Orientalosuchus naduongensis gen. et sp. nov. The combined presence of an enlarged fifth maxillary tooth, prominent preorbital ridges, a large supraoccipital exposure on the skull table, a palatine-pterygoid suture anterior to the posterior end of the suborbital fenestra, and a pterygoid forming a neck surrounding the choana is unique to this species. Unlike previous phylogenies, our parsimony analysis recovers a monophyletic Late Cretaceous to Paleogene East to Southeastern Asian alligatoroid group, here named Orientalosuchina. The group includes Orientalosuchus naduongensis, Krabisuchus siamogallicus, Eoalligator chunyii, Jiangxisuchus nankangensis and Protoalligator huiningensis, all of them sharing a medial shifted quadrate foramen aerum. The recognition of this clade indicates at least two separate dispersal events from North America to Asia: one during the Late Cretaceous by Orientalosuchina and one by the ancestor of Alligator sinensis during the Paleogene or Neogene, the timing of which is poorly constrained.During systematic paleontological surveys in the Na Duong Basin in North Vietnam between 2009 and 2012, well-preserved fossilized cranial and postcranial remains belonging to at least 29 individuals of a middle to late Eocene (late Bartonian to Priabonian age (39–35 Ma)) alligatoroid were collected. Comparative anatomical study of the material warrants the diagnosis of a new taxon,  Alligator spp. and Central to Middle American caimans, as well as many fossil taxa  .In this study, we provide a complete description of this material, which represents the best preserved alligtatorid from the Paleogene of Asia. We demonstrate that Cretaceous-Eocene East to Southeastern Asian alligatoroids form a monophyletic group, here named Orientalosuchina and that Asia was colonized by alligatoroids at least two times independently.The Na Duong Basin is located in northern Vietnam near the border with China . It reprAsiatosuchus-like crocodiles, as well as many fish taxa and two different turtle species occurred sympatrically with the herein described alligatoroid  and, therefore, the heuristic search was repeated until the MPTs were found 50 times during each replicate . The trees retained in the memory were exposed to a second round of tree-bisection-reconnection.http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:08B6F167-AAC7-4184-97BA-B7467D4F036B. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix EusuchiaBrochu, 2003CrocodyliaBenton & Clark, 1988AlligatoroideaBrochu, 2003GlobidontaBrochu, 1999AlligatoridaeBrochu, 2003Orientalosuchina new clade nameOrientalosuchus gen. nov.Orientalosuchus naduongensis sp. nov.Orientalosuchus refers to the Latin word “oriens” for “east” and “suchus” the old Greek word “soukhos” for “crocodile.”Etymology: The name naduongensis” refers to the Na Duong coal mine type locality in northeastern Vietnam.Etymology: The species name “Orientalosuchus naduongensis is diagnosed by the combination of the following characters: notch between the premaxilla and maxilla; dominant maxillary ridge alongside the nasal; the fifth maxillary tooth is the largest maxillary tooth; anterior tip of frontal is acute and projects between the nasal bones; small supratemporal fenestra; large supraoccipital exposure preventing the parietal from reaching the posterior skull table in adults; quadrate foramen aerum lies on the dorsomedial angle of the quadrate; large suborbital fenestrae reaching anteriorly the level of the seventh to eighth maxillary tooth; maxilla-palatine suture forms an obtuse angle and not reaching beyond the anterior end of the suborbital fenestra; palatine-pterygoid suture lies anterior to the posterior end of the suborbital fenestra; pterygoid forms a neck surrounding the choana; dentary tooth row with only 16 teeth; laterally compressed posterior teeth; very small external mandibular fenestra; foramen aerum at the lingual margin of the retroarticular process; axis with a hypapophysis that is located near the center of the centrum; coracoid with a very large glenoid; iliac blade with a rectangular posterior outline and a dorsal indentation; dorsal osteoderms with no or only modest ridge.Diagnosis: Orientalosuchus naduongensis differs from Krabisuchus siamogallicus in having dorsal osteoderms with no or only a modest ridge; an inward-pushed pterygoid around the choana and a neck surrounding the aperture, while the pterygoid surface is flush with the choanal margin in K. siamogallicus; and a very large supraoccipital exposure, preventing the parietal from reaching the posterior edge of the skull.Differential diagnosis: Orientalosuchus naduongensis differs from Eoalligator chunyii in having dorsal osteoderms with no or only a modest ridge; very prominent preorbital ridges; squamosals, that do not extend ventrolaterally to lateral extent of paroccipital process; a very large supraoccipital exposure, preventing the parietal from reaching the posterior edge of the skull; a smooth dorsal surface of the surangular, whereas Eoalligator chunyii has a large sulcus next to the anterior half of the glenoid fossa; and an intersupratemporal bar similarly broad as the supratemporal fenestra, while the bar is strongly constricted in Eoalligator chunyii.Orientalosuchus naduongensis differs from Jiangxisuchus nankangensis in having a deeply curved dentary; a small incisive foramen; prominent preorbital ridges; a palatine-pterygoid suture nearly at the posterior angle of suborbital fenestra; a frontoparietal suture entirely on the skull table ; a very large supraoccipital exposure preventing the parietal from reaching the posterior edge of the skull; anterior maxillary teeth with vertical ridges on their lateral surface; and an intersupratemporal bar that is similarly broad as the supratemporal fenestra (the bar is constricted in J. nankangensis).Orientalosuchus naduongensis differs from Protoalligator huiningensis in having a deeply curved dentary; laterally compressed posterior teeth; a deep notch lateral to the naris; an occlusion pit between the seventh and eighth maxillary teeth with all other dentary teeth occluding lingually, while in Protoalligator huiningensis all dentary teeth are lingual to maxillary teeth; very prominent preorbital ridges; and maxillary teeth with vertical ridges on their lateral surface.Holotype: GPIT/RE/09761; partial skeleton consisting of skull, lower jaws and incomplete postcranial skeleton see .Type locality and horizon: The fossils were recovered from layer 80 of the Na Duong coal mine in northern Vietnam ; Na Duong Formation, Eocene, late Bartonian to Priabonian age (39–35 Ma) .Referred material: A total of 29 individuals represented by incomplete skulls, skull fragments and associated postcranial material, Na Duong Formation, Na Duong coal mine, Vietnam  medial to the fifth tooth of the maxilla.The maxilla-ectopterygoid suture is shifted posterolaterally from the tooth row, preventing the ectopterygoid from contacting the alveoli . A septum seems present at least anteriorly as indicated in a CT-scan of GPIT/RE/09761, but it is unclear if it projects out of the choana or remains recessed within. In adult individuals, the posterolateral margin of the choana is smooth. In the juvenile GPIT/RE/09772, the posterolateral margin seems more concave (notched), but this could be a crushing artifact.Anterolateral to the choana, the pterygoid surface is pushed inward and forms a thin neck surrounding the choana opening in GPIT/RE/09761  and GPITThe ectopterygoid forms the posterolateral border of the suborbital fenestra. Anteromedially, the bone tapers into an acute tip and expands posteriorly, forming a small medial shelf projecting into the suborbital fenestra. Posteromedially to the suborbital fenestra, the ectopterygoid contacts the pterygoid and forms a posterolaterally projecting process that does not reach as far posteriorly as the pterygoid. The ectopterygoid does not abut the maxillary tooth row as seen in GPIT/RE/09730  and dorsThe surface of the anterior process bears many small foramina, whereas the posterior region is decorated with very fine anteroposteriorly oriented lines, visible in GPIT/RE/09761  and GPITThe supraoccipital is the most dorsal bone of the occipital region and has an ornamented dorsal surface. It forms a large trapezoid process on the skull table, which prevents the parietal from reaching the posterior edge of the skull table in adults. In a juvenile (GPIT/RE/09772), the supraoccipital is still large, but the parietal has a minor lateral contact with the skull table.GPIT/RE/09728  offers tThe suture with the exoccipital originates ventrally to the posttemporal fenestra and projects ventromedially resulting in a roughly triangular outline of the bone.The exoccipital is best observable in GPIT/RE/09728 . It shapAlongside the ventrolaterally extending suture with the basioccipital, the exoccipital possesses a relatively short ventrally oriented process. Laterally from the occipital condyle, the caudal aperture of the carotid foramen is visible, and dorsolaterally from the occipital condyle lies the opening for the foramen vagus. The suture with the basisphenoid is not preserved.The basioccipital forms the ventral part of the occipital region and the occipital condyle. Directly ventrally to the condyle, a small foramen is visible in GPIT/RE/09729 , which iThe basisphenoid is anteroventrally located from the basioccipital and projects relatively far ventrally. Its extension on the lateral braincase wall cannot exactly be determined in GPIT/RE/09728 , but it The dentary is best observable in GPIT/RE/09728  and GPITIn total the tooth row consists of 16 teeth . The firThe dentary surface is ornamented with small pits anteriorly and grooves posteriorly. Anteriorly to the external mandibular fenestra, nearly no ornamentation is visible. The symphysis extends to the height of the fifth dentary tooth. The Meckelian groove is preserved as a very narrow canal, nearly closed by the surrounding dentary.The dentary-splenial suture abuts the tooth row at the level of the 13th dentary tooth. The dentary contacts the angular posteroventrally and the surangular posterodorsally. The suture with the angular projects ventrally from the height of the most posterior dentary tooth to the ventral part of the external mandibular fenestra in a bowed line. The suture with the surangular intersects the external mandibular fenestra at its posterodorsal corner. The external mandibular fenestra itself is very small. It has nearly the same