fkh mutants at stage 13, when expression of these genes in WT SGs had disappeared (The ‘decreased’ group of early expressed genes (eight genes) includes those whose expression was significantly reduced or gone in the SGs of  mutants . These gan in WT  and 3C, an in WT . One of stage 13 . Two posappeared  and 3D.fkh mutants   , which eer (bai) . Given pC.elegans Fkh/FoxA homolog PHA-4 is thought to directly activate expression of most or perhaps all pharyngeal-specific genes Since the fkh leads to both decreases and increases in SG gene expression. Our analysis also identified a large number of new Fkh dependent genes, which encode proteins implicated in a wide variety of functions. Furthermore, we found that Fkh regulates the expression of far more genes at late stages than at early stages. This can be explained by a model wherein Fkh regulation of many SG genes is indirect and through its role in maintaining the expression of other SG transcription factors such as CrebA.In summary, our large scale in situ analysis reveals that Fkh plays a major role in maintaining SG fate and physiological activity. Fkh affects the majority of SG genes (59%) and loss of fkh overexpression throughout the embryo arrested SG development and disrupted germband retraction  and 1087 up-regulated genes  in fkh mutants compared to WT  and because the in situ hybridization analysis cannot identify genes that might normally be repressed in the early SG by Fkh. Thus, to identify additional ed to WT . To valifkh loss at this stage did not show significant expression changes in the microarray analysis; seven were downregulated, one was upregulated and 20 were unchanged  and one was linked to lifespan (enrichment score 3.40).DAVID analysis of the genes that were downregulated in ed genes , Table 2fkh embryos, which marked not only the proliferating CNS cells but also the endoreplicating cells of the SGs, midgut, hindgut, and MTs in WT embryos  siblings . Through the detailed analysis of expression changes, we learn that Fkh both upregulates and downregulates SG gene expression and that regulation of many SG target genes is indirect. We also show that Fkh is not sufficient to drive SG development on its own, a finding consistent with a role in maintaining but not specifying the SG fate. Our whole embryo microarray experiments comparing transcripts from early WT and fkh mutant embryos revealed two unexpected findings: (1) that Fkh represses inappropriate expression of terminal differentiation genes in early embryos and (2) that Fkh is required for endoreduplication in the SGs and in other Fkh expressing tissues.In this work, we used genome-wide approaches to identify the transcriptional targets of the fkh versus wild type SGs suggest an alternative paradigm for how FoxA proteins regulate organ-specific gene expression. There are profound differences in how the fly FoxA protein Fkh regulates SG gene expression and how the worm PHA-4 regulates pharyngeal gene expression; however, there are some similarities. Both PHA-4 and Fkh regulate many tissue-specific genes; PHA-4 regulates 100% of pharyngeal genes and Fkh affects expression of the majority of SG genes (59%). Both PHA-4 and Fkh regulate expression of other transcription factors that contribute to tissue formation and/or physiological activity. For example, both proteins activate expression of bHLH proteins that work with them to activate expression of tissue-specific downstream target genes; HLH-6 in the case of PHA-4 The findings from our in situ analysis of pha-4; similar changes in SG gene expression are observed with the loss of Scr, the hox gene that specifies the SG.Our findings also reveal that PHA-4 and Fkh play different roles in the two organs. Fkh plays a major role in maintaining and implementing the SG cell fate. This role is critically important in this organ since the factors that initiate the SG cell fate – Scr, Exd and Hth – disappear shortly after the glands begin to form Finally and importantly, many of the genes affected at late stages are likely to be only indirectly regulated by Fkh. This is certainly the case for most, if not all, of the SG transcriptional targets of the CrebA transcription factor, which mediates high-level secretory capacity by upregulating genes encoding components of secretory organelles Discovering profound differences in both the role and mode of action of FoxA proteins in these two simple models provides alternative paradigms for considering how the mammalian proteins function in the many cell types in which they are expressed and required. Already, studies suggest that mammalian FoxA proteins also function at multiple levels at various tissues. During midbrain dopaminergic (mDA) neuron development, Foxa1 and Foxa2 activate the bHLH transcription factor Neurogenin 2, which is required for cell fate specification Although a comparison between the two large-scale approaches to discovering target genes revealed that the in situ hybridization analysis is a better way to find tissue-specific Fkh targets , a zinc finger DNA binding protein that is negatively regulated by Wingless signaling and that promotes endoreduplication in larval salivary glands through activation of E2F-1 and PCNA expression pathways . The Not mutants . Thus, F6fkh, Df(3L)H99, wR23CrebA, tub-Gal4, UAS-Scr (R.Mann) and UAS-fkh. The UAS-fkh construct was generated by PCR amplification of the fkh ORF from a cDNA clone using primers that introduced an XhoI site at the 5’ end and an XbaI site at the 3’ end of the amplified product. The amplified product was subsequently cloned into XhoI/XbaI cut pUAST fkh construct was introduced into the genome by P-element mediated insertion, with injection services provided by Rainbow Transgenics.The fly strains used in this study include Oregon R, 6Df(3L)H99 fkh homozygous embryos were distinguished from their heterozygous siblings either morphologically or by the absence of lacZ hybridization, which is driven from a Ubx-lacZ insert on the TM6B balancer chromosome. For some of the early expressed genes, 6fkh homozygous embryos were isolated prior to hybridization using a COPAS Select embryo sorter (Union Biometrica). Homozygous embryos were sorted based on the lack of GFP signal driven by the Twi-GFP on TM3 balancer chromosome.Salivary gland genes for in situ analysis were chosen from the expression pattern database Release 2 of the Berkeley Drosophila Genome Project (BDGP). The corresponding cDNA clones were obtained from collections maintained in the laboratories of Allan Spradling or Phil Beachy or were purchased from the Drosophila Genome Resource Center (DGCR). Digoxygenin-labelled antisense RNA probes were generated and hybridizations were carried out as described Embryo fixation and staining were performed as described Late third instar larval salivary gland polytene chromosomes were prepared as described 6fkh mutant embryos sorted by a COPAS Select embryo sorter (Union Biometrica) as previously described Drosophila genome 2.0 chip (Affymetrix). Following scanning, intensity values were normalized . Fkh dependent genes were identified based on a 1.4-fold change in gene expression, with p-value <0.05. This fold change was selected because the change in gene expression is being averaged over the entire embryo, which includes a majority of cells that do not express Fkh. All of our microarray data are MIAME compliant and have been deposited in the GEOarchive, accession number GSE28324.Total RNA was isolated from three independent collections of stage 11 WT and three independent collections of stage 11 6Df(3L)H99 fkh embryos were labeled with BrdU following a modification of the protocol as described WT and Figure S1Volcano plot of gene expression changes between stage 11 WT and fkh mutant embryos. Genes whose expression is significantly downregulated in fkh mutants are shown with blue filled circles and genes whose expression is sifnificantly upregulated in fkh mutants are shown with red filled circles. Genes whose expression changed but with P-values greater than 0.05 are shown with open circles. Highlighted in green are Fkh-dependent SG genes identified by our in situ hybridization analysis.