The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.While cochlear implantation (CI) technology has greatly improved over the past 40 years, one aspect of CI that continues to pose difficulties is the variability of outcomes due to numerous factors involved in postimplantation performance. The electric acoustic stimulation (EAS) system has expanded indications for CI to include patients with residual hearing, and is currently becoming a standard therapy for these patients. Genetic disorders are known to be the most common cause of congenital/early‐onset sensorineural hearing loss, and are also involved in a considerable proportion of cases of late‐onset hearing loss. There has been a great deal of progress in the identification of deafness genes over the last two decades. Currently, more than 100 genes have been reported to be associated with non‐syndromic hearing loss. Patients possessing a variety of deafness gene mutations have achieved satisfactory auditory performance after CI/EAS, suggesting that identification of the genetic background facilitates prediction of post‐CI/EAS performance. When the intra‐cochlear etiology is associated with a specific genetic background, there is a potential for good CI performance. Thus, it is essential to determine which region of the cochlea is affected by identifying the responsible genes. This review summarizes the genetic background of the patients receiving CI/EAS, and introduces detailed clinical data and CI/EAS outcomes in representative examples. Anat Rec, 303:563–593, 2020. © 2020 The Authors.  Cochlear implantation (CI) is the surgical insertion of a device to provide electrical stimulation directly to the auditory nerve Fig. . CI is aGJB2 (29%), followed by SLC26A4 (9%), CDH23 (7%), MYO7A (4%), OTOF (5%), MYO15A (3%), and LOXHD1 (2%), indicating that these deafness genes are typical deafness genes found in the prelingual CI/EAS patients. A further 9% of the patients were diagnosed with syndromic deafness associated with other symptoms. For postlingual CI/EAS patients, a genetic etiology was detectable in ~36% of cases. Although genetic causes were the most common, a number of different kinds of causative genes, including various rare genes, were found to be involved in postlingual CI/EAS patients. The most common causative gene was CDH23 (9%), followed by MYO7A (4%), TMPRSS3 (4%), MYO15A (2%), DFNB31 (1%), ACTG1 (2%), DFNA5 (1%), MYO6 (1%), and CRYM (1%). In the postlingual CI/EAS patients, mitochondrial m.3243A>G (1%) and m.1555A>G mutations (2%) were also found to be involved. Compared to the prelingual group, many genes with autosomal dominant inheritance, such as MYO7A, ACTG1, DFNA5, MYO6, and CRYM, as well as mitochondrial genes reported to cause progressive hearing loss, were found to be involved.Although CI provides a good outcome for the majority of cases, factors affecting the outcomes of CI vary among patients. One reason of such variations is thought to be the heterogeneous cause of hearing loss. The function of the cochlea could be affected by various factors, including genetic factors, viral infections, and congenital anomalies. Among them, genetic factors represent the most common etiology in severe‐to‐profound hearing loss, and might be one of the key determinants of outcomes for CI and electric acoustic stimulation (EAS)  analysis has helped clarify genetic epidemiology; i.e., The cochlea is composed of various types of cells. The coordinated action of various molecules is essential for the normal development and maintenance of auditory processing in the cochlea. A series of studies have clarified the localization of these molecules in the cochlea  was separated, and then total RNA was extracted from each slice and converted to cDNA. Each gene has a specific expression pattern , good CI outcomes may potentially be achieved. Therefore, efforts for the identification of the region of the cochlea involved (either inside or outside the cochlea) should be made by delineating the responsible genes. Regarding the outcomes of CI, the LittlEARS auditory questionnaire was used to assess the auditory behavior development before the operation and at 3, 6, and 12 months after CI. Although scores varied among the patients, the majority of non‐syndromic hearing loss patients with specific deafness gene mutations showed good and rapid development of auditory behavior. In contrast, syndromic hearing loss patients as well as the patients with inner ear anomalies showed comparatively poorer and slower development , was the first gene identified as being involved with non‐syndromic hearing impairment , and there has been a general rule applied to the relationship between mutations and hearing loss; that inactivating mutations (deletion mutations and stop mutations) result in more severe phenotypes compared to those caused by non‐inactivating mutations (missense mutations) , and the parents were found to be carriers for the mutation  to non‐syndromic hearing loss with enlarged vestibular aqueducts , and the parents were found to be carriers for the mutation  had compound heterozygous ion Fig. . Her newion Fig. . CT scanion Fig. . COR evaion Fig.  and she ion Fig. , and a mion Fig. . CI outcSLC26A4 gene. Thereby, a clinician knew that she may experience profound hearing loss, which could potentially worsen. Amplification using hearing aids was found to be insufficient for good language development. Taken together with her etiology of hearing loss (intra‐cochlear etiology), these findings indicated that CI was the recommended intervention. Therefore, a wait‐and‐see strategy was identified as not a good option for this patient. The clinician was also able to provide information regarding the future possible appearance of other symptoms (vertigo and goiter) in association with the mutations in this gene.In this case, genetic testing identified compound heterozygous mutations in the CDH23, a member of the cadherin superfamily, encodes calcium‐dependent cell–cell adhesion glycoproteins, and is expressed in the inner and outer hair cells in the cochlea  or non‐syndromic hearing loss (DFNB12). The phenotype range of DFNB12 is variable from congenital profound hearing loss to adult‐onset high‐frequency involved hearing loss. As shown in our past studies, CDH23 mutations are often identified in patients having recessive inheritance, and one phenotypic feature is the presence of residual hearing , and the parents were determined to have been the mutation carriers  and USH1D   Fig. . He had W]) Fig. , we chosW]) Fig. . TherefoW]) Fig. . As showW]) Fig. , hearingW]) Fig. .MYO7A and MYO15A encode myosin VIIA and XVA, members of the unconventional myosin superfamily of proteins . MYO7A mutations may cause various phenotypes ranging from non‐syndromic hearing loss (DFNA11/DFNB2) to Usher syndrome (USH1B)  for the left ear at the age of 10. Residual hearing was completely preserved six months after the surgery. She uses the natural residual hearing combined with ES, and does not require any acoustic amplification at low frequency. Speech perception test results were dramatically improved   inherited from the parents  Fig. .ACTG1 encodes γ‐actin, the predominant actin isoform in the inner and outer hair cells in the cochlea. Within the hair cells, γ‐actin is an important component of the cuticular plate, adherens junctions, and stereocilia , and his father, brother, and younger son carried the same mutation. Due to progressive hearing loss and insufficient amplification by hearing aids, he received EAS at age 39. His brother showed a similar audiogram configuration from around 15 years old. Residual hearing was preserved and monosyllable tests showed dramatic improvement. Speech perception test results were dramatically improved from 20% to 80% one year after receiving the EAS  DFNB10: congenital or early childhood onset, with severe and prelingual hearing impairment, and (2) DFNB8: a later‐onset ski‐slope type audiogram and progressive postlingual hearing impairment.Tmprss3, hair cells in the organ of Corti were found to be deteriorated at the start of the basal turn, and progressing toward the apex   Fig. . PreservT]) Fig. –E. An imT]) Fig. . After bT]) Fig. .The m.1555A>G mutation in the mitochondrial 12S ribosomal RNA gene is the most common mitochondrial mutation associated with hearing loss  is predominantly expressed in the cochlear inner hair cells, and is essential for synaptic exocytosis at their ribbon synapses  is hearing loss derived from the dysfunction of auditory signals downstream of and/or including the inner hair cells. It is marked by loss of ABR signal yet retention of OAE and/or cochlear microphonics (CM). Now known as “auditory neuropathy spectrum disorder (ANSD),” AN was renamed in 2008 due to being a spectrum of associated symptoms   Fig. . Based oQ]) Fig. . Speech Q]) Fig. .LOXHD1, a known cause of DFNB77, is the gene for the protein Lipoxygenase Homology Domains 1, comprised entirely of 15 PLAT  domains   Fig. . His sisA]) Fig. . His speA]) Fig. .COCH encodes extracellular protein “cochlin” that consists of a single peptide, a late gestation lung protein Lgl1 (LCCL) domain, and two von Willebrand factor A (vWFA) domains   Fig.  Tsukada. In his ely Fig. .The above results suggest that the genes presented may be involved in hearing loss in CI patients. As patients with these mutations showed relatively good auditory performance after receiving CI or EAS, genetic background could be added as one of the factors useful in predicting performance after implantation. Also genetic testing is helpful in predicting future hearing levels; therefore, genetic diagnosis could facilitate decision making for early intervention. Furthermore, genetic testing is potentially useful for evaluating residual hearing, estimating progression, and successful hearing preservation making it valuable in the selection of candidates and electrodes for CI or EAS.The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results.