size as the foramen intermandibularis caudalis  and in form of marks in GPIT/RE/09728 . An antePosteriorly, the splenial meets the surangular, coronoid and angular, as well as it forms the anterior border of the foramen intermandibularis caudalis. The suture with the surangular projects anteriorly until reaching the tooth row. The concave suture with the coronoid is only visible in the right ramus of GPIT/RE/09727 . The antThe surangular is best observable in GPIT/RE/09761  and GPITAnteriorly, the surangular has two processes. The ventral process is slightly shorter than the dorsal one, but they do not differ much in length. Posteriorly, the surangular extends to the posterior end of the retroarticular process. Dorsally, the surangular is slightly elevated, but does not seem to reach to the dorsal tip of the lateral wall of the glenoid fossa (best observable in GPIT/RE/09783). Laterally, the suture with the angular contacts the external mandibular fenestra slightly ventrally to its posterodorsal corner. Lingually, the suture between the surangular and articular cannot be clearly followed within the glenoid fossa, due to deformation. The suture ventrally to the fossa is simple, without any visible lamina. A lingual foramen is not preserved. The surangular-angular suture meets lingually the articular dorsally to the ventral tip of the articular. Posterodorsally to the surangular-coronoid suture, a small foramen is visible in GPIT/RE/09727 .The angular is best observable in GPIT/RE/09761  and GPITThe bone borders most of the foramen intermandibularis caudalis dorsally and ventrally as well as its complete posterior border. The angular-coronoid suture is nearly straight and anteroposteriorly oriented. Ventrally, anteriorly to the height of the glenoid fossa, a relatively large foramen is visible. Another posterior foramen, is only preserved in GPIT/RE/09761 .The coronoid is only well preserved in GPIT/RE/09727 . Its outThe articular possesses the glenoid fossa and the posterodorsally oriented retroarticular process in its medial part. Anteromedially, the process produces a broad shelf. The glenoid fossa is separated from the process by a mediolaterally oriented sigmoidal-shaped ridge with its lateral part being slightly more anteriorly oriented than the medial one. The foramen aerum is visible in GPIT/RE/09727  on the mMost of the teeth attached to the skulls and lower jaws are poorly weathered. Only disarticulated teeth of GPIT/RE/09728 (posterior ones) and GPIT/RE/09761 (a single anterior one)  are wellThe anterior and middle teeth are pointed. In a single well-preserved tooth several relatively dominant vertical ridges are present laterally and weaker ones lingually. This condition is not visible in poorly-preserved teeth, making it unclear if this condition is true for all pointed teeth. Laterally, the teeth are slightly convex, whereas they are concave lingually.Posteriorly, at the 10th maxilla tooth . All of these are crushed and/or covered by a pyritized matrix except for the single vertebra of GPIT/RE/09761 .The neural spine is slightly sloping anteriorly, but the most dorsal tip is not preserved. The spine has a dorsoventrally extending anterior ridge and a posterior groove, with the latter deepest between the postzygapophyses. The hypapophysis is located at the anterior part of the centrum and reaches posteriorly roughly its midpoint. The pre- and postzygapophyses are similarly formed and have oval articular surfaces. The diapophysis initiates above the base of the neural arch, whereas the parapophysis originates ventrally on the centrum. The centrum is concave medially, smooth and without any pits. A lateral foramen is preserved on the right side, slightly posteriorly between the diapophysis and parapophysis. A few smaller foramina are visible posterior to the hypapophysis.A total of 30 dorsal vertebrae are preserved . They are best preserved in GPIT/RE/09761 . The posOnly the first sacral vertebra  is preseA total of 19 caudal vertebrae are preserved . The first caudal vertebra in GPIT/RE/09761  is by faThe rest of the caudal vertebrae are poorly preserved and their neural spines are narrower and originate more on the posterior part of the centrum. Their pre- and postzygapophyses are of similar size and relatively small. The prezygapophyses are very close to each other. Their articulation surfaces point medially. The transverse processes are fragmentarily preserved in the anterior caudal vertebrae and absent in the more posterior ones. The centra gradually elongate and flatten laterally as well as reduce in size posteriorly in the vertebral column. The centrum further has a broad, anteroposteriorly extending groove, which makes the centra even narrower and the articulation facets more oval posteriorly. The ventral side of the centra bears a deep anteroposteriorly projecting sulcus in all caudal vertebrae except for the first one.Three cervical and 10 dorsal ribs are preserved. The cervical ribs  have a hThe shaft of the dorsal ribs  is slighTwo highly weathered right scapulae  is preserved in GPIT/RE/09727. It has a very broad proximal and a broad distal end. The shaft is slightly S-shaped, which could be an artifact of crushing. The right and left ulnae of GPIT/RE/09784 are poorly preserved.The left radiale  is relatIn GPIT/RE/09761, elongated autopodial elements , either There are seven disarticulated phalanges of GPIT/RE/09761 , but we Its proximal end is nearly triangular with three knob-like structures, which are separated by a ridge. Its distal end has two condyles. Their articulation surfaces slope dorsally and ventrally. The condyles are separated from each other by an intercondylar groove. In lateral view, relatively deep collateral ligament fossae are visible on the distal condyles. The shaft has a smooth surface, but bears a foramen on the slightly laterally bent side.The preserved claws of GPIT/RE/09761 differ considerably in length, but cannot be assigned to either manus or pes. They are long, only slightly curved, ventrally flattened, dorsally curved and pointed. The claws of GPIT/RE/09784 belong to the pes based on their position recorded in the field and are partly articulated with the distal phalanges. The better preserved ones are much smaller and stronger curved than the claws of GPIT/RE/09761, but it is unclear to which digit they belong.Both ilia are preserved in GPIT/RE/09761  and GPITThe best preserved ischium is the left one of GPIT/RE/09761 . Its proA single left pubis is preserved in GPIT/RE/09784. Its articulation surface with the ischium is oval and anteroposteriorly elongated. The shaft is very thin and the blade flares strongly anteroposteriorly.The femur of GPIT/RE/09761  is 112.8The tibia and fibula are best preserved in GPIT/RE/09761  and GPITThe left astragalus and calcaneum are preserved in GPIT/RE/09784 and show no noticeable difference from other alligatoroids.Orientalosuchus naduongensis.More than 150 osteoderms  and osteMost of the preserved osteoderms of GPIT/RE/09761 are dorsals. They are nearly square-shaped and possess no, or only a very shallow keel. This is also true for the posterodorsal midline osteoderms of GPIT/RE/09784.Another osteoderm type, probably more posterolaterally located and very well preserved (GPIT/RE/09727) is relatively small and oval. These osteoderms show still weak, but slightly more pronounced keel than the dorsal osteoderms.A single osteoderm of GPIT/RE/09761 is roughly triangular and could belong to the anterolateral region. No keel is visible, but the surface is weathered.Asiatosuchus nanlingensisA total of 20,160 equally optimal trees with a length of 927 steps were recovered, with a consistency index (CI) of 0.292 and a retention index (RI) of 0.759  and 22. Arenysuchus gascabadiolorumAllodaposuchus subjuniperusAllodaposuchus precedensLohuecosuchus mechinorumLohuecosuchus megadontos(199) from the analysis results in the previous sister group relationship outside Crocodylia.Outside Brevirostres, the monophyletic Pachycheilosuchus trinqueiPietraroiasuchus ormezzanoiShamosuchus djadochtaensisPietraroiasuchus ormezzanoi and Shamosuchus djadochtaensis now form a monophyletic group with Acynodon iberoccitanusAcynodon adriaticusHylaeochampsa vectianaIharkutosuchus makadii(196) supports this monophyletic group as all of these taxa, except Hylaeochampsa vectiana, in which the state is unknown, lack a notch between the premaxilla and maxilla as an adult (196-1). Pachycheilosuchus trinquei is the sister taxon to the clade formed by these taxa and all Crocodylia.The polytomy between Crocodylus depressifronsCrocodylus affinisCrocodylus depressifrons + Crocodylus affinis is their short dentary symphysis (49-0), similar to most Crocodylidae, whereas the symphysis is long (49-1) in Asiatosuchus germanicusAsiatosuchus germanicus and a more basal position (142-0). By adding a new character (197), Crocodylus depressifrons and Crocodylus affinis are now drawn closer to Crocodylidae, but it is worth to mention that the character can be only scored for Crocodylus depressifrons .The clade Crocodylus acerBrachyuranochampsa eversoleiCrocodylus acer was moved to a more derived position inside Crocodylinae. The reason for this is, that the scorings for the new characters are identical in Crocodylus acer and Mecistops cataphractus  or posterior (195-2) to the anterior frontal process in Diplocynodontinae .gatoroid . In the muelleri ), as oppNavajosuchus mooki . Inside Orientalosuchina, In the present analysis, a broad scapulacoracoid facet immediately anterior to the glenoid fossa (26-1) and nearly squared dorsal midline osteoderms (39-1) defines Alligatoridae and three characters define Alligatorinae: eight contiguous dorsal osteoderms per row at maturity (40-2), a premaxilla with a deep notch lateral to the naris (86-1) and a longer prefrontal than the lacrimal (130-1).Orientalosuchus naduongensis + K. siamogallicus is 1 and the absolute frequency bootstrap value is 35%. If the poorly preserved Protoalligator huiningensis is removed from the analysis, the absolute frequency bootstrap value of Orientalosuchina goes up to 45% and further removing the also poorly preserved Eoalligator chunyii results in a bootstrap value of 54% for Orientalosuchina and 51% for Orientalosuchus naduongensis + K. siamogallicus.The Bremer support for Orientalosuchina is 1 and the absolute frequency Bootstrap value is 5%. The Bremer support for Globidonta is better resolved in the current analysis compared to previous studies with East and