(TIF)Click here for additional data file.Table S1Summary of the results from the large-scale in situ hybridization analysis of SG genes in fkh mutants. Column 1 indicates the CG assignment, column 2, the gene name if one has been given, column 3, the cDNA used for the in situ analysis, column 4, if expression changed or not, column 5, how expression changed, if it did, column 5, gene function based on Gene Ontology term assignments, and column 6, whether the gene is known to be a CrebA target or not, based on the microarray analysis (XLS)Click here for additional data file.Table S2Fkh-dependent genes identified by the microarray analysis. Column 1 indicates the gene symbol or CG assignment, column 2, the gene name or CG assignment, column 3, fold-change in expression (fkh vs WT), and column 4, p-value (fkh vs WT) based on three independent samples of WT and three independent samples of fkh mutant embryos.(XLS)Click here for additional data file.


The human intestine is a large and delicately balanced organ, responsible for efficiently absorbing nutrients and selectively eliminating disease-causing pathogens. The gut architecture consists of a single layer of epithelial cells that forms a barrier against the food antigens and resident microbiota within the lumen. This barrier is augmented by a thick layer of mucus on the luminal side and an underlying lamina propria containing a resident population of immune cells. Attempted breaches of the intestinal barrier by pathogenic bacteria result in the rapid induction of a coordinated innate immune response that includes release of antimicrobial peptides, activation of pattern recognition receptors, and recruitment of various immune cells. In recent years, the role of epithelial cells in initiating this immune response has been increasingly appreciated. In particular, epithelial cells are responsible for the release of a variety of factors that attract neutrophils, the body’s trained bacterial killers. In this review we will highlight recent research that details a new understanding of how epithelial cells directionally secrete specific compounds at distinct stages of the inflammatory response in order to coordinate the immune response to intestinal microbes. In addition to their importance during the response to infection, evidence suggests that dysregulation of these pathways may contribute to pathologic inflammation during inflammatory bowel disease. Therefore, a continued understanding of the mechanisms by which epithelial cells control neutrophil migration into the intestine will have tremendous benefits in both the understanding of biological processes and the identification of potential therapeutic targets. Most microbes encountered are non-pathogenic, and the human intestine is host to trillions of harmless or beneficial bacteria. The challenge for the gut immune system is to detect and defend against pathogenic invaders, while protecting commensals and host cells from a potentially damaging inflammatory response. Neutrophils are phagocytic innate immune cells that provide a first line of defense against bacterial infection [reviewed in ]. These Leukocytes migrate to their sites of activity via a carefully regulated multistep adhesion cascade .During inflammation of the intestinal mucosa, neutrophils that migrate across the endothelium and through the extracellular matrix to the base of the epithelial layer must undergo an additional transepithelial migration step in order to reach the lumen. Epithelial cells form a tight barrier whose permeability is regulated by the apical junction complex, which consists of proteins from adjacent cells interacting to form the tight junctions and adherens junctions [reviewed in ]. CrossiIn addition to acting as the physical substrate for transepithelial migration, epithelial cells play a critical role in migration by producing chemotactic signals that recruit neutrophils out of the vasculature. CXC chemokines are the main class of chemoattractant ligands that recruit neutrophils and include CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, also known as IL-8, the prototypical neutrophil-attracting chemokine. These CXC chemokines bind to the two IL-8 receptors on the neutrophil surface, CXCR1 and CXCR2, with varying affinities and specificities . CXC che4 (LTB4) or the C5a complement fragment. It seems therefore that IL-8 is an intermediate-stage chemoattractant, whereas C5a and LTB4 are “end-stage” chemoattractants that guide neutrophils to their final destination , which then recruits neutrophils into the lumen  and LXB4. Alternatively, epithelial cells expressing 12-lipoxygenase generate 12-HETE from arachidonic acid, which is then acted on by leukocyte 5-lipoxygenase to generate LXA4 and LXB4 HPETE by 12-lipoxygenase, and 12-(S)HPETE is then converted to HXA3 by hepoxilin synthase (3 is facilitated by multidrug resistant protein 2 (MRP2), an ATP-binding cassette transporter that is localized specifically to the apical surface of IECs , are also able to trigger 12-lipoxygenase and MRP2 dependent synthesis and secretion of HXA3 to drive neutrophil transepithelial migration  are necessary for the induction of HXA3-mediated neutrophil migration, and trans-expression of the AAF subunits was sufficient to confer an inflammatory phenotype on non-pathogenic E. coli , and RvE1 binding can induce anti-inflammatory effects (3 biogenesis. Known anti-inflammatory cytokines such as IL-10 are other potentially interesting candidates. It is clear that a major area of continuing research interest lies in understanding how secretion of pro- and anti-inflammatory mediators by epithelial cells is regulated during inflammation. These studies will contribute to our further understanding of how the epithelium plays a critical role in initiating and sustaining the inflammatory immune response, with potential therapeutic implications for a wide variety of inflammatory conditions.A critical question, then, is what are the pathways that normally regulate HXAiewed in , 60]. Thry cells , 62. It  effects , 63. It The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Asthma is characterized by lung inflammation caused by complex interaction between the immune system and environmental factors such as allergens and inorganic pollutants. Recent research in this field is focused on discovering new biomarkers associated with asthma pathogenesis. This review illustrates updated research associating biomarkers of allergic asthma and their potential use in systems biology of the disease. We focus on biomolecules with altered expression, which may serve as inflammatory, diagnostic and therapeutic biomarkers of asthma discovered in human or experimental asthma model using genomic, proteomic and epigenomic