Therefore, a rabbit milk replacer (RMR) was formulated in this study for comparison with these commercial products. Early weaned rabbits at 18 days of age were fed daily with RMR, KMR or MMR until 36 days after birth, while a commercial pelleted diet and water were provided at an amount exceeding the normal intake. The results indicated that it is possible to use RMR as a milk replacer for rabbits without serious adverse consequences. However, the RMR group presented a lower trend in nutrient digestibility than the other groups, although there was no statistical significant difference. Therefore, prebiotics and/or probiotics should be added to RMR formulations to improve this parameter.A milk replacer must be given as the main diet to young rabbits that are separated from their mothers before they reach weaning age (31–35 days). This procedure, which is a rescue protocol, allows them to survive. Moreover, the early separation of young rabbits before weaning prevents negative consequences in lactating rabbits, which is beneficial to pet rabbit producers. Kitten (KMR®: KMR) and a mammal milk replacer (Zoologic® Milk matrix 30/52: MMR). Thirty-six native crossbred rabbits aged 18 days were divided into three experimental groups , fed RMR, KMR or MMR daily until they were 36 days old and euthanized at 38 days, while a complete pelleted diet and water were provided ad libitum. No statistically significant differences were observed in growth performance parameters, water intake, faecal weight, nutrient digestibility, internal organ weight, caecal pH, caecal cellulose activity, number of faecal pellets and amount of crude protein intake (p > 0.05). Caecal amylase activity in the KMR group and caecal protease activity in the RMR group were higher than in the MMR group (p < 0.05). The villus height and crypt depth of the MMR group were greater than in the RMR and KMR group (p < 0.05). In conclusion, it is possible to feed RMR to early weaning rabbits without serious adverse effects. However, probiotics and/or prebiotics should be supplemented in milk replacers and their benefits studied.Early weaned rabbits should be fed using a milk replacer in order to survive. Therefore, a rabbit milk replacer (RMR) was developed and compared with a kitten milk replacer (KMR The size of the pet market has increased sharply in recent years and was estimated to be around 131.7 billion US dollars in 2016 [Generally, rabbits are weaned at 31–35 days of age by rabbit producers in Thailand and other countries ,5 and beSylvilagus audubonii) and eastern cottontail rabbits (S. floridanus), for which the mortality rate was 26–59% [Commercial rabbit milk replacer remains lacking or unavailable in some countries. For this reason, kitten milk replacer (KMR) has been suggested as a substitute . Howevers 26–59% . The higs 26–59% . Therefos 26–59% . Moreoves 26–59% . TherefoThis study was conducted following standard guidelines at the animal experimental unit, Faculty of Veterinary Medicine  and was approved by the Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (ACKU62-VET-037).®; Pet-Ag Inc., Hampshire, IL, USA); and (3) rabbits fed mammal milk replacer . Rabbits were placed in a stainless cage (35 cm × 35 cm × 35 cm) with controlled room temperature, light and humidity at 20 ± 2 °C, 16L:8D and 75 ± 10%, respectively. The experiment was conducted for 20 days until the rabbits reached 38 days of age. At the end of the experiments, one rabbit per replicate was euthanized by intraperitoneal injection with pentobarbital sodium at 100 mg/kg  [Thirty-six 18-day-old native crossbreed rabbits with initial body weights of 134 ± 6.31 g/head (mean ± standard deviation) were taken from a local rabbit farm . Rabbits were randomly separated into three experimental groups with equal numbers of each sex (six replicates per group and two rabbits per replicate) containing: (1) rabbits fed rabbit milk replacer, which was formulated in this study (RMR); (2) rabbits fed kitten milk replacer (KMR France)  and the ad libitum throughout the experiment. The KMR and MMR powders were diluted with warmed water at a 7:13 ratio and homogenized. The RMR consisted of two parts: a hydrogenated palm fat part and a mixed dried powder part, containing all ingredients except hydrogenated palm fat and polyoxyethylene (80) sorbitan monooleate. For RMR preparation, hydrogenated palm fat was heated in an 800 W microwave for 60 s, changing from solid to liquid form as a result. The dried mixed powder part was then mixed with warm water. Subsequently, the two parts were homogenized and polyoxyethylene (80) sorbitan monooleate was added as an emulsifier. The ratio of mixed dried powder to hydrogenated palm fat to warm water was 24.5:17.5:78. The dilution ratio was selected based on equal dry matter content between the milk replacers and the solubility of the milk mixture.The formulation of the RMR, including the chemical composition of the milk replacer, complete pelleted diet and rabbit milk, is illustrated in The animals were weighed at 18, 24, 30 and 36 days of age, whereas average daily feed intake (ADFI), average daily weight gain (ADG), feed conversion ratio (FCR), water intake and weight of faeces output were evaluated at 19–24, 25–30 and 31–36 days of age. The apparent digestibility of dry matter, organic matter, ether extract and crude protein was conducted at 23–27 and 31–35 days of age and contained six replicates/groups. The procedures for feeding, faecal collection, chemical analysis and calculation were in accordance with . BrieflyThe internal organ weight and the body weight of the euthanized rabbits were determined. The duodenal part of the small intestine was fixed in 10% buffered formalin for further villus morphometric evaluation. Briefly, small pieces of middle duodenum after fixation were processed, embedded in paraffin, sectioned at 7-µm thicknesses by means of a rotary microtome  and stained by haematoxylin and eosin method. Villi height and crypt depth were evaluated under a microscope using an image analysis programme . Caecal pH was measured directly using a Crison MicropH 2001 pH meter . The caecal content was immediately placed in sterile plastic tubes under ice for enzyme preservation and kept at −20 °C for further analysis of caecal enzyme activity. w/v). The homogenates were centrifuged at 18,000× g for 30 min at 4 °C to obtain the supernatant used to evaluate amylase, protease and cellulase activity. Amylase and cellulase activity were assayed according to [The crude enzyme extracted from the caecal content was extracted by homogenized caecal content with phosphate buffer solution (pH 7) at a 1:5 ratio  was used to compare the different types of milk replacers (fixed factors) for internal organ characteristics, caecal pH, caecal digestive enzyme activities and duodenal histology, whereas the growth performances, water intake, faeces excretion, apparent digestibility, number of faecal pellets and amount of protein intake were analyzed by two-way mixed analysis of variance, with treatment groups or age serving as the between-subjects or the within-subjects factor, respectively. Duncan’s multiple range test was used for post hoc analysis. Differences were considered statistically significant at  package .p > 0.05). The age increment was correlated with increased body weight, ADFI, ADG, FCR, water intake, faeces excretion and crude protein intake (p < 0.05), whereas apparent digestibility did not affect dry matter, organic matter or ether extract (p > 0.05). However, the crude protein digestibility of rabbits at 31–35 days old was lower than rabbits at 23–27 days old (p < 0.05). The rabbits fed KMR and MMR displayed higher nutrient digestibility in both age ranges compared with rabbits fed RMR, but there was no statistically significant difference (p > 0.05). Rabbits in RMR, KMR and MMR were received the crude protein from milk daily at 2.23, 2.61 and 1.62 g dry matter/head. No deaths, morbidities or clinical signs were observed in rabbits during the experimental period. Moreover, a rabbit in each replicate was not euthanized at the end of the experiment and remained alive until it reached two months of age. In addition, there were no problems of milk perception and palatability in any group in this experiment, because the rabbits sucked milk directly from the syringe without any force feeding.The effects on performance, apparent