Southeastern Asian alligatoroids included . Statist194, 195, 197 and 199, results in a similar phylogeny in most non-globidontan taxa as in former analyses .In all trees, the single synapomorphy of Orientalosuchina is a short dentary symphysis extending to the height of the fourth to fifth alveolus (49-0) as opposed to the long symphysis (49-1) of for example, J. nankangensis, Eoalligator chunyii, Protoalligator huiningensis and Orientalosuchus naduongensis + K. siamogallicus form a polytomy. In those trees in which Protoalligator huiningensis is ancestral, the presence of a notch between the premaxilla and maxilla (196-0) is a further synapomorphy for the group.Inside Orientalosuchina, Eoalligator chunyii is recovered as the most basal taxon, the monophyly of Orientalosuchina is further supported by seven synapomorphies:If (1) a truncated surangular around the lateral wall of the glenoid fossa (67-1); (2) the medial position of the foramen aerum on the retroarticular process (70-0) and (3) the quadrate (177-0); (4) a very short anterior palatine process, not reaching the anterior end of the suborbital fenestra (115-1); (5) the quadratosquamosal suture extends dorsally along the caudal margin of the external auditory meatus (148-1); (6) the squamosal extends ventrolaterally to lateral extent of paraoccipital process (159-1); and (7) the 11th dentary tooth is the largest one after the fourth one (200-0).Further synapomorphies may diagnose Orientalosuchina including: (1) the posterior lateral edges of the palatines are parallel (120-0); (2) the lacrimal makes broad contact with the nasal without any sign of a maxillary process (128-0); (3) the anterior tip of the frontal is acute (131-0); (4) the postorbital does not contact the quadrate and quadratojugal at the mediodorsal angle of the infratemporal fenestra (143-0); and (5) the angular-surangular suture lingually originates near the dorsal border of the external mandibular fenestra and is straight (201-1). However, given that the corresponding anatomical regions are unknown in the basally branching and poorly preserved taxa these now define a more inclusive clade.Consistent with their basal divergence and age, orientalosuchians retain several crocodylian plesiomorphies, which may explain their recovery inside Crocodyloidea in previous analyses . These cOrientalosuchus naduongensis and Jiangxisuchus nankangensis share a very similar pterygoid around the choana opening (123-2), which strongly resembles the morphology of Osteolaemus tetraspisVoay robustus. The choanal region in Krabisuchus siamogallicus (though not intact) looks like the pterygoid surface is flush with the choanal margin (123-0). In Eoalligator chunyii and Protoalligator huiningensis the pterygoid is not preserved. The pterygoid forming a neck around the choana is a condition unknown for any other alligatoroid and could be a further synapomorphy for Orientalosuchina with a potential reversal in K. siamogallicus.Orientalosuchus naduongensis and Eoalligator chunyii. The axis is, unfortunately, not preserved in any other members of Orientalosuchina. A shifted axial hypapophysis among Crocodylia is otherwise only present in Diplocynodontinae and Crocodylus depressifrons.A further potential synapomorphy for the group is the axial hypapophysis located towards the centrum of the axis body (15-1) in Orientalosuchus naduongensis + K. siamogallicus has no synapomorphies in all trees, but has five in some trees including (1) the intersupratemporal bar being similarly broad as the supratemporal fenestra (199-1); (2) the dentary curves deeply between the fourth and 10th dentary alveoli (50-1); (3) a palatine-pterygoid-suture situated far from the posterior angle of the suborbital fenestra (118-1); (4) the frontoparietal suture lies entirely on the skull table (150-2); and (5) the anterior and medial teeth have dominant vertical ridges on their labial surface (198-1) and to a lesser extent on their mesial side. Such strong ridges are only present in few crocodylians , but not in other member of Alligatoroidea.The clade of K. siamogallicus as having unusually long lacrimals, reaching the premaxilla and as a result preventing the maxilla from contacting the nasal. These sutures are more likely the preorbital ridges (97-1) also present in Orientalosuchus naduongensis. These ridges may be a further autapomorphy for Orientalosuchus naduongensis or a synapomorphy for Orientalosuchus naduongensis + K. siamogallicus. Prominent preorbital ridges are otherwise only present in Mourasuchus atopusOsteolaemus tetraspis and Crocodylus porosus, but these structures are more prominently developed in Orientalosuchus naduongensis.Orientalosuchus naduongensis is characterized by a very large supraoccipital exposure on the skull table (160-3), whereas the exposure is large (160-2) in Eoalligator chunyii and K. siamogallicus, but small (160-0) in J. nankangensis. The state of this character for Protoalligator huiningensis is unknown. A very large supraoccipital exposure is otherwise found in some Caimaninae, but in most of those taxa, except for Globidentosuchus brachyrostris, the supraoccipital is not trapezoid-shaped but block-shaped.Protoalligator huiningensis from the phylogenetic analysis results in a sister taxon relationship between J. nankangensis and Eoalligator chunyii, forming a sister group to Orientalosuchus naduongensis + K. siamogallicus. A close relationship between the two Late Cretaceous/early Paleocene species J. nankangensis and Eoalligator chunyii was also recently found in the analysis of Omitting Stangerochampsa mccabei + Brachychampsa spp. + Orientalosuchina is supported by: (1) a dorsally pojecting naris (81-1), (2) a large incisive foramen, intersecting the premaxillary-maxillary suture (88-2), (3) a maxilla with posterior process between lacrimal and prefrontal (128-2) and (4) a frontoparietal suture making modest entry into the supratemporal fenestra (150-1).In some trees, the monophyly of Brachychampsa spp. + Orientalosuchina is supported by: (1) the fifth maxillary alveolus as the largest (93-1) and (2) a large supraoccipital exposure on the skull table (160-2).The monophyly of Diplocynodon spp. and Leidyosuchus canadensis), the fourth and fifth maxillary teeth have the same size (93-3), whereas it is the fourth tooth (93-2) in members of Alligatoridae. The only exception is the Late Cretaceous/early Paleocene Bottosaurus harlani, although position of this taxon inside Caimaninae is questionable .The monophyly of Brachychampsa montana and Orientalosuchina than between Brachychampsa montana and Brachychampsa sealeyi seems unlikely and could be an artifact due to the poor preservation of the latter.A closer relationship between N. mooki and Ceratosuchus burdoshi compared to Alligatoridae in this study can be explained by: (1) a straight dentary (50-0), (2) the presence of a large incisive foramen (88-1), and (3) a relatively flat skull in lateral view (193-0) unlike in the monophyletic group consisting of Arambourgia gaudryi  a surangular-dentary suture intersecting the external mandibular fenestra at the posterodorsal corner (64-1) and (2) an anterior jugal process extending anterior to the frontal (195-0). In Wannaganosuchus brachymanus, Arambourgia gaudryi and Hassiacosuchus haupti, the anterior jugal process lies at the same height as the anterior frontal process (195-1).The monophyly of Alligator is polyphyletic with Alligator prenasalis (Alligator mcgrewiAlligator species due to a long dentary symphysis (49-1). They further differ in: (1) an anterodorsally projecting naris (81-0), (2) a lingual foramen for the articular artery and alveolar nerve perforating the surangular/angular suture (69-1) and (3) a maxilla bearing a broad shelf extending into the suborbital fenestra (112-1). The last two characters, however, can also be found in other Alligator species .In the present phylogeny, enasalis  + AlligaEocaiman caverensisGlobidentosuchus brachyrostris and Culebrasuchus mesoamericanus, which are found in an ancestral position in the current analysis, with Globidentosuchus brachyrostris as the most basal caiman.Globidentosuchus brachyrostris results mainly from (1) the splenial participating in the mandibular symphysis (54-0), (2) a concavoconvex frontoparietal suture (151-0) and (3) a trapezoid-shaped supraoccipital exposure on the skull table (202-0). Globidentosuchus brachyrostris and Culebrasuchus mesoamericanus both differ from most of other Caimaninae in: (1) having an angular-surangular suture contacting the external mandibular fenestra at the posterior angle (60-0) and (2) an exoccipital terminating dorsally to the basioccipital tubers (176-0) in Culebrasuchus mesoamericanus, whereas this is unknown for Globidentosuchus brachyrostris. Considering the Miocene age of both taxa, either they have a ghost lineage reaching back to the Cretaceous, or their basal position is an artifact due to incompleteness of those taxa.This basal position of Bottosaurus harlani was found in a polytomy with Paleosuchus spp.. In the present analysis, however, Tsoabichi greenriverensis(202). In Tsoabichi greenriverensis and Paleosuchus spp., the large supraoccipital exposure is triangular (202-1), while it is block-shaped (202-2) or unknown for the remaining caimans, including all recent species.As in Asiatosuchus nanlingensis and the Maoming alligatoroid. The Maoming taxon , other than Alligator sinensis into a phylogeny, followed by Eoalligator chunyii and Protoalligator huiningensis) and Jiangxisuchus nankangensis).The Chinese alligator, resolved . Martin Alligator sinensis and their morphology as well as their Cretaceous origin  strongly suggest that they represent a more basal monophyletic clade of alligatoroids. East and Southeastern Asia was, therefore, colonized by alligatoroids twice: once by Orientalosuchina during the Late Cretaceous and once by the sinensis lineage during the Cenozoic  molecular divergence date estimates and fossil dates of crown-Alligator are in conflict, (2) pan-sinensis in Asia cannot be traced back further than the Pliocene 58–31 Ma  as opposlligator . Given tBeringia  but a reOrientalosuchus naduongensis, as the sister taxon to Krabisuchus siamogallicus from the Eocene of Thailand. Together they form a monophyletic extinct basal East to Southeastern Asian alligatoroid clade of Late Cretaceous origin that also included Jiangxisuchus nankangensis, Eoalligator chunyii and Protoalligator huiningensis. The current phylogeny supports at least two different