approaches for gene and protein expression profiling. These include high-throughput technologies such as state of the art microarray and proteomics Mass Spectrometry (MS) platforms. Emerging concepts of molecular interactions and pathways may provide new insights in searching potential clinical biomarkers. We summarized certain pathways with significant linkage to asthma pathophysiology by analyzing the compiled biomarkers. Systems approaches with this data can identify the regulating networks, which will eventually identify the key biomarkers to be used for diagnostics and drug discovery. Asthma is a chronic immunological disorder of lung characterized by reversible airway obstruction, airway inflammation and increased airway hyperresponsiveness in response to provocative challenge. Physiological changes of the disease include the accumulation of inflammatory cells, especially the eosinophils, goblet cell metaplasia of lung epithelium with a mucus secreting phenotype , SERPINs  and CarboxypeptidaseA3. These three genes have not been studied earlier in detail however they have the potential of being used as asthma biomarkers. Chemokine ligands are potent attractants of Th2 lymphocytes at the site of lung inflammation in atopic asthma Lukacs, . SERPINSProteomic approaches are widely used to identify the expression level and modification of proteins to understand the pathophysiology of asthma. Proteomic signatures of lung parenchyma, BAL fluid, Immune cells (CD3+T cells or CD4+ T cells) from human or animal model have been used in different studies after experimental allergen challenge or after natural exposure to inhalant allergens. The advancement of proteomic techniques from earlier 2D gel based approach to recently more advanced LC-MS/MS based analysis resulted in precise identification of candidate proteins involved in asthma inflammation. In Table AMcases are human chitinases induced via Th2 specific IL13 mediated pathway in aeroallergen challenged lung epithelium and macrophages as means of host defense. Th2 inflammation in asthma can be improved by targeted neutralization of these human chitinases  form complex and inhibits macrophage activation and immunoglobulin synthesis by lymphocytes. Its homodimer also acts as a chemotactic agent for leukocytes and has pro-inflammatory activity on endothelial cell and inflammatory cells , is highly expressed in the upper airways and nasopharyngeal regions and thought to be involved in inflammatory responses to irritants in the upper airways , the chromatin structure  and by small non-coding RNAs. It was found that reduced Histone Deacetylase (HDAC) activity and increased Histone acetyl transferase (HAT) activity jointly promotes the expression of multiple inflammatory genes associated with asthma, however inhaled steroids reduce HAT activity to the normal level  of fold change, validated further by low-throughput techniques such as PCR, blotting and hold significantly close association with asthma pathophysiology.We have compiled the asthma biomarkers from different approaches including genomics, proteomics and epigenetics and have found little overlap amongst them as shown in Figure Furthermore, we have listed fifteen genes in Table The most significant pathway triggering asthma has been the adipocyte signaling pathway. A few significant genes such as ACSL3, IL13, IL9, IL4, IL2, IL10, IFNA1, SOCS1, PON1, APOB, SOCS3, SCD, and NR1D1 were found to be the component of this pathway and associated with asthma pathogenesis  remarkable overlap in symptoms of other pulmonary diseases, (ii) high interindividual and interpopulation variation at genetic level leads to changes in the uniformity of molecular marker, and (iii) absence of discriminative molecular markers, specific to atopic asthma, since most of the biomarkers currently used or in clinical trial are indicative of asthmatic inflammation irrespective of atopic background. Some of the common features of asthma exacerbation are eosinophilic inflammation, collagenitis, mucus deposition and extracellular matrix formation. However, these are common characteristics of other lung inflammations such as Chronic Obstructive Pulmonary Disease (COPD) or, non-allergic asthma. Therefore, the genes involved in these phenotypes may also be induced in all kinds of lung inflammations. To develop diagnostic markers exclusively for “allergic asthma” it is necessary to identify upstream components of the molecular pathways initiated immediately after allergen sensitization. Researchers can use these biomarkers for screening and risk assessment before the disease assumes severity by (i) identifying polymorphisms in wide population and (ii) correlating them with the alteration of signaling pathways that ultimately lead to allergic asthma. Since application of single biomarker approach to asthma research may not be realistic, newly identified biomarkers can be integrated in a multidimensional way to strengthen the treatment. Our mini review is focused on biomarker discovery by systemic approach using high-throughput “OMICS” platforms including genomics, proteomics and epigenetics and further some of them are well-studied in low-throughput experiments. Application of systems biology as a discipline provides a way to investigate the pathophysiology of asthma by giving a closer look to the system components, its dynamics and response to any kind of perturbation in the population level. Systemic approaches may emerge as a promising strategy to zoom into the global mechanism and identify features specific to asthma for developing better diagnostics and therapeutics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The increasing prevalence of allergic diseases in childhood in the last decades could be linked to concomitant dietary changes, especially with the modified and lower consumption of fruit, vegetables and minerals. The consumption of these foods by pregnant women and children in the first years of life seems to be associated with a reduced risk of asthma and related symptoms. Foods that can prevent the development of wheezing through their antioxidant effects contain vitamin C and selenium; blood levels of these elements correlate negatively with the risk of wheezing. Intake of vitamin E during pregnancy also appears to be correlated with a reduced risk of wheezing for the unborn child. Similarly, low intake of zinc and carotenoids by pregnant women is associated with an increased risk of wheezing and asthma in childhood. Fiber also has anti-inflammatory properties and protective effects against allergic diseases such as atopic dermatitis and asthma. The consumption of fat influences the development of the airways. Populations in Western countries have increased their consumption of n-6 PUFAs and, in parallel, reduced n-3 PUFAs. This has led to decreased production of PGE2, which is believed to have a protective effect against inflammation of the airways. Conflicting hypotheses also concern vitamin D; both an excess and a deficiency of vitamin D, in fact, have been associated with an increased risk of asthma. Further studies on the role of these substances are necessary before any conclusions can be drawn on a clinical level.La crescente prevalenza negli ultimi decenni delle malattie allergiche in età pediatrica potrebbe essere legata a concomitanti cambiamenti nella dieta, in particolare alla minore e modificata introduzione di frutta, verdura e minerali. Il consumo di questi alimenti da