digestibility, amount of faeces excretion and crude protein intake from rabbits fed the different milk replacers are shown in p > 0.05). Caecal pH was not influenced by the differences in milk replacers (p > 0.05). Caecal amylase activity in the MMR group was lower than in the KMR group (p < 0.05), whereas greater caecal protease activity was observed in the RMR group compared to the MMR group (p < 0.05). Cellulase activity was not affected by the treatments (p > 0.05). Respectively, the shortest villus and the shallowest crypt depth were found in the RMR and the KMR groups compared to the MMR group (p < 0.05).The consequences for internal organ weight, caecal pH, duodenal wall histology and digestive enzyme activities between the groups are compared in p < 0.001); however, a sharp drop occurred in all study groups at 35 days of age, followed by another increase. The largest number of faecal pellets existed in the MMR group compared to the RMR group . A significant interaction between the fixed factors (age and treatment group) was identified (p < 0.05). Generally, the same increasing trend was observed in all groups, except for the sharp rise in the number of faecal pellets in the MMR, RMR and KMR groups at 22–23, 24–26 and 34–36 days of age, respectively.The average number of faeces pellets from two rabbits of each replicate in the experiments is illustrated in The RMR was formulated according to the profile of rabbit milk; therefore, its chemical composition was the most similar to the composition of rabbit milk . KMR conFat in rabbit milk represents the major energy sources for rabbits , whereasA previous report has shown that the gut microbial community of rabbits plays an important role relating to the efficiency of nutrient fermentation, productive performance and heath condition . A steriLactobacillus sp., Enterococcus faecium, Bifidobacterium bifidum, Pediococcus adicilactici) and prebiotics (fructooligosaccharide) in the KMR formulation contributed to higher enzyme activity, whereas RMR and MMR did not contain these supplements. Such findings are in accordance with another research study [Caecal enzyme activity was another indicator representing microbial activity. The amount of caecal amylase and protease in the RMR and KMR groups was higher than in the MMR group. Fermentation products from probiotic bacteria and prebiotics play an important role in supporting the development of a normal flora in rabbits . Supplemch study . A highech study . Thus, pNo statistically significant difference in growth performance was observed in this study. However, a trend existed that can be explained in detail. The lowest FCR was observed in the RMR group as a consequence of the lower ADFI and the higher ADG. This may have been due to the appropriate crude protein and fat proportion in the RMR, which promoted lower adaptation following the milk replacer in the first period. However, the highest ADFI was observed in the KMR group as the lowest energy density because the feed intake was stimulated by a physiological function until the animal had obtained its daily energy requirement . TherefoLower growth performance and survival rates were reported in the group fed with KMR compared with the group fed with rabbit milk from lactating does . UnfortuDifferences in efficiency between the RMR developed in this study and commercial milk replacers (KMR and MMR) used in 18-day-old rabbits were revealed in the current study. Based on the results, it was possible to use RMR as a milk replacer for 18-day-old rabbits and to wean at 36 days of age, this not providing any adverse consequences for final body weight, ADG, FCR, ADFI, nutrient digestibility, internal organ characteristics, caecal pH, amount of faeces excretion and crude protein intake. Lower nutrient digestibility was observed in the RMR group without statistically significant differences. Therefore, probiotics and/or prebiotics can be supplemented in formulations to promote a suitable microbial community and to provide benefits in terms of growth performance and nutrient digestibility.


Peteinosaurus zambelli and Macronychoptera, a clade here named Zambellisauria (clade nov.), as well as for a monophyletic and early diverging Preondactylia. Some analyses also support the existence of a clade that falls as sister-taxon to the zambellisaurs, here named Caviramidae (clade nov.). Furthermore, some support has been found for a monophyletic Austriadraconidae at the base of Pterosauria. Somewhat surprisingly, Lagerpetidae is recovered outside of Ornithodira sensu stricto, meaning that, based upon current definitions at least, pterosaurs fall within Dinosauromorpha in this analysis. However, fundamental ornithodiran interrelationships were not the focus of this study and this particular result should be treated with caution for now. However, these results do further highlight the need for broader taxon and character sampling in phylogenetic analyses, and the effects of outgroup choice on determining ingroup relationships.The pterosaurs first appear in the fossil record in the middle of the Late Triassic. Their earliest representatives are known from Northern Hemisphere localities but, by the end of the Jurassic Period, this clade of flying reptiles achieved a global distribution, as well as high levels of diversity and disparity. Our understanding of early pterosaur evolution and the fundamental interrelationships within Pterosauria has improved dramatically in recent decades. However, there is still debate about how the various pterosaur subgroups relate to one another and about which taxa comprise these. Many recent phylogenetic analyses, while sampling well from among the known Triassic and Early Jurassic pterosaurs, have not included many non-pterosaurian ornithodirans or other avemetatarsalians. Given the close relationship between these groups of archosaurs, the omission of other ornithodirans and avemetatarsalians has the potential to adversely affect the results of phylogenetic analyses, in terms of character optimisation and ingroup relationships recovered. This study has addressed this issue and tests the relationships between the early diverging pterosaur taxa following the addition of avemetatarsalian taxa and anatomical characters to an existing early pterosaur dataset. This study has, for the first time, included taxa that represent the aphanosaurs, lagerpetids, silesaurids and dinosaurs, in addition to early pterosaurs. Anatomical characters used in other recent studies of archosaurs and early dinosaurs have also been incorporated. By expanding the outgroup taxa and anatomical character coverage in this pterosaur dataset, better resolution between the taxa within certain early pterosaur subclades has been achieved and stronger support for some existing clades has been found; other purported clades of early pterosaurs have not been found in this analysis—for example there is no support for a monophyletic Eopterosauria or Eudimorphodontidae. Further support has been found for a sister-taxon relationship between  Pterosaurs were a diverse, disparate and highly specialised group of terrestrial reptiles that represent the oldest set of vertebrates currently understood to have achieved powered flight . OriginaThe earliest pterosaurs were generally small bodied animals, with toothed upper and lower jaws and usually an elongated tail , 2019. IWithin Pterosauria, which currently comprises the same set of taxa as the clade Pterosauromorpha see, , there ePreondactylus buffarinii and Austriadactylus cristatus. This pairing of Preondactylus buffarinii and Austriadactylus cristatus concurs with the findings of Peteinosaurus zambellii and Eudimorphodontoidea, a topology also supported by The recent phylogenetic analysis by Peteinosaurus zambellii in the analysis of Eudimorphodon ranzii within Lonchognatha, and finds no evidence for a close relationship between Eudimorphodon ranzii, Carniadactylus rosenfeldi and Arcticodactylus cromptonellus  is present in theropods and ornithischians, whereas state (1) describes the condition in ‘basal’ pterosaurs, herrerasaurs, sauropodomorphs, silesaurids, lagerpetids and Marasuchus lilloensis. The full list of characters added to the data matrix is given in the In addition to the new taxa, 27 new anatomical characters were incorporated into the dataset of Of the additional characters, 108 and 