dispersals from North America to Eastern Asia: one during the Late Cretaceous  and a second during the Cenozoic (Alligator sinensis lineage). Improved fossil calibrations and taxon sampling will be vital for further constraining the timing and resolving the climatic/paleogeographical context of these dispersals.Parsimony analysis finds the new late Eocene taxon from Vietnam, See (51) Largest dentary alveolus immediately caudal to fourth is (0) 13 or 14, (1) between 11 and 14 and a series behind it, (2) 11 or 12, (3) no differentiation, (4) behind 14, (5) 10. (modified from Comments: According to Hassiacosuchus haupti) were scored with (1) despite the fact that their largest tooth was not the 13 or 14 because they also possess enlarged posterior teeth. In addition, taxa with enlarged posterior teeth that are compressed instead of globular were furthermore excluded previously from this state. For instance, in Orientalosuchus naduongensis, Bottosaurus harlani, Procaimanoidea utahensis  as in recent Alligator spp. or Caiman spp., but here we score them as (1).We rephrased character state (1) of tahensis  and Procdea kayi  the rear(193) Skull in lateral view relatively flat (0) or wedge-shaped (1) (new character).Comments: A triangular- or wedge-shaped skull in lateral view occurs in some short snouted taxa. The character is not fully correlated with the presence of an anterodorsally positioned external narial opening because some fossil taxa with an anterodorsally positioned naris have a relatively long and flat snout (e.g. Navajosuchus mooki). The character is somewhat problematic to score due to the often ocuring dorsoventrally deformation in most crocodilian fossils. In most taxa, the skull shows a sudden increase in height between the orbits giving the it a slide-like outline in lateral view. In wedge-shaped taxa, however, the snout region is nearly straight triangular from the narial opening to the skull table, clearly visible in some basal alligatorines like Hassiacosuchus haupti.Orientalosuchus naduongensis are dorsoventrally compressed due to deformation, making a reliable scoring for this character impossible. The snout, however, looks more elongated than that of the typical wedge-shaped short snouted alligatorines. Krabisuchus siamogallicus as having a similar morphology as H. haupti and the head of Kr-C-007 indeed looks wedge-shaped, although the preservation is suboptimal and it could be an artifact due to the apparent postmortem deformation.All speicmens of (194) Nasal bone does (0) or does not (1) reach to the level of the orbita (new character).Comments: In Alligator, the nasal usually reaches the level of the orbits, but not in Alligator sinensis in which the nasal terminates anterior to the orbits in the herein analyzed individuals . Hassiacosuchus haupti and Arambourgia gaudryi have a far posteriorly reaching nasal bone, while it is shorter in all other basal alligatorines and Navajosuchus mooki. In crown-group Caimaninae, only Melanosuchus niger  Anterior process of jugal extends anterior (0), lies at the same level as (1), or well posterior to the anterior process of frontal (2).  in Hassiacosuchus haupti, Arambourgia gaudryi, Navajosuchus mooki, Wannaganosuchus brachymanus and Procaimanoidea kayi. In crown-group Caimaninae, the process always reaches anterior to the frontal, except for Paleosuchus palpebrosus and Melanosuchus niger, in which the process lies on the same height as the frontal. For Orientalosuchina, only Orientalosuchus naduongensis could be reliably be scored as state (1).Among Globidonta, the jugal extends anteriorly to the frontal (0) in polyodon , Allogna(196) Notch between the premaxilla and maxilla present (0) or not present (1) in adult individuals (new character).Comments: A few characters in the matrix are referring to an early ontogenetic state of a taxon . The reason for this is potential changes during ontogeny like the development of a notch between the premaxilla and maxilla late in ontogeny in Caiman crocodilus  was reviewed based on published work and if the scoring was solely based on adult individuals, they were rescored as (?) and the previous scoring was added to the new character (196).(197) Sutural contact of the exoccipitals dorsal to the foramen magnum (0) long, at least half the height of the foramen magnum, (1) short, shorter than half the height of the foramen magnum, or (2) no sutural contact between the exoccipitals (new character).Comments: In recent Crocodylus and Alligator species, the supraoccipital does not project far ventrally, resulting in a relatively long suture between the two exoccipitals. In for example, Osteolaemus tetraspis and some globidonts  the supraoccipital projects further ventrally and the sutural contact between the exoccipitals becomes smaller, until it is only roughly half as long as the maximal height of the foramen magnum. Unfortunately, in many fossil taxa, the occipital region is too poorly preserved or not figured to reliably score this character, but it could be valuable to understand wheter some basal Alligator species show a short suture and if there are differences between basal Alligatorinae taxa. Among recent Caimaninae, in Paleosuchus palpebrosus and Melanosuchus niger the suture is long and it is short in Caiman crocodilus and Caiman latirostris .irostris . In OrieAllodaposuchus precedens and Borealosuchus sternbergii (In two non-Brevirostres taxa (rnbergii ) the sup(198) Anterior maxillary teeth without (0) or with (1) ridges on their lateral surface (new character).Comments: In many crocodilians, the maxillary teeth bear fine striae, but in Orientalosuchus naduongensis and Krabisuchus siamogallicus there are marked dorsoventrally running ridges present on the labial surface of the anterior teeth, . Dominant ridges are also present in the tomistomine Maomingosuchus petrolica and in Allodaposuchus precedens and Allodaposuchus subjuniperus.(199) Intersupratemporal bar (0) as or near as broad as the supratemporal fenestra, (1) at least twice as broad as the supratemporal fenestra, (2) around half the broadness of the supratemporal fenestra or (3) constricted, less than half the broadness of the supratemporal fenestra (new character).Comments: The intersupratemporal region differs among crocodilians, but most commonly, the region is either similarly broad as the supratemporal fenestra or constricted, especially in longirostrine taxa. The level of constriction was divided in two states (2 and 3). A markedly broad state is only present in few taxa, especially in crown-group Caimaninae in which the supratemporal fenestrae are overgrown, but it is not completely correlated with (152) as a broad intersupratemporal bar is also present in Procaimanoidea kayi, which has open fenestrae.Brachychampsa montana, Ceratosuchus burdoshi and Eoalligator chunyii, which have a constricted bar.Other globidonts mostly have an intersupratemporal bar as broad as the fenestra or a slender, but not constricted one. Exceptions are (200) If largest dentary alveolus is between 11th and 14th and a series behind it, is it the (0) 11th, (1) 12th, or (2) 13th to 14th (new character).Comments: This character is only applicable for taxa with (51-1).(201) Surangular-angular suture lingually originates (0) near the ventral border of the external mandibular fenestra, (1) near the dorsal border of the external mandibular fenestra and straight, (2) near the dorsal border of the external mandibular fenestra and bowed (new character).Comments: In most crocodylians, the surangular-angular suture lingually originates near the ventral border of the external mandibular fenestra (201-0). In Orientalosuchina, however, the suture originates near the dorsal border of the external mandibular fenestra and is notably straight (201-1). The state is, however, unknown for Eoalligator chunyii. The only other taxon showing this morphology is Maomingosuchus petrolica. In Stangerochampsa mccabei and Voay robustus  Large to very large supraoccipital exposure on skull table is (0) trapezoid, (1) triangular, or (2) block-shaped (new character).Comments: In most crocodylians, the supraoccipital exposure on the skull table is either small (160-0) or absent (160-1). In some species, however, the exposure is either large (160-2) or very large, excluding the parietal from the posterior edge of skull table (160-3). Among species with a large to very large exposure, the shape can differ markedly. In Orientalosuchina, except for Jiangxisuchus nankangensis, which only has a small exposure, the supraoccipital is trapezoid. This also true for Stangerochampsa mccabei, Brachychampsa spp., Bottosaurus harlani and Globidentosuchus brachyrostris. In most Caimaninae, on the other hand, the shape is either triangular, for example, in Paleosuchus spp. or block-shaped like in Caiman spp.. This character is only applicable for taxa with large to very large supraoccipital exposure on the skull roof.10.7717/peerj.7562/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7562/supp-2Supplemental Information 2Character changes.Click here for additional data file.10.7717/peerj.7562/supp-3Supplemental Information 3List of characters.Click here for additional data file.10.7717/peerj.7562/supp-4Supplemental Information 4Postcranial measurements.Click here for additional data file.