parte delle donne in gravidanza e dei bambini nei primi anni di vita sembra essere associato ad un ridotto rischio di asma e di sintomi correlati. Gli alimenti che possono prevenire lo sviluppo di respiro sibilante (wheezing) attraverso i loro effetti antiossidanti contengono vitamina C e selenio; i livelli ematici di questi elementi sono correlati negativamente con il rischio di wheezing. Inoltre l'assunzione di vitamina E durante la gravidanza sembra essere correlato con un rischio ridotto di respiro sibilante per il nascituro. Allo stesso modo, basso apporto di zinco e di carotenoidi in donne in gravidanza è associata ad un aumentato rischio di wheezing e asma nell'infanzia. Anche le fibre hanno proprietà anti-infiammatorie ed effetti protettivi contro le malattie allergiche come la dermatite atopica e l’asma. Il consumo di grassi influenza lo sviluppo delle vie aeree. Le popolazioni dei paesi occidentali hanno aumentato il loro consumo di n-6 PUFA e, parallelamente, ridotto n-3 PUFA. Ciò ha portato alla diminuzione della produzione di PGE2, che si ritiene abbia un effetto protettivo contro l'infiammazione delle vie aeree. Ipotesi contrastanti riguardano la vitamina D, sia un eccesso che una carenza di vitamina D, infatti, sono stati associati ad un aumentato rischio di asma. Ulteriori studi sul ruolo di queste sostanze sono necessari prima di trarre conclusioni sul piano clinico. The prevalence of allergic diseases in Western countries has shown a growing trend since the early 60s, with a growth rate ranging from 25% to 75% per decade -4, when The modern diets of people living in industrialized countries have changed in terms of nutrient composition and food quality. Studies show that in Great Britain between 1967 and 2005 there was a decrease in the consumption of fruit, vegetables and saturated fats (butter), and an increase in the consumption of fruit juices, low-fat spreads and vegetable oils . The flaRecently, the interest of researchers has focused on the nutrition of pregnant women and children in the earliest years of life, on the assumption that diet may play a central role in the development of the airways and the immune system, possibly already in the womb, through epigenetic mechanisms -13. IndeIn this review we analyze various micronutrients and their possible association with atopic diseases.One of the most recent developments has been the recognition of the epigenetic effects of dietary folate on the development of asthma. Animal models demonstrated that folate supplementation during pregnancy induced hypermethylation (suppression) of regulatory genes in lung tissue, leading to the subsequent development of allergic airway disease . In humaThe observation that the increased prevalence of asthma has risen along with lower intake of antioxidants in the diet led Seaton et al. in 1994 to assume that the population had become more susceptible to asthma due to reduced antioxidant intake . The proAntioxidants may also affect the first interaction between antigens and the immune system . VitaminIn 2001 Shaheen et al.  demonstrSeveral observational studies have linked antioxidants with asthma and have investigated the possible effects of supplements on the treatment of asthma. However, the evidence that emerges from these studies is weak, due to possible bias and limitations, because cross-sectional and case–control studies predominate . The limRegarding vitamin C, a Cochrane review concluded that there is not enough evidence to recommend supplements in the treatment of asthma . A more With regard to selenium, it was found that in Europe selenium intake with foods and consequently its blood levels has dropped; the cause is likely due to the increased consumption of European wheat, which is less rich in selenium compared to that of North America, as well as to changes in the technology of bread making .Devereux, in a birth cohort study of 1924 mothers and their children, reported that high levels of selenium in maternal plasma at 12 weeks of gestation and in the newborns' umbilical cord blood were associated with a reduced risk of wheezing at 2 years of age, but not with asthma at 5 years . The EngAlthough zinc is not properly considered an antioxidant, Litonjua and Devereux in their cohort studies reported that low levels of zinc in the diet of women during pregnancy were associated with an increased likelihood that their children would develop wheezing and asthma in childhood ,49.Dietary fiber and oligosaccharides (acting as prebiotics) are important in promoting gut colonization. These non-digestible dietary components ferment to produce short-chain fatty acids (SCFAs) with anti-inflammatory properties . Modern In 1997 Black and Shape observed that in Western countries the intake of saturated fats (butter and lard) had dropped in recent decades in favor of polyunsaturated fatty acids omega 6 (n-6 PUFA) contained in vegetable oils  and margarine. This is probably a consequence of a coronary heart disease campaign by public health authorities . At the Several studies have assessed the effects of omega-6 and omega-3 in the diet on outcomes of atopic diseases. Some studies have focused on concentrations of polyunsaturated fatty acids in the breast milk of atopic mothers versus non-atopic mothers. These studies showed lower concentrations of AA in the breast milk of atopic mothers compared with healthy controls -63, althWijga et al  reportedin vivo production of TNF-α by mononuclear cells [Even if other factors, such as cytokines may be involved -76, dietar cells . Anotherar cells . Other iar cells , 616 chiar cells . It coular cells , in a raar cells . Concernar cells .There are two conflicting hypotheses linking vitamin D to an increasing incidence of asthma and allergic diseases, according to the so-called "paradox of vitamin D". Both an excess (resulting from supplementation) and a deficiency (due to low solar exposure and the inability to compensate with diet) of vitamin D have been associated with an increased risk of asthma and allergies in Western countries . Three bFurther studies are necessary to clarify the role of vitamin D in the induction of immune tolerance and the subsequent risk of developing allergic diseases.From an epidemiological point of view allergic diseases and asthma have been strongly associated with modification of nutrients in the diet , a reduction of n-3 PUFAs (polyunsaturated fatty acids) and an increase in n-6 PUFAs, and vitamin D supplementation or deficiency. However, there are several observational studies whose results are often contradictory and non-conclusive. Data about the effects of supplementation with these nutrients during pregnancy or early life are particularly lacking. Therefore, we suggest that further studies on the role of these substances are necessary before any conclusions can be drawn on a clinical level.PUFA, Polyunsaturated Fatty Acids; PGE-2, Prostaglandin E-2; AA, Arachidonic Acid; FFQ, Food Frequency Questionnaire; ALPSAC, Avon Longitudinal Study of Parents And Children; SCFAs, Short-Chain Fatty Acids; DGLA, Di-Homo-Gamma-Linolenic Acid; ALA, Alpha-Linolenic Acid; EPA, Eicosapentaenoic Acid; DHA, Docosahexaenoic Acid.The authors declare that they have no competing interests.DG Peroni and AL Boner designed the review and revised with GL Piacentini the final version of the manuscript. B Bonomo and S Casarotto generated the draft version of the paper. All authors approved the final version of the paper.