109 were treated as ordered, following k-values) set to 3, 5, and 10 . However, despite the agreement between Raeticodactylus’ filisurensis and Caviramus schesaplanensis to be members of this more ‘derived’ clade /Preondactylia + (Caviramidae + Zambellisauria))Preondactylus bufarinii and Quetzalcoatlus northropi and all their descendants , 9 (0-->1), 16 (1-->2), 22 (0-->1), 39 (0-->1), 40 (0-->1), 58 (0-->1), 60 (0-->1), 70 (0-->1), 74 (0-->1), 75 (0-->1), 91 (0-->1), 94 (0-->1), 117 (0-->1), 120 (2-->0).Preondactylus bufarinii, together with Austriadactylus cristatus, forms the earliest diverging clade within Pterosauria—a monophyletic Preondactylia—this definition for Pterosauria encompasses the same set of taxa as Pterosauromorpha, sensu Remarks: The definition given by Node 5. Preondactylia, sensu Preondactylus bufarinii + Austriadactylus cristatus)Preondactylus bufarinii and Austriadactylus cristatus.Definition: The least inclusive clade that includes Character support: 11 (0-->1), 48 (0-->1), 49 (0-->1).Remarks: Often found to be the earliest diverging members of Pterosauria , or earlUnnamed clade (Caviramidae + Zambellisauria)Character support: 45 (1-->0), 47 (1-->0), 77 (0-->1), 95 (0-->1).Preondactylus bufarinii + Austriadactylus cristatus that is, all pterosaurs more ‘derived’ than Preondactylia. This clade has consistently been found among studies Arcticodactylus cromptonellus and Caviramus schesaplanensis (new).Definition: Node-based—the least inclusive clade that includes Caviramus schesaplanensis—one of two anchoring member taxa used in the node-based definition for the clade, as outlined above.Etymology: For Character support: 3 (0-->1), a skull that is curved down caudally; 12 (0-->1), a jugal process of the maxilla that is subtrapezoidal, tapering to a point only distally, and a proximal part with parallel dorsal and ventral margins; 43 (0-->1), a dentition that has tri- to quinticuspid tooth crowns; 81 (0-->1), a wing phalanx two that is as long as the ulna.Remarks: This clade has been recovered in the recent analysis of Node 6. Austriadraconidea, sensu Arcticodactylus cromptonellus + Austriadraco dallavecchiai + Seazzadactylus venieri)Arcticodactylus cromptonellus and Austriadraco dallavecchiai, sensu Definition: The least inclusive clade that includes Character support: 103 (1-->0), 120 (0-->1).Seazzadactylus venieri; as in the analysis of Remarks: This clade was named by Unnamed clade Character support: 11 (0-->2), 21 (0-->1), 65 (0-->1), 76, (0-->1), 80 (0-->1).Remarks: This clade was also recovered in the analysis by Node 8. Zambellisauria (new clade) (Peteinosaurus zambelli + Macronychoptera)Peteinosaurus zambelli, D. macronyx, Pterodactylus antiquus and Q. northropi (new).Definition: Node-based—the least inclusive clade that includes Peteinosaurus zambelli was named; Peteinosaurus zambelli being one of the two taxa chosen as an anchor in this cladistic definition. Eudimorphodon ranzii, a well-known and important early pterosaur and putative member of Zambellisauria , more than three sacral vertebrae; 59 (0-->1), filiform processes of the caudal zygapophyses present in caudal vertebrae.Peteinosaurus zambelli as sister-taxon to Eudimorphodontoidae, within a monophyletic Eopterosauria—a hypothesis that has fallen out favour in more recent studies and is not supported in any of the analyses carried out in this study.Remarks: This clade is consistently recovered by most modern analyses  and in aNode 9. Macronychoptera (Dimorphodontidae + Lonchognatha)Character support: 63 (1->2), 64 (0->1), 65 (0->1)Node 10. Dimorphodontidae (D. macronyx + Caelestiventus hanseni)Character support: 7 (3-->2), 8 (0-->1), 10 (0-->1), 12 (0-->2), 20 (0-->1), 26 (0-->1), 35 (0-->1), 41 (0-->1), 48 (0-->1), 52 (0-->1), 96 (1-->0), 118 (1-->2).D. macronyx and Caelestiventus hanseniRemarks: As in the analyses of Node 11. LonchognathaCharacter support: 70 (1-->2), 80 (0-->2), 88 (0-->1)Campylognathoides to be ‘less derived’ than Eudimorphodon species (see below).Remarks: The composition of Lonchognatha and its possible sister-taxon relationship with Dimorphodontidae has been fairly consistently recovered in recent analyses , althougSeazzadactylus venieri. This is similar to the result obtained in the first analysis, although the position of Austriadraconidae is different in both the reduced and full second analyses , the earliest diverging clade of pterosaurs are the Austriadraconidae—however, this clade was not found to contain Seazzadactylus venieri in this analysis. The clade made up of Austriadactylus cristatus and Preondactylus buffarini, named above as Preondactylidae, then forms the sister-taxon of the clade of Peteinosaurus zambelli + Macronychoptera, named above as Zambellisauria. In searches with k at or above 10 however, the structure of the tree once again changes, with the preondactylids once again falling out as the most stem-ward of the clades in Pterosauria and, as in some of the previous analyses of this study, Austriadraconidae was found to contain Seazzadactylus venieri , or Austriadraconidae (when using TBR branch swapping or implied weights with k < 10).What this study has also demonstrated is how using different approaches to phylogenetic analysis can produce substantially different results when it comes to the interrelationships within Pterosauria. The earliest diverging clade of pterosaurs has been found to be either the Preondactylia (in analysis one and in implied weights analyses for Peteinosaurus zambellii and the various members of Macronychoptera. This clade is now consistently recovered in most phylogenetic analyses and is strongly supported in the results of these analyses. Should Peteinosaurus zambellii be recovered in a much more stem-ward position in the future, for example if future analyses resurrect Eopterosauria, with Peteinosaurus zambellii contained within it, the definition for Zambelliasauria would then encompass the same set of taxa as Pterosauria and, as a result, would become obsolete. However, in the emerging consensus on early pterosaur relationships in this part of the tree, the clade Zambellisauria remains well-supported and distinct for now.However, in spite of this uncertainty, some clades have been consistently recovered throughout the various analyses of this study, and many too have been supported in previous analyses . This woPeteinosaurus zambellii and Eudimorphodon ranzii, which have a tendancy to ‘bounce around’ the tree, so long as the Autriadraconids are not found to be either more or less ‘derived’ than the small clade containing Carniadactylus rosenfeldi, ‘Raeticodactylus’ filisurensis, and Caviramus schesaplanensi. It is only in hypotheses in which Austriadraconids fall as the earliest diverging members of Pterosauria (e.g. analysis two of this study; Under the definition given for Caviramidae, as also given above, in scenarios in which a distinct monophyletic group is found to be more ‘derived’ than Preondactylia but outside of the clade containing all other, more ‘derived’ pterosaurs see , the namEudimorphodon ranzii is a particularly difficult taxon in terms of its position, having been recovered in a range of ‘derived’ and more ‘basal’ positions within the tree, in the both the analyses of this study and previous recent studies. With the addition of more taxa and more characters, and as more phylogenetic analysis techniques are turned on the question of pterosaur systematics, such problems of placing difficult to classify taxa may yet be resolved.It would also appear from the results of these analyses, and other recent works, that the higher-level interrelationships between pterosaur taxa are becoming more stabilised. Clades such as Dimorphodontidae, Lonchognatha, Novialoidea, Caelidracones, Monofenestrata, Pterodactyloidea, and Anurognathidae are all consistently found in these analyses, and only the composition of each varies a little between them. In particular Pterosaur interrelationships have been shown to vary between analyses, with the fundamental interrelationships that are recovered being dependant upon the method of analysis, the character choice and taxon choice. The addition of more appropriate avemetatarsalian outgroup taxa to the early pterosaur dataset of 10.7717/peerj.9604/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9604/supp-2Supplemental Information 2Click here for additional data file.