High variability exists in individual susceptibility to develop overweight in an obesogenic environment and the biological underpinnings of this heterogeneity are poorly understood. In this brief report, we show in mice that the vulnerability to diet-induced obesity is associated with low level of polysialic acid-neural cell adhesion molecule (PSA-NCAM), a factor of neural plasticity, in the hypothalamus. As we previously shown that reduction of hypothalamic PSA-NCAM is sufficient to alter energy homeostasis and promote fat storage under hypercaloric pressure, inter-individual variability in hypothalamic PSA-NCAM might account for the vulnerability to diet-induced obesity. These data support the concept that reduced plasticity in brain circuits that control appetite, metabolism and body weight confers risk for eating disorders and obesity. Changes in lifestyle and in the availability and quality of food largely explain the worldwide epidemic of obesity . HoweverProtocols including manipulation of animals (registration number 853.01) were reviewed and approved by our local Institutional Animal Care and Use Committee , and were in strict accordance with the European Community guidelines (directive 86/906). Experiments were carried out with 2 months-old male C57Bl/6JOla mice from Harlan Laboratories. Mice were housed individually, fed either a standard diet  or a customized highly palatable high-fat diet . Free access was given for food and water. Recordings of food intake and body weight were done manually at 9:00 h. For tissue collection, mice were killed between 14:00 and 16:00 h after a 6-h fast.After sacrifice, brains were quickly removed and immersed in cold PBS solution. A 2 mm coronal slice was cut with a brain matrix. The slice was laid on a 6% agarose block and the MBH was dissected under stereomicroscope and cold-light illumination using a scalpel. Tissues were immediately frozen in liquid nitrogen and stored at −80°C until experiments.g and supernatants were collected for PSA-NCAM assay using an enzyme-linked immunosorbent assay kit  and for protein assay .Tissues were lysed and homogenized in RIPA lysis buffer using the TissueLyser system  and 5 mm stainless steel beads (Qiagen). The homogenates were centrifuged 5 min at 5,000 Post-hoc analyses were done when main effects reached significance. Before comparison, Bartlett's and Shapiro–Wilk's tests were applied to check equality of variances and to evaluate the normality of the distribution, respectively.All data are expressed as means. Error bars indicate standard errors of the mean (SEM). Multiple comparisons between groups were carried out by one- or two-way ANOVA using Prism 5.0 software . http://phenome.jax.org/). It is well-known that high-energy foods cause transient overeating in most of the mice, which corresponds to an acute increase in energy intake during a few days , HFD-tolerant and HFD-intolerant individuals according to their feeding response to 1-week HFD and then we examined the concentration of PSA-NCAM in the medio-basal hypothalamus (MBH) collected after this oral fat tolerance test. Intolerant mice showed significant lower levels of hypothalamic PSA-NCAM in comparison to tolerant mice . As a reference, the constitutive level of PSA-NCAM in the MBH of control mice fed a standard diet was 508.5 ± 31 ng/mg protein (n = 16). The level of PSA-NCAM in the MBH of intolerant mice did not differ from the constitutive value. These data suggest that tolerant mice might have higher ability to mobilize PSA-NCAM to trigger synaptic plasticity in the hypothalamus than intolerant mice.The polysialic acid-neural cell adhesion molecule (PSA-NCAM) is a cell-surface molecule that promotes various plasticity-related changes in brain circuits . We receIn this study, we measured the feeding response to 1-week HFD in mice. We found variability in the behavioral response with high responders who failed to rapidly normalize their energy intake during the metabolic challenge. High responders also gained more weight when they consumed the hypercaloric diet for a long time. Thus, the 1-week HFD challenge may be considered as a Dietary Fat Tolerance Test (DFTT) for identifying mice prone to nutritional obesity. Similar predictive tests have been used in the past showing that short-term response to HFD constitutes a phenotypic trait of metabolic flexibility which provides information on vulnerability to metabolic diseases –11. The The DFTT might be relevant for investigating intrinsic factors that produce maladaptive eating disorders and vulnerability to obesity before the clinical onset of the disease. Some of these factors have been already evidenced, including efficiency of peripheral endocrinological and metabolic processes , 26, 27,Gne and St8sia4 genes, respectively. One can propose that these genes might be differentially upregulated on HFD depending on individuals. Gene polymorphism association studies and epigenetic research on these specific genes in murine models and in humans are thus promising research to increase our knowledge on obesity susceptibility.After the DFTT, intrahypothalamic PSA-NCAM content was elevated in HFD-tolerant mice, while it remained low and similar to the constitutive level in HFD-intolerant mice. This result suggests that inter-individual variability exists in the ability to upregulate PSA-NCAM production in the hypothalamus in response to the metabolic challenge, and that PSA-NCAM production was stimulated in HFD-tolerant mice. The source of variation of PSA-NCAM expression between HFD-tolerant and -intolerant animals is not known. Similar intrinsic differences in healthy animals have been already reported, linking the level of prefrontal PSA-NCAM to the susceptibility for addiction-related behaviors . InterinIn several models of neuropsychological disorders, the loss of neural PSA-NCAM alters physiological defenses, exacerbates symptoms or worsens the disease , 37–40, Future research is now needed to understand molecular basis of inter-individual heterogeneity in hypothalamic PSA-NCAM level and to identify life stressors that influence the expression of this factor in the hypothalamus.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Fu-Zheng) herbs is a representative type of traditional Chinese medicine (TCM) widely used for cancer treatment in China, which is in contrast to pathogen eliminating (Qu-Xie) herbs. However, the commonness in the biological basis of health-strengthening herbs remains to be holistically elucidated. In this study, an innovative high-throughput research strategy integrating computational and experimental methods of network pharmacology was proposed, and 22 health-strengthening herbs were selected for the investigation. Additionally, 25 pathogen-eliminating herbs were included for comparison. First, based on network-based, large-scale target prediction, we analyzed the target profiles of 1446 TCM compounds. Next, the actions of 166 compounds on 420 antitumor or immune-related genes were measured using a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS2. Furthermore, the structural information and the antitumor activity of the compounds in health-strengthening and pathogen-eliminating herbs were compared. Using network pharmacology analysis, we discovered that: (1) Functionally, the predicted targets of compounds from health strengthening herbs were enriched in both immune-related and antitumor pathways, similar to those of pathogen eliminating herbs. As a case study, galloylpaeoniflorin, a compound in a health strengthening herb Radix Paeoniae Alba (Bai Shao), was found to exert antitumor effects both in vivo and in vitro. Yet the inhibitory effects of the compounds from pathogen eliminating herbs on tumor cells proliferation as a whole were significantly stronger than those in health-strengthening herbs (p < 0.001). Moreover, the percentage of assay compounds in health-strengthening herbs with the predicted targets enriched in the immune-related pathways  were significantly higher than that in pathogen-eliminating herbs (p < 0.05). This finding was supported by the immune-enhancing effects of a group of compounds from health-strengthening herbs indicated by differentially expressed genes in the HTS2 results. (2) Compounds in the same herb may exhibit the same or distinguished mechanisms in cancer treatment, which was demonstrated as the compounds influence pathway gene expressions in the same or opposite directions. For example, acetyl ursolic acid and specnuezhenide in a health-strengthening herb Fructus Ligustri lucidi (Nv Zhen Zi) both upregulated gene expressions in T cell receptor signaling pathway. Together, this study suggested greater potentials in tumor immune microenvironment regulation and tumor prevention than in direct killing tumor cells of health-strengthening herbs generally, and provided a systematic strategy for unveiling the commonness in the biological basis of health-strengthening herbs in cancer treatment.Health-strengthening ( China has a long history of using traditional Chinese medicine (TCM) for treating cancer . A largeHuangdi Neijing). Therefore, an in-depth exploration on the antitumor effects exerted by health- strengthening herbs is meaningful and urgently needed. TCMs in the Chinese national medical insurance catalogue (2017) for oncology treatment are officially divided into two categories, including antitumor TCM and adjuvant TCM for tumors, which contain 40 TCM prescriptions in total [Sijunzi decoction in colorectal cancer [Shenqi Fuzheng injection in colorectal cancer and breast cancer [Shenling Baizhu San in gastric cancer [Buzhong Yiqi decoction in colorectal and lung cancer [Shenqi Fuzheng injection, Danggui Buxue decoction, Huangqi Jianzhong decoction, and Liu-wei-di-huang pill) revealed their protective effect on immune functions in cancer therapy [Liu-wei-di-huang pill, Jianpi Yiqi decoction, and Yishen Gukang decoction [Traditionally, TCM adopts a relative and holistic point of view in cancer treatment. The clinical treatment strategy by strengthening health reflects the characteristics of focusing on regulatory effects instead of the antagonistic effects of TCM, and embodies the classical therapeutic theory that “pathogenic-qi cannot invade the body if health-qi remains strong” in the Canon of Internal Medicine  pathways involved in the antitumor or immunological activities of the TCM compounds are identified [To approach the systematic mechanisms of TCM compounds for cancer treatment, we combined network pharmacology prediction methods with HTSentified . Taking Together, by combining target prediction and high-throughput assay, this study proposed a systematic overview on the biological basis underlying the pharmacological effects of health strengthening herbs in cancer treatment. Despite the need for further investigation, it was indicated that health-strengthening herbs may provide researchers with a valuable candidate library for tumor immune regulatory and tumor preventive drug development.Due to the complex composition of TCM and the lack of corresponding target records, the potential target lists of TCM compounds were obtained by using a computational prediction method, drugCIPHER-CS . Literat2 assay. We examined whether a predicted target was directly or indirectly related with the biomolecules in the literature or DEGs in the HTS2 assay. The indirect relationship was established if the predicted target was in the upstream in a KEGG pathway of the biomolecules in the abstracts or DEGs, or if they were related by protein–protein interaction (PPI) in the HPRD, BIND, IntAct, MINT, or OPHID database [2 assay. The cover rate was calculated as 2 experimental results, which demonstrated that the