In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome. Alternative splicing is a fundamental molecular process in eukaryotes, where it not only greatly increases the diversity of proteins that the genome can encode According to their protein-coding capacities, the gene isoforms could be divided into two distinct classes: messenger RNAs (mRNAs) which are translated into proteins, and the noncoding RNAs (ncRNAs), which function at the RNA level. Previous studies have indicated that most of the human genome is likely to be transcribed, generating a complex network of diverse transcripts that includes a large amount of ncRNAs It is worth noticing that multi-exon genes could encode different isoforms through alternative splicing and different isoforms have different protein-coding potential. In addition, gene expression usually exhibit temporal and spatial specificity. Therefore, the same gene could generate different isoforms, even encoding both protein-coding and noncoding isoforms in different conditions or stages. RNA-Seq provides tremendous opportunities to reveal these diversities and the peculiar specificities of the human transcriptome To more comprehensively illustrate the complexity of human transcriptome, we investigated the diverse expression features of human known genes at both gene level and isoform level based on two RNA-Seq datasets generated from human brain tissues and 10 mixed cell lines. We first inferred the isoforms encoded in brain and cell lines, and then assessed the protein-coding capacities of all those expressed isoforms. Next, we compared the gene expression profile between brain and cell lines, and revealed that there are great differences in expression for those expressed genes between gene level and isoform level, implying that only the isoform level rather than gene level is able to accurately reflect the expression profiles of human genes. We also identified hundreds of long ncRNAs in brain or cell lines , and found a significant number of human genes in brain and cell lines could encode both protein-coding and noncoding RNAs through alternative splicing. Our results show that most long ncRNAs from brain and cell lines contain conserved elements across 46 vertebrates, or 33 placental mammalians, or 10 primates. Through RT-PCR we further validated that several long ncRNAs were differentially expressed in breast cancer cells versus normal breast cells, or between lung cancer cells and normal lung cells. In addition, those differentially expressed long ncRNAs are significantly correlated with a number of breast cancer or lung cancer related genes.Our study used two transcriptome sequencing datasets from two reference RNA samples established by the MicroArray Quality Control (MAQC) project Read Per Kilobase of transcript per Million mapped reads) as threshold, and obtained a total of 28,571 transcripts in brain from 16,818 UCSC annotated genes and 32,714 transcripts in cell lines from 18,431 UCSC annotated genes (We first mapped the brain and cell line reads onto the human reference genome (hg19) and junction sequences using bowtie ed genes  and S2. ed genes . Among tWe then investigated the number of exons contained in each transcripts and the number of isoforms encoded by each gene in brain and cell lines. About 93.88% transcripts in brain and 94.73% transcripts in cell lines contained two or more exons . AccordiIn brain and cell lines, 11,654 expressed genes are in common, while 5,164 (30.71%) expressed genes only detected in brain and 6,777 (36.77%) expressed genes only detected in cell lines according to our threshold . InteresTo further explore the different expression profiles between brain and cell lines, we compared the composition of all their expressed isoforms, and surprisingly found that only 7,492 isoforms  are the same between brain and cell lines, 21,079 (73.78%) isoforms  are expressed only in brain, 25,222 (77.1%) isoforms  are expressed only in cell lines . About tPrevious evidences have indicated that some genes can encode bifunctional RNAs, with their certain splicing isoforms acting as regulatory noncoding RNAs, and other specific splicing isoforms being translated into a protein, such as LXRB/LXRBSV isoform pair To better characterize the expression features of those long ncRNAs which transcribed from the 146 brain and 185 cell line genes that encode bifunctional RNAs, we further compared the exon structures of those long ncRNAs with their gene annotations on the reference genome. We found that 13 brain long ncRNAs contain introns, 79 brain long ncRNAs with new alternative exon boundaries or new exons and 84 brain long ncRNAs were produced by the exon skipping mechanism; 17 cell line long ncRNAs contain introns, 90 cell line long ncRNAs with new alternative exon boundaries or new exons and 110 cell lines long ncRNAs belong to the alternative splicing events of exon skipping. Those long ncRNAs which contain introns or with new alternative exon boundaries or new exons might result from the intron retention or alternative splice site usage or the incomplete annotation of their genes on the reference genome To gain insights into the evolutionary conservation of those long ncRNAs identified in brain and cell lines, we investigated the base-by-base phastCons scores see  and searWe further explored the expression profiles of long ncRNAs in human cancers. We first aligned the probes of Human Genome U133 Plus 2.0 Array from Affymetrix to the brain and cell line long ncRNAs to find whether any probe can represent the expression of our identified long ncRNAs. Using the exact and unique match criteria, 56 probes with no corresponding gene symbols could be aligned to 56 brain long ncRNAs and 59 probes for cell line long ncRNAs. Then we detected the differential expression of those probes in two human Gene Expression Omnibus (GEO) datasets, one is about breast cancer We then chose those five brain long ncRNAs  which were differentially expressed in both breast cancer and lung cancer as the candidates to validate our results with RT-PCR. All but one long ncRNAs were obviously differentially expressed in breast cancer cells versus normal breast cells , two long ncRNAs (one was up-regulation and the other one was down-regulation) were apparently differentially expressed between lung cancer cells and normal lung cells . These rIn this study, we investigated the diverse expression profiles of present human known genes based on two RNA-Seq datasets from human brain tissues and a variety of cell lines. We found that under our selection criteria, the majority of (more than 60%) the expressed genes in both brain and cell lines generated only one isoform with relative higher level that can be detected, whereas a portion of the expressed genes could encoded more than five isoforms. In those expressed genes of brain and cell lines, a number of them could encode both protein-coding and noncoding RNAs through alternative splicing, implicating the intricacy of gene transcription and post-transcriptional