Taekwondo athletes had lower LBM and jumping power per LBM. RFD was similar between groups at 30–50 ms, but higher for throwers at 80–250 ms. RFD adjusted for VL thickness was higher in taekwondo athletes at 30 ms, but higher in throwers at 200–250 ms. These results suggest that lower body LBM is correlated with RFD in power trained athletes. RFD adjusted for VL thickness might be more relevant to evaluate in power athletes with low LBM, while late RFD might be more relevant to evaluate in athletes with higher LBM.The rate of force development (RFD) is vital for power athletes. Lean body mass (LBM) is considered to be an essential contributor to RFD, nevertheless high RFD may be achieved by athletes with either high or low LBM. The aim of the study was to describe the relationship between lower-body LBM and RFD, and to compare RFD in taekwondo athletes and track and field (T&F) throwers, the latter having higher LBM when compared to taekwondo athletes. Nine taekwondo athletes and nine T&F throwers were evaluated for countermovement jumping, isometric leg press and leg extension RFD, vastus lateralis (VL), and medial gastrocnemius muscle architecture and body composition. Lower body LBM was correlated with RFD 0–250 ms ( Explosive strength is of great importance for performance in several power-demanding sports, such as sprints, long and high jumps, athletic throws, and martial arts. Explosive strength may be evaluated with the rate of force development , whicRFD is influenced by different factors at early <100 ms) and late phases (>100 ms) from the onset of muscle contraction ,7,8. The00 ms andMuscle architectural characteristics, fascicle length, pennation angle, and muscle thickness, may also influence RDF ,14,16. PThe aim of the study was to describe the relationship between lower-body lean mass and RFD and to compare lower-body RFD in taekwondo athletes and track and field throwers, the latter having higher lean body mass when compared to taekwondo athletes. For this purpose, maximal isometric leg press and leg extension RFD curves were recorded from taekwondo athletes and track and field throwers of national and international level. It was hypothesized that (1) lower body lean mass would be associated with RFD in both athletic populations, but also (2) that they would differ in early and late RFD characteristics. Muscle architecture was also evaluated to provide further insight into the muscle morphology origin of the anticipated differences in RFD between the two athletic groups.All of the measurements were performed within 20–30 days before the national championship for each athletic group. Athletes visited the laboratory on four different occasions. On day 1, they gave their written consent to participate in the study, and their lower-extremity dominance and anthropometry were evaluated. Familiarization with the countermovement jump (CMJ), isometric leg press and leg extension RFD and maximal isometric force/torque were also performed. On day 2, three days later, athletes visited the laboratory for muscle architecture evaluation and another testing familiarization. On day 3, three days later, they performed the testing session. On day 4, three to four days after the testing session, body composition analysis was performed (see description below). All of the procedures were approved by the local university ethics committee.Nine male taekwondo athletes  and nine male track and field throwers  gave their written consent to participate in the study after being informed about the experimental procedures as well as the risks and benefits of this research. Four of the taekwondo athletes were champions in the previous three years within separate weight classes. The rest of the taekwondo athletes placed second or third in the national championship or the national cup competition in the recent years. All of the taekwondo athletes participated in international competitions during the previous two years. The group of track and field throwers included two javelin throwers (mean performance: 79.8 ± 1.8 m), four shot putters (mean performance: 15.6 ± 0.9 m), and three hammer throwers (mean performance: 70.5 ± 4.3 m). The javelin throwers were the national champions in junior and senior categories, respectively, and were also among the best eight throwers of the European championship in the respective age categories, during the previous two years. Two of the shot putters placed first and second of the last years’ national outdoor championship while the others ranked 9th and 11th. One of the hammer throwers was among the eight best of the European championship under 23 years, while the other two placed 3rd and 4th at the national championship, during the previous year. After detailed oral and written description of the procedures, they gave their written consent to participate in the study. All of the procedures were approved by the School of Physical Education & Sports Science, National and Kapodistrian University of Athens Ethics Committee (registration number: 1039/14/02/2018).Taekwondo athletes were following a training program that was designed to develop technical skills and increase both their anaerobic and aerobic capacity to face the competition needs as recently described . Resista−1) = (Max force − Body weight) × [(Time of maximum force − Time at which force reach the body weight)−1]] during the push off phase (Time of maximum force − Time at which force reach the body weight) [n = 13), coefficient of variation (CV) = 8.1%. Countermovement jumps were performed on a force platform  with hands on hips with elbows pointing out. Briefly, after a short warm up on a stationary bicycle, athletes performed three sub-maximal CMJ’s with progressively higher intensity. Then, athletes performed three CMJ’s with maximum intensity, instructed to jump as high as possible. Two-minute rest was allowed between attempts. Data from the force platform were recorded and analysed  to calculate the maximum vertical jump height, the power output, the velocity, and the average rate of force development [ARFD (N·s weight) ,30. The 100ms was used for statistical analysis. Rate of force development was calculated as the mean tangential slope of the force–time curve in specific time windows of 0–30, 0–50, 0–80, 0–100, 0–150, 0–200, and 0–250 ms, relative to the onset of contraction which was set at 2.5% of the difference between baseline and maximum force; e.g., RFD = DForce/DTime [n = 13). Fifteen minutes after the CMJs, leg press maximum isometric force and RFD were evaluated. Throwers were seated on a custom-made completely rigid leg press chair made of steel with rigid plywood boards and placed both their feet on the force platform , which was positioned perpendicular on a concrete laboratory wall. Knee angle was set at 120° and hip angle was set at 100° ,30,31. Ace/DTime . Early Rce/DTime ,8,32,33.−1) from the force-time curve, as described in the previous paragraph, while the torque was corrected for the effect of gravity on the lower leg [100ms was used for statistical analysis. The ICC for the leg extension maximum isometric force and RFD, were calculated during the pilot study and were: ICC = 0.98, , CV = 12%, and ICC = 0.93, , CV = 10%, respectively (n = 11). Fifteen minutes after the isometric leg press test the leg extension test was performed. The athletes were positioned on a custom-made steel chair. Hip angle was set at 110° and initial knee joint angle was set at 70° (0° = full extension), ,32. Strap = 0.001), fascicle angle , and fascicle length  were determined on two consecutive days by the same investigator (n = 36), during the pilot study.B-mode ultrasound images were obtained during the morning hours from vastus lateralis (VL) and medial gastrocnemius (GM) of the dominant leg using a 38-mm linear probe . Extended-Field-Of-View mode was used to obtain panoramic images along the fascicle length, according to previous instructions ,35. For n = 13.Τotal body scans were obtained with dual X-ray absorptiometry . All of the measurements were analysed using the LUNAR radiation body composition program (LUNAR Radiation Body Composition Program). Analyses included fat mass, bone mineral density (BMD), and lean body mass (LBM) for total body, arms, trunk, and legs, according to manufacturer instructions. The ICC for body composition analysis was determined from two different researchers ICC = 0.98, , CV = 9%, η2) were also calculated . Pearson’s (r) product moment correlation coefficient was used to explore the relationships between variables. Standard multiple regression analysis was performed, including both groups of athletes. Because of the relative small sample size (n = 18) adjusted R squared was used for the interpretation of the multiple regression analysis results as the percentage explained variation [p ≤ 0.05. All statistical analyses were performed using SPSS version 17.0 software .All data are represented as mean ± SD. The comparisons between the two groups were also presented as percentage differences between them. One-way ANOVA was used to compare performance and biological variables between taekwondo athletes and track and field throwers. Effect sizes , as well as total lean mass  and bone mineral density  lower CMJ height, lower CMJ power , lower CMJ power relative to body mass , lower CMJ maximum velocity , lower leg press, and leg extension maximum isometric force/torque  and leg extension torque , as well as VL fascicle angle , when compared to throwers. Taekwondo athletes had lower body mass  as compared to throwers, while throwers had higher leg press RFD at 200 and 250 ms , while leg extension sequential RFD expressed per VL thickness was lower for taekwondo at 30–50, 50–80, 80–100, and 150–200 ms, respectively  expressed per percentage of maximum volunteer contraction (%MVC) than throwers at all time windows  as well as leg press RFD100ms , RFD200ms , RFD250ms , and leg press maximum isometric force . Also, in taekwondo athletes, lean mass of the legs was significantly correlated with leg extension RFD250ms  and leg extension maximum isometric torque . In contrast, no significant correlations were observed between lean body mass and performance for the throwers. When all athletes were considered as a group, lean mass of the lower extremities was significantly correlated with leg press RFD80ms  while GM muscle thickness was correlated with leg press maximum isometric force , in taekwondo athletes. VL fascicle length was significantly correlated with leg press RFD250ms  in throwers. The linear combination of VL muscle thickness and VL fascicle length could significantly predict the power performance in CMJ and the RFD between 80–250 ms, as well as maximum isometric force/torque for both leg press and leg extension  lower body lean mass was significantly correlated with RFD in power-trained athletes having large lean body mass differences, (2) taekwondo athletes had greater early RFD when this was expressed relative to total lean mass or vastus lateralis muscle thickness, as compared to track and field throwers, and (3) throwers performed better in late RFD when compared to taekwondo athletes both in absolute values as well as relative to total lean mass or vastus lateralis thickness. Unfortunately, in the current study neither the neural components nor muscle fiber composition were assessed, therefore the contribution of these components to the early phase of force rise was not explored. However, it is reasonable to assume that longitudinal taekwondo training adaptations induced in response to fast muscle actions performed with relative low external resistance  might hConversely, the delivery phase in track and field throws ranges between 150–250 ms ,39,40,41When RFD was expressed relative to total lean mass and legs lean mass, taekwondo athletes had higher early RFD than throwers in leg press but not in leg extension (no significant difference between groups for the leg extension). Although leg extension is commonly used to assess RFD, it is not regularly performed during training by either taekwondo athletes or track and field throwers. Thus, specific neural adaptations may not have developed for both groups for the leg extension, which might explain these results. In contrast, the multi-articular leg press exercise is a movement regularly performed by athletes either as specific leg press or in a broader sense in jumping, squatting, etc. Neural adaptations may be more applicable in leg press, which might explain the closer link with RFD relative to muscle mass/thickness. 100–250ms have been recently reported in young well-trained track and field throwers [When all the athletes were considered together, lean body mass was significantly correlated with late RFD. The close link between muscle mass and late RFD has been predicted in earlier studies where training-induced increases in late RFD correlated with chronic increases in muscle mass ,10,11,13throwers . HoweverThe role of muscle architecture in power performance and the RFD has been debated. Some studies have shown a link between fascicle length or fascicle angle and muscle power ,28,42,43Unfortunately, it was not possible to evaluate the electromyographic activity in this study. This might have provided further insight into the current results, especially about the neural input in the early phase of the RFD curve. Another limitation of this study was the lack of information regarding the muscle fiber composition of the athletes. Previous studies have shown a higher percentage of vastus lateralis area that is covered with type II muscle fibers in track and field throwers ,48. AlthIn conclusion, the current results reveal a significant correlation between lower body lean mass and RFD in power-trained athletes having large inter-individual differences in lean body mass. The track and field throwers are stronger than taekwondo athletes and they have more power in countermovement jumping. When the RFD is expressed relative to lean body mass, taekwondo athletes perform better that the throwers in the initial 30–50 ms after the initiation of an explosive contraction, which might be related to a higher neural input. In contrast, track and field throwers perform better relative to their lean muscle mass in the late part of the RFD curve. In practice, the current results suggest that taekwondo athletes and coaches should regularly measure the lower body RFD, especially the early part of the force rise (<100 ms), as well as the lean body mass to evaluate the lower body power adaptations induced with training. The importance of the early RFD in taekwondo athletes revealed in this study suggests that these athletes should implement explosive actions with moderate to low external resistance  in their training. In contrast, in sports demanding explosive movements against higher external loads, such as the track and field throws, training may mainly focus on increasing the later part of the force rise of the force-time curve (>150 ms) and laboratory testing of the RFD may also focus on this part of the curve. For these athletes, development of lean body mass seems to be an important factor for the late RFD.