HTS2 assay may be an alternative method for exploring the novel biological functions of TCM compounds.After literature mining, we obtained the biomolecules mentioned in the abstracts related to the compounds in database ,37,38,39 results A indicatRadix Scutellariae (Huang Qin), were supported with literature or assay evidence. As for the other 13% of the potential targets of wogonin predicted by drugCIPHER-CS, some of them were connected to the biomolecules in the literature or the DEGs in the HTS2 assay by an additional indirect mapping, as shown in In p-values of the targets in 26 cancer hallmarks (immune-excluded) and immune-related KEGG pathways. The literature verification of the pathways with HTS2 or enrichment evidence was conducted manually by reading the papers in the PubMed database. If a significantly enriched pathway was related to the bioactivity records in the published papers in the PubMed database [2 assay results, then it would be considered as a predicted related pathway with supports from literature records or HTS2 assay results. As shown in 2 assay) was set to examine the robustness of the literature verification results. Our results indicated that the cut-off values had little influence on the precision rate for the related pathways determined using an HTS2 assay, and the recall rate scaled from 97% to 72% as the cut-off changed. The precision and recall rate of the significantly enriched KEGG pathways  adjusted) were approximately 60% and 70%, respectively, which indicated the reliability of the pathway prediction results. By comprehensively considering both the precision and recall results, we selected the pathways with three or more DEGs in the HTS2 assay as the ones with support from the assay data for further investigation.Next, to further analyze the mechanism of the action of the compounds, we performed KEGG pathway enrichment analysis based on the target prediction results. We checked the enrichment database  or inclu2 assay. As shown in p < 0.001). The structure similarity analysis was conducted by calculating Tanimoto coefficients [Historically, TCM encompasses a two-way philosophy in cancer treatment in that it is involved in both health strengthening and pathogen elimination. TCMs applied in curing cancer are classified as health-strengthening or pathogen-eliminating herbs according to their therapeutic effects. However, the biological functions of the two types of anti-cancer TCMs have not yet been elucidated. Therefore, we hoped to identify the regulated pathways of both types of TCM herbs by taking advantage of network pharmacology prediction and the HTSficients  between 2 assay, including 67 compounds in health-strengthening herbs, 66 compounds in pathogen-eliminating herbs, and 33 compounds in both types of herbs  replicates were also added in the wells as controls. After obtaining the gene probe reads, we performed gene expression normalization by using 18 stable genes in in colorectal cancer  database [Among the 1446 TCM compounds applied in target prediction, 166 compounds were used in the HTSof herbs D. In the2 assay after the manual adjustment.To explore the inhibitory effects on proliferation in the different cell lines of compounds from health-strengthening and pathogen-eliminating herbs, we analyzed the half-maximal inhibitory concentration (IC50) and the half-maximal inhibitory concentration on cell growth (GI50) data of the TCM compounds collected from the ChEMBL database or literature. The median IC50 (or GI50) was calculated as the median number of all the human cell line specific experiments in ChEMBL and literature records after the TCM compound treatment. These two metrics were considered as the same and were merged in the analysis. Even though the compounds from pathogen-eliminating herbs exhibited the lower median IC50s (or GI50s) than compounds from health-strengthening herbs in 2 assay suggested that some compounds may regulate immune and cancer hallmarks (immune-excluded) pathways simultaneously. As shown in 2 assay were reduced after treatment with several compounds in the two types of herbs, while the gene expressions in T cell receptor signaling pathways were upregulated after treatment with several TCM compounds from the two types of herbs. The percentage of the experimental compounds in health-strengthening herbs with predicted targets enriched in the immune-related pathways was significantly higher than that in pathogen eliminating herbs  . In 2 assay results were listed. These results indicated that TCM herbs, no matter their therapeutic classification, may exhibit therapeutic effects in cancer treatment via both immune regulation and other antitumor functions.In 2 assay results. For instance, ginsenoside Re, a compound in Radix Ginseng (Ren Shen), was suggested to exert an NK cell activation effect by upregulating the expression of genes involved in degranulation and NK cell-related cytokines release.NK cells recognize and kill tumor cells containing the mutated gene fragments . In the 2 assay results revealed that several health strengthening herbs may comprehensively increase the level of antigen presentation in tumor cells by promoting intracellular synthesis of main histocompatibility complex class I (MHC-I) molecules, improving antigen processing efficiency, and combining tumor cell biopeptides with MHC-I molecules (Cortex Cinnamomi (Rou Gui), was indicated to upregulate antigen processing and MHC biosynthesis related genes, as shown in Antigen processing and presentation plays an important role in tumor immunity . In the olecules B. For ex2 results of the TCM compounds that were predicted to regulate the same pathway. Next, we examined the expression data of the genes in the predicted regulated pathways. After the integration of the HTS2 results from the TCM compounds and the gene information in the KEGG pathways, we concluded that the TCM compounds in the same herb may have interactions in several patterns in regulating the same KEGG pathway. As shown in 2 assay results. For instance, kaempferol and kaempferide are two compounds from the health-strengthening herb Fructus Corni (Shan Zhu Yu). After analyzing the HTS2 assay results of kaempferol and kaempferide, we discovered that they have similar molecular patterns for inhibiting cell cycles, as shown in Radix Paeoniae Alba (Bai shao), were indicated to influence gene expression in the mitogen-activated protein kinase (MAPK) signaling pathway in the opposite directions  and Radix Sophorae flavescentis (Ku Shen) are two typical health-strengthening and pathogen-eliminating herbs that are widely used for cancer treatment [Radix Paeoniae Alba and Radix Sophorae flavescentis in the HTS2 assay. The target enrichment results of the pathways with the assay evidence are shown in Radix Paeoniae Alba, including 1,2,3,4,6-pentagalloylglucose, albiflorin, coumarin, galloylpaeoniflorin, paeoniflorin, and paeonol , and cancer hallmarks pathways   C. For inZi-Yin) herbs and qi-tonifying (Yi-Qi) herbs. In this research, as a first step, we treated health-strengthening herbs as a whole rather than the subcategories for the following network pharmacological analysis. Several biological processes may be involved in the regulatory effects of health-strengthening medicine, including some immune-related bioactivities. This finding is in accord with the previous studies on the comprehensive anti-tumor mechanisms of nuciferine, a compound from a yin-nourishing health-strengthening herb, Nelumbo nucifera Gaertn (He Ye) [Health-strengthening medicine is seen as a representative application of the classical philosophy of TCM in cancer therapy. It is reported that, unlike Western medicine, which may exhibit direct killing effects on tumor cells, health-strengthening medicine is developed to treat cancer by systematically regulating the tumor microenvironment ,52,53,54 (He Ye) . Additio (He Ye) ,57. Thes2 assay results, we analyzed the potential bioactivities of compounds from health-strengthening herbs. Several pathogen-eliminating herbs were also included in the research paradigm for comparison. Our prediction and assay results suggested that compounds from both types of TCMs may regulate both immune and cancer hallmarks (immune-excluded) pathways. This finding was consistent with the multitarget characteristic of TCM compounds. We further investigated the differences between these two types of TCMs and discovered that the overall inhibitory effects of the compounds in pathogen-eliminating herbs on tumor cells were significantly stronger than those in health-strengthening herbs. The results were consistent with the traditional understanding that pathogen-eliminating herbs tend to target the tumor directly. However, the traditional therapeutic advantages of health-strengthening herbs on the immune system need to be further explored by adopting immunological experimental results, and our high-throughput assay was conducted on tumor cells. Additionally, we revealed a group of compounds from the same herb that influences pathway gene expression in the same or different directions. The compounds with opposite influences on pathway gene expression may be explained by the different underlying mechanisms in cancer treatment. Therefore, it was suggested that they may be applied for treating different types of tumors.Here, based on the target prediction and the HTSRadix Paeoniae Alba (Bai Shao) was predicted to impact several pathways that are significant in tumor development, including T cell receptor signaling pathway, B cell receptor signaling pathway, cell cycle, and mTOR signaling pathway. The regulatory effects were further supported by the HTS2 gene expression profile of the related genes in the pathways after the treatment with galloylpaeoniflorin. In fact, the antitumor effects of Radix Paeoniae Alba and its total glucosides were found in recent research, and the mechanism may be related to the inhibitory effects on the cell cycle of tumor cells [In addition, galloylpaeoniflorin, a compound from a health-strengthening herb or cells ,59. NotaIn this work, we divided cancer-related pathways into two categories: immune pathways and other cancer hallmarks for separate investigations. The distinction was made for the following reason: in the aspect of clinical treatment, immunotherapy and other approaches  are distinguished treatment options available in cancer treatment . That waTo be mentioned, previous studies indicated that health-strengthening herbs may exert their therapeutic