RNA processing. We also found that although most of the expressed genes in human brain and cell lines are the same, but their expressions are significantly different at the isoform level, and only less than 30% of the isoforms are identical between brain and cell lines. Besides that, some expressed genes only generated protein-coding or noncoding transcripts in one sample but not in another. Our results reveal that the significant difference for gene expression profiles between brain and cell lines is not only relevant to the sequence compositions of their isoforms, but also associated with the protein-coding capacities of their isoforms. Furthermore, when taking the expression level of each isoform into consideration, the variations for gene expression will inevitably increase more dramatically. Accordingly, only based on the isoform level, can we accurately recognize the gene expression pattern and estimate the gene expression profiles in various biological conditions and different tissues, such a fine scale will definitely improve our ability to gain new important insights into the functionalities for each gene on the human genome.Therefore, to comprehensively explore the differences between different tissues, we need to consider isoforms the genes generated in addition to identify the expressed genes. Due to the complex alternative splicing in mammalians, the actual expression level of multi-exon genes is not only determined by the number of distinct isoforms they encoded, but also related to expression abundance of each isoform. Thus, the expression level of a gene might not be present differential expression in two samples, but it does not mean that this gene encodes the same isofoms in both samples. Because multi-exon genes could encode multiple different isoforms through alternative splicing and different isoforms would result in the same expression level of the gene between the two samples. Furthermore, the multi-exon genes would even encode both protein-coding and noncoding isoforms. Different isofroms of the same gene could play different roles with the temporal and spatial specificity We also found that the majority of brain and cell line long ncRNAs contain the regions similar to the phastCons-predicted conserved elements across 46 vertebrates, or their 33 placental mammal subset, or their 10 primate subset. Consequently, although the entire transcripts of most long ncRNAs lack strong conservation, yet highly conserved elements are preserved in them. In addition, some conserved element fragments of vertebrates, placental mammals and primates in the long ncRNAs are not similar to each other, suggesting that those conserved element fragments provide the structural base for the corresponding long ncRNAs to play special roles in vertebrates, placental mammals or primates. We also observed that some long ncRNAs are differentially expressed between breast cancer cells and normal breast cells, or between lung cancer cells and normal lung cells, and those differentially expressed long ncRNAs are significantly correlated with several identified breast cancer or lung cancer related genes. The results indicate that those differentially expressed long ncRNAs may directly or indirectly influence the expression of the cancer related genes. Our findings further imply that long ncRNAs could be involved in human cancers and play important roles in the pathogenesis of human diseases. In conclusion, these results indicate that long ncRNAs are not transcriptional “noise”, but might be functional macromolecules and they might have special relationship with cancer related genes.Ab initio reconstruction of the human transcriptome is crucial for the identification of those long ncRNAs which transcribed from the intergenic regions, With the improvements of sequencing technologies and the algorithms for ab initio reconstruction of the transcriptome, it will enable us more comprehensively and more thoroughly to comprehend the human transcriptome.Our study suggests that to comprehensively investigate the overall expression profiles of human genes, it is important to consider the following aspects: the number of distinct isoforms encoded by each gene; the protein-coding capability of each isoform; the composition of the isoforms generated by alternative splicing and the expression levels of those isoforms and etc. Although our comparison is based on human brain and 10 mixed cell lines, it is anticipated that the results could also be applied to the comparison among different tissues or cell lines. Current annotated genes on the human reference genome are mainly about protein-coding genes and known stable noncoding RNAs, such as tRNAs, snRNAs and snoRNAs, only a few of those annotated genes could encode long noncoding RNAs . However, previous transcriptomics studies have indicated that a huge number of transcripts in mammals do not seem to encode proteins but function as noncoding RNAs instead Two transcriptome sequencing datasets were generated from two reference RNA samples established by the US FDA-led MicroArray Quality Control (MAQC) project http://www.ncbi.nlm.nih.gov/geo) under accession number GSE30222.These two samples were prepared using the standard Illumina mRNA-Seq protocol and reagents. The sequencing data were single end reads, with ∼59.46 million and ∼53.24 million of sequencing reads in 35 bp length for cell line and brain samples, respectively. Those two datasets from this study have been submitted to the NCBI Gene Expression Omnibus (http://genome.ucsc.edu/) and junction sequences using bowtie The brain and cell line reads were first aligned onto the human reference genome hg19 . The phastCons scores can be interpreted as probabilities that each base is in a conserved element, given the assumptions of the model and the maximum-likelihood parameter estimates We separately downloaded the base-by-base phastCons To investigate the disease association between long ncRNAs and human cancers, we first aligned the probes of Human Genome U133 Plus 2.0 Array onto the brain long ncRNAs to find the probes that can represent the expression of our identified long ncRNAs using the exact and unique match criteria. Then we detected the differential expression of those probes in two human Gene Expression Omnibus (GEO) datasets: GSE10810 which was about breast cancer Document S1This file records all the related supporting information mentioned in the main text.(DOC)Click here for additional data file.Table S1Significantly correlated genes of the long ncRNAs that differentially expressed in breast cancer.(XLS)Click here for additional data file.Table S2Significantly correlated genes of the long ncRNAs that differentially expressed in lung cancer.(XLS)Click here for additional data file.File S1Transcript expression values, coordinates and protein-coding capacities in brain.(TXT)Click here for additional data file.File S2Transcript expression values, coordinates and protein-coding capacities in cell lines.(TXT)Click here for additional data file.