Three-dimensional (3D) models are increasingly used to help surgeons, guiding them through the complex hepatic vasculobiliary anatomy. The biliary tract is a relatively untapped territory with only a few case reports described in medical literature. Our aim is to present an innovative 3D reconstruction methodology for biliary imaging and surgical planning, applied to a case of iatrogenic biliary stricture, with fusion of segmented CT and MRI images.A selected case of Bismuth type III iatrogenic biliary stenosis for 3D planning. CT and MR studies were acquired with dedicated protocols for segmentation. Two radiologists performed segmentation and 3D model post-processing, fusing both imaging techniques to faithfully render the anatomical structures. Measurements of anatomical landmarks were taken in both the CT/MRI and the 3D model to assure its accuracy and differences in measurement were calculated. The 3D model replicates anatomical structures and pathology with high accuracy, with only 2.2% variation between STL, CT and MRI measurements. The model was discussed with the surgical team and used in the surgical planning, improving confidence in this delicate procedure, due to the detailed prior knowledge of the patient's anatomy.Three-dimensional reconstructions are a rapidly growing area of research with a significant impact in the personalized and precision medicine. The construction of 3D models that combine vascular and biliary anatomy, using different imaging techniques, respectively CT and MRI, will predictably contribute to a more rigorous planning of complex liver surgeries. Three-dimensional (3D) printing is a rapidly growing area of research with a significant impact in personalized and precision medicine. The design of 3D models and 3D printing have been developed for different medical purposes.3D models, often printed, are widely used as surgical aids in orthopedic and maxillofacial surgery due to the relative ease in bone segmentation . Liver mA systematic review by Perica et al.  confirmeComplete biliary stenosis resulting from iatrogenic injury can be life-threatening. Surgical repair with Roux-en-Y hepaticojejunostomy is the gold standard therapy as it ensures biliary outflow and reconstitutes bilio-enteric drainage. Given the high density and frequent anatomic variability of vasculo-biliary structures in the liver hilum, meticulous preoperative planning is crucial. This requires excellent preoperative imaging, usually with multiple modalities, such as computed tomography (CT) and magnetic resonance—with cholangiopancreatography (MRCP). However, these different modalities will provide distinct features that need three-dimensional integration by the surgical team to plan the approach .For instance, the arterial and portal vascular anatomy are reliably demonstrated by the CT-angiography, while biliary anatomy is best depicted by MRCP. An integrated approach of these images would be extremely helpful as it would provide a roadmap to this challenging anatomical area, further compounded with adhesions and inflammatory changes.We present a case of a patient with an iatrogenic biliary stricture in which we used an innovative 3D reconstruction methodology, fusing images from two distinct modalities, one for vascular illustration (CT) and other for biliary tree anatomy demonstration (MRCP). We believe that this approach can be used in other contexts of complex surgical, endoscopic and interventional radiology procedures, particularly in cases of benign or malignant biliary stenosis, as well as in liver transplantation.A 50-year-old male patient presented to the emergency department with jaundice, fever (39 °C) and choluria in the previous 2 days. The patient had undergone cholecystectomy for cholecystolithiasis and Mirizzi syndrome two years earlier Fig. . He repoHe also had personal medical history of Graves' disease, ulcerative colitis and clear cell carcinoma of the kidney .The laboratory studies showed elevation of liver enzymes, mainly gamma glutamyl transpeptidase (GGT) and total and direct bilirubin, of 789 U/L [N: < 55U/L], 10.1 mg/dL [N:0.3–1.2 mg/dL] and 6.7 mg/dL [N:0.1–0.3 mg/dL], respectively, and a slight elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). C-reactive protein was also elevated .The patient was admitted for medical treatment of cholangitis and underwent imaging tests to characterize the bile ducts, with suspicion of iatrogenic/postinflammatory biliary stenosis.A MRI was performed with MRCP protocol and with hepatobiliary contrast, confirming dilatation of the intrahepatic bile ducts with an abrupt stop near the biliary confluence, in close contact with the first portion of the duodenum. In the hepatobiliary phase, contrast was excreted to the duodenal bulb and no contrast was present in the choledochal duct, seemingly confirming hepaticoduodenal fistula. Stenosis of the main biliary tract was confirmed, with approximately 3 cm in length Figs. , 3.Fig. The patient was diagnosed with Bismuth type III stenosis after cholecystectomy, associated with hepaticoduodenal fistula. Given the young patient age and after discussion in a multidisciplinary setting, the patient was offered surgical reconstruction. Abdominal triphasic CT was also performed to detect potential associated vascular injury and to obtain a preoperative anatomical mapping. The imaging protocols used in MRI and CT are detailed below.3  with the following scanning protocol: detector collimation 64-slice × 0.625 mm with gantry rotation of 500 ms, 120 kVp and 134 mAs, a slice thickness of 0.625 mm with resulting voxel size of 0.625 × 0.625 × 0.625 mmThe MRI was performed in a 3 T machine  using axial and coronal HASTE; axial in-phase and out-of-phase T1-weighted imaging; axial T2-weighted fat saturated sequence; 3D colangiographic sequence; axial DWI b-50-100-800 and a precontrast and postcontrast T1-weighted 3D VIBE after 10 mL endovenous gadoxetic acid injection on arterial, portal, transitional and hepatobiliary phases. The segmentation was performed in the hepatobiliary phase, a 3D Dixon sequence, 20 min after injection, with a 320 × 320 matrix and field of view (FOV) of 320 × 280  format were exported to a separate workstation with IntelliSpace Portal, version 9  for post-processing and segmentation.Two radiologists trained in 3D printing performed segmentation of MR and CT images. The liver, portal and hepatic veins were segmented using in the portal venous phase, the coeliac trunk and hepatic artery using the arterial phase imaging. The intrahepatic bile ducts were segmented in both the CT  and the hepatobiliary phase of the hepatic MR. Regarding the segmentation techniques, both manual and semi-automatic segmentation techniques were used with different methods in order to faithfully render the anatomical structures including thresholding technique, edge detection, region growing and multiple slice interpolation. The larger volume structures, such as the liver and portal vein, were initially segmented with semi-automatic selection tools of edge detection and thresholding, the contours were defined and adjusted manually. The smaller caliber vessels and biliary ducts were segmented step-by-step, with high precision selection tools requiring manual delineation using region growing technique.The final dataset was exported into Standard Tessellation Language (STL) to be viewed and processed with the Meshmixer 3.5 . Using the CT STL as a reference, the