effects in cancer treatment in multiple aspects  ,17,18,19According to the analysis results in this study, health-strengthening herbs may exhibit both immune-regulatory and antitumor effects. Taking into consideration the generally weaker antitumor effects (IC50 or GI50) in vitro of compounds in health strengthening herbs than that in pathogen-eliminating herbs in public records, the high percentage of selected compounds in health-strengthening herbs related to immune-related pathways, and the good safety of health-strengthening herbs, it was indicated that health-strengthening herbs may have more pharmacological potential in preventing tumors and improving a tumor-immune microenvironment, compared to directly killing tumor cells.2 may significantly promote the parallel processing of candidate compounds and genes, and has been applied in drug screening [2 assay on one cell line, HCT116, and it does not represent cells of the immune system. Therefore, most of the results in this manuscript are hypothesis-generating and more experimental studies are needed to further explore the bioactivities regulated by TCM compounds. Still, the research strategy in this study does provided avenues for large-scale experimentation. Hopefully, our study may help reveal the biological basis of health-strengthening herbs, a characteristic herb type which has been used for a long period by TCM practitioners, and shed light on the future researches on anti-tumor drugs by unveiling the wisdom of TCM.In contrast to previous studies, our study features the usage of high-throughput computational and experimental methods for a more comprehensive understanding of underlying mechanism of health-strengthening herbs. HTScreening . HoweverBuzhong Yiqi decoction, and Sijunzi decoction) (Zi-Yin) herbs and qi-tonifying (Yi-Qi) herbs, and the 25 pathogen-eliminating herbs include categories, such as heat clearing and detoxifying (Qing-Re-Jie-Du) herbs, and blood activating and stasis dissolving (Huo-Xue-Hua-Yu) herbs, according to the traditional classification based on TCM efficacy. Together, 1446 TCM compounds with PubChem records were collected, including 655 compounds from health-strengthening herbs, 667 compounds from pathogen-eliminating herbs, and 124 compounds from both types of herbs.We collected compound information of 47 TCM herbs  that are widely used in cancer therapy from the Chinese national medical insurance catalogue and commonly used prescriptions  . The 22 2 assay, public assay data, and in vitro and in vivo experiments. Taking advantage of the prediction and analysis results, we analyzed the molecular functional patterns of health-strengthening herbs  for further analysis. The interval was necessary because the number of the related literature varied greatly, and this would have impacts on the following analysis . All the related abstracts of the selected compounds were downloaded, and text-processing codes were programed for extracting the biomolecules mentioned in the abstracts. If a biomolecule co-occurred in the abstract with the compound name, then we considered that the biomolecule was related to the compound.2 assay results.The results from the literature mining were then verified via a manual examination by deleting the false positive responses. Then, the results were used as the verification set for target prediction and HTSThe potential targets of the TCM compounds were predicted by drugCIPHER-CS  using Ma2 results were collected. If a predicted target is directly or indirectly related to a biomolecule which co-occurred with the compound in the literature or to a DEG in the HTS2 assay after treatment with the compound, it is considered to be supported by the literature mining and HTS2 assay. The indirect relationship was established if the predicted target in in the upstream in a KEGG pathway of the biomolecules in the abstracts or DEGs, or if they are related via PPI. The cover rate was calculated as Moreover, to measure the reliability of target prediction results, biomolecules mentioned in the literature and DEGs in HTSp-values of the pathways to see if they were significantly enriched. The enrichment p-values of 26 pathways from cancer hallmarks and immune-related pathways in the KEGG database were examined. The enrichment analysis was performed using RStudio v1.1.447 and an open source programming language, Ruby 2.3.0. The significantly enriched KEGG pathways  were retained for further research.We performed the KEGG pathway enrichment analysis for the predicted targets of the TCM compounds applied in cancer therapy in order to identify their biological functions. We used a hypergeometric test for enrichment analysis. We performed target enrichment under the background of 13388 human proteins and checked the CACTVS substructures in PubChem were adopted in the chemical space analysis. In the analysis, we used 881-dimensional substructure binary vectors to encode the investigated TCM compounds. A PCA analysis was conducted using the princomp function in the R packages stats v3.2.2 under the environment of RStudio v1.1.447 [Network visualization was performed using Cytoscape v3.6.0  . For vis2 at 37 °C. Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin were applied for the incubation.HCT116 , B16F10 (a melanoma cell line), MCF-7 (a breast cancer cell line), and NCI-H460 (a lung cancer cell line) cells were obtained from the cell center of Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS and PUMC). The cells were cultured in an incubator with 5% CO2 assay is a high-throughput screening strategy that enables a large-scale and quantitative analysis of gene transcriptional profiles in cells [2 assay, approximately 3000 HCT116 colorectal cancer cells were seeded in each well of a 384-well plate for 24 h. After that, the TCM compounds library was added to the cells for another 24 h, including eight DMSO replicates as negative controls. The HTS2 assay was then conducted to obtain the transcriptional profiles of the designed gene probes. The cells were lysed in GentLys buffer . The instrumentation of the HTS2 assay was an automated liquid handling system, which contained the Agilent Bravo automated liquid handling platform  and the Agilent bench robot . By RNA annealing, selection and ligation, the instrumentation automatically performed the HTS2 assay. Pooled pairs of oligonucleotides targeting selected gene probes by streptavidin-magnetic beads and the biotinylated oligo-dT were used. Then, the paired oligonucleotides were ligated using T4 DNA ligase and were amplified using Polymerase Chain Reaction (PCR). Using unique bar-coded primer in Illumina flowcells, the HTS2 assay allowed a high-throughput transcriptional profiling of up to 1400 genes from 2000 samples.The HTSin cells . In the 2 assay were performed considering their recorded biological activity data and the offer lists provided by the suppliers. The bioactivity data (IC50 and GI50) of the TCM compounds were collected from ChEMBL or via manual literature searching. The median IC50 and GI50 of a compound were achieved via calculating the median number in all human cell line specific experiments in ChEMBL and literature records. The two metrics, IC50 and GI50 were used as the same metrics in the analysis. Then, we chose the compounds with an adequate antitumor activity (median IC50 or GI50 < 100 µM) as candidate compounds for the HTS2 assay. The candidate compounds were further selected considering the product availability of the suppliers.The selection of the TCM compounds for the HTS2 assay . Detailed information about the compounds applied in the HTS2 assay , supplier, and purity) was presented in The TCM compounds were dissolved in DMSO. The concentrations were preliminarily set as the median IC50 and were adjusted afterwards based on the cell survival rate using MTS assay for the HCT116 cells to meet the standard for the HTS2 assay. The gene set contained immune-related and other antitumor-related genes. The selection of the 420 genes were achieved in three steps. First, the genes were selected from databases and by predictions to form a cancer-related gene lists, including genes in pathways in cancer (hsa05200) in KEGG, genes in colorectal cancer (hsa05210) in KEGG, the colorectal cancer related genes in OMIM (MIM Number: 114500) [2 assay was a colorectal cancer cell line.A total of 420 genes were selected to form a gene set for the HTS 114500) , the tar 114500) ,72. The 2 assay results.Then, we referred to the public gene expression profiles in the GEO database for selecting a reliable set of genes with adequate expression levels. Here, we selected two profiles of samples from patients with colorectal cancer (GSE44076 and GSE44861) and two profiles of the HCT116 cell line (GSE53295 and GSE53965) for measuring the gene expression. A gene was selected if it had at least three of the above profiles in its expression data and if the expression ranked 10% to 60% in the detected gene set in at least one profile. Additionally, we selected 30 housekeeping genes that meet the following standards: (1) it was not in the cancer-related gene lists achieved in the first step; (2) the expression of the gene was detected in the four gene expression profiles , and was not a DEG in any one of these profiles; and (3) the expression of the gene ranked 10% to 60% in the gene sets of at least one profile. The moderate ranking was to ensure that the gene expression was neither too high or too low, which may impair the credibility of the HTS2 assay, including 18 housekeeping genes. Sequences of 10 probes used in the HTS2 assay were provided in At last, the probes for the genes were designed, and 420 genes with efficient probes, were selected for the HTS2 assay after treatment with DMSO and several TCM compounds were provided in 2 assay, which represent the abundance of genes. The HTS2 data was normalized by the expression of 18 stable housekeeping genes. The normalized gene expression was computed with raw reads of the gene after the drug treatment and the median number of raw reads of 18 housekeeping genes after the drug treatment.First, the reads were mapped to the probe sequences, and three mismatches for each were permitted. The raw experimental data of HTSSecond, to identify the DEGs for each TCM compound, we calculated the fold change of the tested genes as the normalized gene expression after the drug treatment divided by the median number of normalized gene expression after the eight DMSO replicates treatments. For each TCM