Osteogenesis imperfecta (OI) is an inherited connective tissue disorder characterized by bone fragility, multiple fractures and significant chest wall deformities. Cardiopulmonary insufficiency is the leading cause of death in these patients.Seven patients with severe OI type III, 15 with moderate OI type IV and 26 healthy subjects were studied. In addition to standard spirometry, rib cage geometry, breathing pattern and regional chest wall volume changes at rest in seated and supine position were assessed by opto-electronic plethysmography to investigate if structural modifications of the rib cage in OI have consequences on ventilatory pattern. One-way or two-way analysis of variance was performed to compare the results between the three groups and the two postures.1 compared to predicted values, on condition that updated reference equations are considered. In both positions, ventilation was lower in OI patients than control because of lower tidal volume (p<0.01). In contrast to OI type IV patients, whose chest wall geometry and function was normal, OI type III patients were characterized by reduced (p<0.01) angle at the sternum (pectus carinatum), paradoxical inspiratory inward motion of the pulmonary rib cage, significant thoraco-abdominal asynchronies and rib cage distortions in supine position (p<0.001).Both OI type III and IV patients showed reduced FVC and FEVIn conclusion, the restrictive respiratory pattern of Osteogenesis Imperfecta is closely related to the severity of the disease and to the sternal deformities. Pectus carinatum characterizes OI type III patients and alters respiratory muscles coordination, leading to chest wall and rib cage distortions and an inefficient ventilator pattern. OI type IV is characterized by lower alterations in the respiratory function. These findings suggest that functional assessment and treatment of OI should be differentiated in these two forms of the disease. Osteogenesis imperfecta (OI) is a genetically heterogeneous group of congenital disorders of collagen synthesis characterized by brittle bones leading to different levels of skeletal deformities and frequent multiple fractures.OI exhibits a broad range of clinical severity, ranging from multiple fracturing in utero to normal adult stature and a low fracture incidence Life expectancy in OI type IV is similar to general population, while in type III is reduced Although it is reasonable to postulate that structural alterations of the chest wall, namely spinal and rib cage deformities, may contribute to cardiopulmonary problems in severe OI, few authors reported data on deformities and pulmonary function The hypothesis of the present paper is that structural modifications of the rib cage due to OI have important consequences on ventilation at rest in terms of chest wall function. To test this hypothesis we noninvasively measure kinematics and volume changes of chest wall in OI patients in two different positions in order to show possible differences between the moderate (type IV) and the most severe (type III) forms and to find possible correlations between chest wall deformities, pulmonary function and ventilatory pattern.The research protocol was approved by the local ethics committee of the Scientific Institute E.Medea, Bosisio Parini, Lc, Italy  and written informed consent was obtained from all patients (or parent). We obtained informed consent from the next of kin on the behalf of the minors/children participants involved in the study.Twenty-two patients affected with osteogenesis imperfecta were recruited for this study. The diagnosis of OI was made on the basis of the following clinical and radiological findings: susceptibility to frequent fractures from the mildest trauma, varying degrees of short stature, progressive skeletal deformities, blue sclerae, dentinogenesis imperfect, joint laxity, early loss of hearing, genetic and/or biochemical analysis with heterozygosity of dominant mutations in one of the two genes (COL1A1 and COL1A2) encoding the chains of type I collagen, with quantitative (mild or moderate forms) or qualitative  defect of the synthesis of type I collagen Seven patients were classified as type III, the most severe form of OI and 15 patients as type IV.Twenty six healthy subjects were recruited as control group.Two type III patients were under nocturnal non-invasive ventilation; 7 patients  were not ambulant; one type IV patient showed basilar impression; one type IV patient had costal fractures secondary of a car crash occurred three months before the study.1), subdivision of lung volumes  by the nitrogen washout technique were performed . Spirometric and lung volumes predicted values were computed using gender, age and height, according to the most commonly used equations of Quanjer et al Measurements of forced vital capacity (FVC), forced expiratory volume in one second (FEV2) was measured using a digital pulse oximeter . Pulse oxymeter was positioned by specialized personnel and only recordings longer than 8 h were considered as acceptable, otherwise measurements were repeated during the following night.Nocturnal oxygen saturation  Chest wall was divided into three compartments: pulmonary rib cage (RCp), under the action of rib cage muscles, abdominal rib cage (RCa), under the insertional action of diaphragm and abdomen (AB), under the action of the diaphragm and the expiratory abdominal muscles.CW) and its compartments  were noninvasively measured by Opto-Electronic Plethysmography. The volume of the entire chest wall  between volume variations of VRC,P and VAB waveforms. ΦTA was derived from the ratio of the distance m delimited by the intercepts of VRC,P (y-axis)-VAB (x-axis) loop on a line parallel to the x-axis at 50% of ΔVRC,P divided by ΔVAB (s) and computed as ΦTA = sin−1(m/s) RC,P and VRC,A waveforms (ΦRC). By convention a positive angle means that RCp expansion is leading on RCa (or AB) expansion; on the contrary negative angles describe the reverse situation.Dynamic thoraco-abdominal asynchronies were quantified by determining the phase shift angle .Labored Breathing Index (LBI) Radiographic measurements included standard standing antero-posterior and lateral views of the entire spine. Thoracic scoliosis was measured using the Cobb method. If two curves were present, the largest thoracic curve was used.Anthropometric, spirometric and lung function data of the three groups were compared using a one-way Analysis of Variance with disease as independent factor. To compare ventilatory pattern data between the groups in the two postures, a two-way Analysis of Variance was performed with posture and disease as independent factors. Post-hoc tests were based on Holm-Sidak method. Data in tables are expressed as mean±standard deviation, in figures as mean±standard error. Significance was determined by p<0.05.Anthropometric characteristics of OI type III, OI type IV and controls are reported in All patients were characterized by severe or moderate scoliosis (Cobb Angle>20°). Average Cobb angle values were >40° in both OI types and no differences were observed.Spirometric parameters, lung volumes and nocturnal saturation data are shown in Spirometry and lung volume data were not available on patients