MRI STL was initially superimposed using three-dimensional Cartesian coordinates and then manually adjusted to achieve a reliable overlap, using reference points placed along the right and left hepatic ducts, the posterior branch of the right hepatic duct and on the convergences of more distal sectoral ducts on both CT and MR STLs.The original (CT) model and the fused model (CT + MR) were reviewed jointly by the radiologists and the surgical team, concluding that the bile ducts were more accurately segmented using MRCP than CT, due to its superior contrast resolution. During the editing process, some deformities and free objects were removed and the borders were smoothed Fig. .Fig. 53DMeasurements of anatomical landmarks were taken in both the CT/MRI and the 3D model to assure its accuracy. The structures were measured three times by both Radiologists for each technique and the mean value was used. The structures measured were: the right and left hepatic duct, main portal vein, proper hepatic artery and right and left branches of the hepatic artery. Differences in measurements of the diameter of the aforementioned structures were calculated between both MR, CT and STL Fig. , using tmentclass2pt{minimThe patient had an interesting anatomical variation, an accessory hepatic vein apparently draining S6, which was also segmented and included in our model. This however did not present any clinical consequences.The ensuing 3D models were discussed and analyzed with the surgical team and used in the planning of the surgery, emphasizing its usefulness for the mapping of vascular relations with the stenosis and biliary tree Fig. .Fig. 73DDuring surgery, after dissection of adhesions, the hepaticoduodenal fistula was identified and the duodenal orifice closed. Thanks to the preoperative 3D reconstructions, the right hepatic artery was found and only minimally dissected away from the biliary convergence, thus avoiding devascularization of the bile ducts. The hilar plate was lowered, and the biliary convergence was dissected in a densely inflamed hepatic hilum, with the usual anatomical landmarks obscured Fig. . After cThe patient made an uneventful recovery and was discharged five days after surgery. On pathology, the liver biopsy was negative for PSC. Three months after surgery the patient is well and symptom free, with normal liver biochemistry.Most benign biliary strictures are iatrogenic, following cholecystectomy or liver transplantation. The main symptoms include jaundice, pruritus, and darkened urine, weight loss, fever, nausea and vomiting. Complications such as ascending cholangitis, abscess formation and sepsis can occur .Benign biliary strictures may be located according to the Bismuth classification Fig. . Type I Bile duct injury resulting in stenosis can be treated by iterative percutaneous transhepatic dilation, with lasting patency of the biliary duct. Biodegradable stents may also prove to be an interesting treatment option for benign biliary strictures, potentially offering better technical results and quality of life for patients , 12.Endoscopic retrograde dilation and stenting is another option. However, in this particular case, due to the young age of the patient and the presence of a long stenosis and associated fistula, a surgical approach was preferred after multidisciplinary discussion. The presence of the fistula may explain the late presentation after cholecystectomy, and the previous episodes of cholangitis, since it would allow an intermittent drainage of bile to the duodenum. As usual in iatrogenic bile duct injuries, associated vascular injury should be suspected and can also be responsible for late stenosis, due to ischemic cholangitis .When preparing for a complex biliary reconstruction, the surgical team is dependent upon optimal preoperative imaging. This should provide the vascular and biliary anatomy, the relationship of these structures, as well as detect potential frequent anatomical variations. However, integrating the vascular and biliary anatomy is difficult since both are best depicted in two distinct exams, CT and MR. This complex case beautifully illustrates the way in which investment in 3D models for surgical planning proved to be essential. The surgeons reported that the additional planning eased the surgical approach and improved confidence in this delicate procedure, due to the detailed prior knowledge of the patient's anatomy, especially in the close relations between the biliary tract and the vascular structures at the level of a densely inflamed hepatic hilum. In Bismuth type III injuries the main bile duct is usually only supplied by the hilar component of the epicoledochal arterial plexus, since the usual 3 and 9 o’clock marginal arteries are interrupted . This maOur 3D model replicates anatomical structures and pathology with high accuracy between STL, CT and MRI without significant discrepancy between measurements of the selected anatomical landmarks. A recent systemic review by Perica et al.  highlighThe design of 3D models and 3D printing have been developed for different medical purposes, strengthened by the growth of imaging techniques and software for the acquisition, processing and segmentation of the image in Radiology, especially applied to CT and MRI .The construction of 3D models that combine vascular and biliary anatomy, using different imaging techniques, respectively CT and MRI, will predictably contribute to a more rigorous planning of complex liver surgeries, such as hepatic resection, living-donor transplantation or, as in the present case, biliary reconstruction surgery. In hepatobiliary surgery, anatomical variations are the norm instead of the exception, and surgical mishaps can have serious consequences.There is scientific evidence that supports the benefits of 3D models in clinical practice, with reduced surgical time, and subsequently anesthetic time; reduction in the number and severity of complications, reduced hospitalization time and, in turn, lower costs with each patient; improvement in the precision of surgeries by better preoperative planning, resulting in better outcome; technical improvement through the possibility of simulation with appropriate models and also improved communication between healthcare professionals and patients and family members –19.Research on 3D generated models in biliary pathology is still limited, with only a few cases reported in the literature and no large-scale studies to accurately assess its true clinical value. However, our 3D model was considered to be of great value for surgical planning.Our 3D model replicates anatomical structures and pathology with high accuracy, with only 2.2% variation between STL,CT and MRI measurements.A 3D printed model could not be obtained as we do not yet possess the adequate printing equipment in our institution. However, it is the authors opinion that the added value of a printed model would not be significant, in this case. Furthermore, the development and availability of new virtual reality technologies in the near future may limit the need for printed models, in some instances.We believe that 3D models of the biliary tract can become valuable tools in daily surgical practice. In the current medical landscape, where a “one size fits all” approach is increasingly outdated, these tools can help take into consideration patient-specific needs and provide a much more personalized approach to hepatobiliary interventions.