compound, genes with fold change > 2 were considered DEGs.Third, to evaluate the reliability of the transcriptional profile, we calculated the Pearson correlations among the normalized transcriptional data after treatment with the eight DMSO replicates. The results are demonstrated in For exploring the antitumor effects of galloylpaeoniflorin, we further performed an MTT cell viability assay on different tumor cells. The HCT116, B16F10, MCF-7, and NCI-H460 cell lines were seeded in a 96-well plate before the drug treatment. Various concentrations of galloylpaeoniflorin dissolved in DMSO and ethanol respectively were added to the cells after incubation for 24 h. To assess the IC50s of the cell lines, the MTT cell viability assay was conducted after incubation for another 120 h. The IC50s were achieved by fitting the dose-response curve. The MTT assay was done once in triplicate.3, the mice were randomly separated into three groups and were administered an oral dose of galloylpaeoniflorin (40 mg/kg/day or 80 mg/kg/day) or vehicle control (1 × solution with cremophor EL/ethanol/water (12.5:12.5:75)). The mice were sacrificed at the end of the treatment period. The tumor volume was measured and weighted for the analysis.Twenty-one six-weeks-old BALB/c/nu nude female mice obtained from the Vital River Laboratories  and were used for the xenograft experiments. H22 cells were injected into the left flank of the mice. When the tumor volume reached the size of 100–250 mmThe animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals. The work was approved by the Animal Care Committee of Chinese Academy of Medical Sciences and Peking Union Medical Colleges  (Permit Number: SYXK 2015-0025).t-tests in p < 0.05, ** p < 0.01, and *** p < 0.001.Data are shown as the means ± standard deviation (SD) E and med2 experiment, and some low-throughput assays. The expression levels of 420 genes, associated with tumor growth and immune functions, were detected after a parallel treatment of 166 TCM compounds. By combining evidence from different sources, we helped further uncover the biological basis of health-strengthening medicine. We concluded that health-strengthening herbs, might regulate both immune-related and antitumor pathways, similar to pathogen-eliminating herbs. A typical case was demonstrated by Radix Paeoniae Alba (Bai Shao), a health-strengthening herb widely used in TCM cancer treatment. Galloylpaeoniflorin, a compound from Radix Paeoniae Alba, was predicted to regulate several essential biological processes in cancer development, and its antitumor effect was preliminarily proven both in vivo and in vitro. Additionally, some TCM compounds in the same herb were indicated to regulate pathway gene expression with similar or different patterns, suggesting the urgent need for further in-depth studies on TCM prescriptions. For instance, acetyl ursolic acid and specnuezhenide, two compounds in a health strengthening herb Fructus Ligustri lucidi (Nv Zhen Zi), both upregulated gene expressions in T cell signaling pathway in HTS2 assay. In summary, this study provided a new research strategy for explaining the biological basis of health-strengthening herbs, and further suggested the tumor immune regulatory and tumor preventive potentials of health-strengthening herbs.In conclusion, in this study, we performed a network-based analysis of health-strengthening medicine by integrating a series of methods, including target prediction, literature mining, the HTS


In the United States, post-cardiac arrest debriefing has increased, but historically it has occurred rarely in our pediatric intensive care unit (PICU). A fellow-led debriefing tool was developed as a tool for fellow development, as well as to enhance communication amongst a multidisciplinary team.A curriculum and debriefing tool for fellow facilitators was developed and introduced in a 41-bed cardiac and medical PICU. Pre- and post-intervention surveys were sent to multidisciplinary PICU providers to assess effectiveness of debriefings using newly-trained leaders, as well as changes in team communication.p < .0001). Providers reported frequent identification and discussion of learning points increased from 32% pre- to 63% post-intervention. In the 12 months post-intervention, 62% of providers agreed that the overall quality of communication during arrests had improved, and 61% would be more likely to request a debriefing after cardiac arrest.Debriefing occurred after 84% (63/75) of cardiac arrests post-intervention. Providers in various team roles participated in pre-intervention (129 respondents/236 invitations) and post-intervention (96 respondents /232 invitations) surveys. Providers reported that frequently occurring debriefings increased from 9 to 58%, pre- and post-intervention respectively (The introduction of a fellow-led debriefing tool resulted in regularly performed debriefings after arrests. Despite post-intervention debriefings being led by newly-trained facilitators, the majority of PICU staff expressed satisfaction with the quality of debriefing and improvement in communication during arrests, suggesting that fellow facilitators can be effective debrief leaders. Since high quality cardiopulmonary resuscitation has been shown to improve return of spontaneous circulation and survival rates after arrests, strategies to improve resuscitation quality have become important areas of research –3. StrucStructured debriefings have been shown to increase the feasibility of introducing a debriefing program , 8. SpecTo better identify targets for improvement in teamwork and medical management of life-threatening events occurring in the PICU, we designed a fellow-driven debriefing program. The main intervention for this project was the creation and implementation of two tools to standardize debriefing: the Data Sheet and the Debriefing Tool. The Data Sheet Fig. a createdPICU fellows, as they consistently attend all arrests in the PICU where attendings are not overnight in house, were selected as the formal debriefing leaders. The fellows were introduced to the Data Sheet and Debriefing Tool during fellow orientation. Fellows also attended a yearly faculty-led debriefing workshop to refresh debriefing skills. The workshop included simulated scenarios and role-playing as debrief leaders, as well as orientation to the Debriefing Tool.The study of this intervention took place at an urban, quaternary care hospital with a 41 bed PICU, caring for all critically-ill pediatric medical, surgical and cardiac patients . Debriefing was expected after every arrest, occurred in the PICU team room, and, whenever possible, occurred within the same nursing shift as the event. All resuscitation team members were invited to participate in debriefings .The debriefing facilitator, typically the fellow who had participated in the arrest, began by welcoming the participants, followed by an assurance that the discussion was occurring without judgment and that the goal was to improve team resuscitation skills. The facilitator next asked the group for general thoughts about the resuscitation. Finally, the facilitator selected two to three focus areas (topic suggestions listed at the top of the Debriefing Tool) and led the team through Gather, Analyze, Summarize Cycles addressing each area. As the debriefings were intended to occur directly after the arrests, the debriefing was not data-driven. Discussion points were recorded in the Debriefing Tool for later analysis. For the purpose of this study, topics most frequently discussed during the debriefings were analyzed using the Team Emergency Assessment Measure (TEAM) framework, a validated tool to evaluate medical resuscitation . The DatThrough a series of Plan-Do-Study-Act (PDSA) cycles Fig. , the DatSurveyMonkey.com). After completing the survey, staff members were invited to enter a voluntary raffle for a $20 gift card. Responses to survey questions were analyzed. Descriptive statistics were used to analyze the quantitative data from arrest sheets; categorical questionnaire responses were analyzed using 2-tailed Fisher’s exact tests. A p value of < 0.05 was considered significant.Faculty provided monthly feedback to the PICU fellows regarding their compliance with debriefing and use of the Debriefing Tool. Additionally, a survey (consisting of 24 multiple-choice questions and one free-text response) was emailed to PICU providers in February 2015 (pre-intervention) and May 2016 (post-intervention) to gather data from multidisciplinary team members  regarding their attitudes and experiences surrounding debriefing and communication. Participation in the surveys was voluntary and anonymous and conducted via an online website .Overall survey response rate was 55% (129 respondents out of 236 invitations) for the pre-intervention survey and 41% (96 respondents out of 232 invitations) for the post-intervention survey. The responder role did not differ significantly between the two surveys (Table p < 0.0001). The most frequently reported debriefing leader pre-intervention was the PICU attending (45%), whereas post-intervention, the PICU fellow (71%) was reported as the most frequent debriefing leader. Additional questions in the post-intervention survey assessed changes in provider practice related to debriefings  also increased post-intervention. Perceived debriefings occurring at least frequently increased from 9 to 58%, pre- and post-intervention respectively , detecting a change in mortality rate would likely not be feasible due to the historically low mortality rate in our PICU. Finally, although there was an increase in learning point identification, the perceived frequency of learning point dissemination remained low post-intervention. Improving the dissemination of learning points from debriefing will be the focus of ongoing quality improvement efforts.The development and implication of a debriefing curriculum may be an effective tool in leadership development for fellow facilitators, as well as a means to improve communication amongst interdisciplinary team members in the PICU. The simplicity and low cost of adapting scripts for fellow debriefing leaders could be applied to other clinicians, such as nurses or residents, and to events other than cardiac arrest; its use may extend far beyond the intensive care unit. Given the proper support in the form of a scripted debrief tool, fellow facilitators develop valuable leadership skills and may provide a means to improve teamwork and communication throughout the hospital.