younger than 9 years (2 type III and 6 type IV) due to their limited collaboration.1 values  within the normal range, with data of OI type III unexpectedly higher than predicted. Using more recent equations of Kuster et al 1 (%pred) became significantly lower in OI patients, particularly in OI type III. No differences were found using the two different predicted equations for TLC, RV and FRC. Finally, although OI type III patients showed a more pronounced tendency to decrease oxygen saturation (SpO2) during the night compared to OI type IV, this was not significant.According to the most commonly used equations of Quanjer et al T), are summarized in T and breathing frequency components were different, being lower the former and higher the latter in OI patients. No differences were found between type III and type IV. In the seated position minute ventilation was lower in OI patients than controls due to lower VT. Only in OI type III patients, breathing frequency was higher than controls.Minute ventilation and its two components, breathing frequency and tidal volume  and lower compared to OI type IV and controls in supine. In OI type IV patients VRC,P contribution was lower than controls only in seated position. Similarly, the VRC,A contribution to VT was reduced in OI type III patients compared to both OI type IV and controls in both postures. Hence, VAB contribution to VT was higher in patients with OI type III than the other two groups both in supine seated position.Patients with OI type III were characterized by reduced contributions of rib cage compartments to VTA was lower in OI type III patients than OI type IV and controls, both in supine and seated position. ΦRC was lower in OI type III patients than OI type IV and controls, but only in supine position. Accordingly, the LBI was higher in OI type III patients than OI type IV and controls, in both positions. In OI type III patients only, ΦTA, ΦRC and LBI were significantly different between seated and supine position.Patients with OI type III were characterized by high levels of dynamic thoraco-abdominal asynchronies as shown in It is known that patients with osteogenesis imperfecta are characterized by bone fragility, frequent fractures, bowing of the long bones, growth disproportionately distributed affecting predominantly trunk height more than the other trunk dimensions, collapsing of thoracic vertebrae resulting in a more horizontal position of the ribs and sternal deformities The present study provides for the first time a detailed description of respiratory function in the two non-lethal more severe types of OI. An important original aspect of the present study is that, in addition to standard spirometry, we used the opto-electronic technique to noninvasively assess chest wall geometry, rib cage deformities and thoraco-abdominal kinematics in two different postures.RC,P to tidal volume was still significantly lower than controls. These results suggest that in OI type III patients the deformed rib cage  alters the normal action of the intercostal muscles and requires the diaphragm to compensate for their reduced contribution to tidal volume. An additional indication that in these patients diaphragm function seems to be preserved is that they do not show significant nocturnal oxygen desaturation.Several anthropometric parameters are significantly different between OI patients and controls. Only type III patients, however, are characterized by significantly lower angles at the xiphosternal level, both in the sagittal and in the transversal plane. We believe that this altered geometry characterizing pectus carinatum has important consequences in terms of volume variations of the chest wall compartments during breathing. In fact, in six out of seven patients with OI type III, paradoxical inward movement of the pulmonary rib cage was detected during spontaneous breathing at rest in supine position. In seated position, no paradoxical motion was present, however, the percentage contribution of VRC,P to tidal volume (TA) and rib cage (ΦRC) asynchronies (It can be hypothesized that the reduced angle between the ribs and the sternum modifies the action of the intercostal muscles and alters the “bucket-handle" motion of the ribs in relation to their fulcrum, the sternum l volume , but alschronies  were sigThe rib cage asynchronies and the paradoxical inward motion of the pulmonary rib cage observed in association with the expansion of the abdominal rib cage in OI type III in the supine position also suggest that in these patients rib cage distortions are significant and therefore a substantial fraction of the force developed by the diaphragm on the rib cage would go into distorting it, and only a small fraction into changing chest wall and lung volume. This is an inefficient way to breathe An implication of these findings is that rib cage deformities, rather than scoliosis which was present in both OI types, is the determinant of the altered breathing pattern in type III. The correlation between spirometric abnormalities and severe scoliosis in OI has been previously reported Another original aspect of the present study is the evaluation of the ventilatory pattern. In general, patients with severe osteogenesis imperfecta breathe with rapid and shallow breathing in both positions. The reduced ability of the rib cage compartment to expand is probably the cause of the reduced tidal volume and the concomitant increased breathing frequency could represent the compensatory mechanism to try to guarantee normal minute ventilation. However, this mechanism is not fully effective in the seated position, as shown by significantly lower minute ventilation in OI patients.The relatively low number of patients could represent a limitation of this work, even if these patients behave very homogeneously. Despite these limitations, we believe that the findings of the present study might have important implications for the functional evaluation and the treatment of OI patients in clinics.1 higher than normal. Similarly, Takken et al 1 values even in children with OI type I, the mildest form of the disease. These high values of FVC and FEV1 are probably artifactual, at least in OI type III, because it is known that cardio-pulmonary insufficiency and respiratory infections represent the leading cause of death in OI. In contrast, the equations proposed by Kuster et al 1, expressed as percentage of predicted values, significantly lower than controls.As recently pointed out by Stanojevic et al In conclusion, in patients with severe OI a direct relationship exists between the structural modifications of the rib cage and the pattern of volume variations during breathing. Patients with OI type III are characterized by pectus carinatum and inspiratory paradoxical inward motion of the pulmonary rib cage during quiet spontaneous breathing in supine position, associated to a high level of asynchrony between the three chest wall compartments. On the other hand, pectus carinatum, paradoxical motion and significant asynchronies between chest wall compartments are not features of type IV patients. These findings suggest that functional assessment and treatment of OI should be differentiated in these two forms of the disease.