Dromedaries have been essential for the prosperity of civilizations in arid environments and the dispersal of humans, goods and cultures along ancient, cross-continental trading routes. With increasing desertification their importance as livestock species is rising rapidly, but little is known about their genome-wide diversity and demographic history. As previous studies using few nuclear markers found weak phylogeographic structure, here we detected fine-scale population differentiation in dromedaries across Asia and Africa by adopting a genome-wide approach. Global patterns of effective migration rates revealed pathways of dispersal after domestication, following historic caravan routes like the Silk and Incense Roads. Our results show that a Pleistocene bottleneck and Medieval expansions during the rise of the Ottoman empire have shaped genome-wide diversity in modern dromedaries. By understanding subtle population structure we recognize the value of small, locally adapted populations and appeal for securing genomic diversity for a sustainable utilization of this key desert species. Sara Lado et al. report a fine-scale analysis of genetic diversity in dromedary species across Asia and Africa. Using a genome-wide approach, the authors are able to infer patterns of migration following domestication, which follow known historic caravan routes, and identify major population expansions and bottlenecks through history. Camelus dromedarius) has a special position in human migration and trading1. Its physiological adaptations to harsh and dry environments allowed humans to traverse hostile lands such as deserts like no other livestock, including the horse2. Reversing a historian’s observation “the sea unites rather than divides”3 to the desert, dromedaries facilitated the expansion of civilizations5. Their superior and unique features predestined dromedaries for the use as military animals, and for the advance of international trading along ancient caravan routes, such as the Silk Road and Incense routes7. Interspecific hybridization between dromedaries and the closely related two-humped Bactrian camels (Camelus bactrianus) produced even more robust and enduring animals with an aptitude for the extreme climatic conditions of the Silk Road9. Nowadays, first generation hybrids and their backcrosses are valued in many countries for increased milk or wool production10, as well as in famous camel wrestling events11. These remarkable commercial networks in human history facilitated domestic animal exchange across large geographical distances and acted as gene-flow corridors, not only for camels but also for other livestock12. While camels were the chosen animals for transportation, horse movements along the complete network of the Silk Road mainly occurred in forms of tributes and gifts13.As one of the most recently domesticated animals , the dromedary (Protylopus) of camelids emerged in the North American savannah during the Eocence (~45 Mya). After their split into New (Lamini) and Old World (Camelini) camels around 15 Mya, the ancestors of the Old World camels crossed into Eurasia via the Bering Land Bridge (~6.6 Mya) and further diverged into one- and two-humped camels . Early-domestic dromedaries  cohabited the coastal Southeast of the Arabian Peninsula for nearly one millennium together with wild specimens, which did not survive the beginning of the Common Era (CE)19. The early dispersal of modern dromedaries from the Arabian Peninsula to the Levant, North Africa, South Asia, and finally to Australia (introduced in the late 19th century)20 was followed by cross-continental back-and-forth movements along historic trading routes. This led to a blurring of genetic stocks21 culminating in a panmictic dromedary population at the mitochondrial DNA level1. Previous studies using a limited number of microsatellite markers and mtDNA detected only weak population structure in the global dromedary population22. As few genomic studies have been completed with the dromedary, this major ungulate species has been left out of the livestock genomic revolution. However, two draft reference genomes at the scaffold level have been released24, as well as a draft reference genome at the chromosome level25, which will facilitate further genomic investigations.The early progenitors , and we decided to remove these samples from downstream analysis due to potential introgression from Bactrian camel (Supplementary Data We performed double-digest restriction site associated DNA (ddRAD) sequencing on 122 dromedary DNA samples from 18 countries  and observed (HO = 0.25 ± 0.17) heterozygosities in the global dromedary population . Separating the samples according to their continental origins, both Asian  and African dromedaries  showed similar genomic diversity. The mean HE  and inbreeding coefficients  were higher in Asian than African dromedaries, but mean HO  was not different between continents, according to Welch′s t test. Complete diversity and inbreeding values are given in Supplementary Table HE = 0.22–0.32)26 or cattle (HE = 0.24–0.30)27, we consider the genome-wide diversity in dromedaries as moderate at the best. Several bottlenecks during the last glacial period  and during domestication left modern dromedaries with a minimum of only six maternal lineages1 and limited genome-wide diversity. This will have implications on future intensification of breeding and genomic selection in dromedaries from regions with increasing desertification.With 22,721 SNPs, we estimated expected , and indices between dromedaries from different countries  were lowest in geographically close populations  and increased with geographic distance (Pakistan/Tunisia: FST = 0.0328)  revealed that most of the variation, ~94.3%, is explained within individuals  <0.05) between African and Asian dromedaries . In addition, we examined  regions 200 kbp upstream and downstream of the FST-outlier loci and detected fifty-three genes related to a number of biological functions .We screened for loci deviating from neutraliy using BayeScan 2.1Hadhana, and is one of the twelve recognized dromedary ecotypes in Saudi Arabia, limited to mountain regions in the South of the Arabian Peninsula, Al-Baha32. In this case, the geographic accessibility might have an important role in the observed genetic distinctiveness. A possible explanation for the close relationship of Hadhana and African dromedaries might be the historic sea route from Jiddah in Saudi Arabia to Aydhab and Port Sudan. On the western coast of the Red Sea existed a trading route connecting the Horn of Africa to Petra and Damascus via Port Sudan, Aydhab and Myos Hormos, near today’s Kosseir  and incorporated continental information (Africa/Asia) for each sample Fig. . Princip.3% Fig. . While t33  from two to seven confirmed the already observed differentiation between African and Asian dromedaries (K = 2), the clustering of the Saudi Arabian Hadhana breed with Africa (K = 4), the separation of Kenyan and Hadhana individuals, and the higher number of distinct clusters on the Asian continent (K = 7). We find a more homogenous gene pool in African animals with the exception of the East African group1 6. Even earlier dispersal of taurine cattle along the Northern coast of Africa and through the Mediterranean sea to Europe was described during the Bronze age35.To formally test our qualitative observations of weak population structure among African and Asian dromedaries Figs. , 3, we vert Fig. . This paert Fig. 1,7. The 36  over time with an Extended Bayesian Skyline approach (EBS)40 , we tested whether the dromedary populations from the two continents experienced a similar demographic history. First, we investigated the global population and second, African and Asian individuals separately. With the latter approach, we accounted for a potential confounding effect of population structure for the inference of NE40. Due to the observed substructure in Kenyan and Hadhana individuals, we excluded these two populations from the continental groups.To complete our understanding of the moderate genome-diversity observed in the global dromedaries, we investigated the demographic history and inferred effective population size  characterized by climatic cycles, and the Galerian Mammal Age (1.2–0.60 million ybp), which influenced the distribution and evolution of biota and resulted in some species being adapted to arid, cold climates43. Moreover, this timeframe also overlaps with the maximal diversity of the family Camelidae , supporting the adaptation of the dromedary ancestor to environmental changes with an expansion of its population during the middle Pleistocene transition44. Population expansion was followed by a drastic decline in NE beginning 700,000 ybp until the dromedary population collapsed during the last glacial period 45. This is a finding shared by previous Old World9 and New World camelid23 studies and those focusing on Late Quaternary Megafauna46.Irrespective of the continental origin, all inferences showed similar patterns of an initial population expansion from one million ybp until ~700,000 ybp Fig. . Our gen16. Previous BSP analysis using mtDNA likewise did not show a population decline during the time of domestication1. It is possible that the detection of a bottleneck with the EBSP analysis related to domestication has been superimposed by the drastic decrease in NE ending ~30,000 ybp , followed by the extension to Southern Asia and the Red Sea coasts47.After the Pleistocene bottleneck, the dromedary population slowly increased until reaching a stable Our study shows that assessing the evolutionary history of species using genome-wide approaches allows detailed inferences of population structure, migration, and potential signals of environmental adaptation. As the movements of dromedaries parallel those of humans, knowledge on dromedary spatial genetic signatures also sheds light into past human history. We detected genetic admixture across continental populations (Asia and Africa), which highlights the strong anthropogenic influence on these animals.48, and in many other (livestock) species50. With a genome-wide dataset we detected patterns of high effective migration in the global dromedary population and revealed pathways of dispersal after domestication, mirroring ancient caravan routes  or cultural nature. Domestic animals, and in particular camels, have been linked to this process of human development and were essential for its success. By establishing trading routes and reusing them over millennia, corridors of gene flow were opened that shaped genetic diversity and structure not only in dromedaries, but also in Bactrian camelstes Fig. . While tDromedaries and Bactrian camels are the most important livestock species in desert areas; their impact on land and water resources for food production is less than that of any other livestock. With increasing desertification and global climate change their importance will grow even more. For this reason, it is essential to understand the demographic history that has shaped modern dromedary populations. The genome-wide diversity present today, which we have characterized in this study, is a result of genetic shuffling due to historic and recent movements along trading routes. This constant mixing might have led to a unique genetic makeup that could make camels more resilient to global environmental changes, and that needs to be preserved.We selected a total of 122 dromedary samples from a pool of previously extracted DNA collected during projects supported from the Austrian Science Foundation, FWF P24607-B25 (PI: P.B.) and the European Research Council, EU ENPI CBC MED PROCAMED I.B.1.1/493 (PI: E.C.) under all legal requirements. The samples originated from 18 countries, which were representative of the species’ distributional range. In view of potential hybridization between one- and two-humped camels known to occur specifically in Central Asian regions, we included a Bactrian camel to control for introgression and as outgroup for phylogenetic analysis. Detailed information about all samples is provided in Supplementary Data C. dromedarius genome assembly (NCBI accession: GCA_000803125.1)24 highlighted SphI-BstYI as the best combination of restriction enzymes able to produce DNA fragments between 400 and 530 bp. ddRAD barcoded libraries were pooled and sequenced on an Illumina HiSeq 2500 in high output mode using 125 bp paired-end reads. Initial raw data analysis as well as SNP calling was performed by IGA Technology Services in-house bioinformatics pipeline. Briefly, all reads were trimmed to 110 bp, and quality controlled reads were aligned to the North African dromedary genome assembly (NCBI accession: GCA_000803125.1) using BWA-MEM51. ddRAD reads were then processed with Stacks v.1.3552. Out of nearly half a million RAD loci, 88,836 SNPs passed the imposed filtering criteria. The pstack module was run with a minimum coverage of 3 reads to call a haplotype, while SNPs were called using a bounded model to highest = 0.5 and alpha = 0.05. Cstacks and sstacks modules were run with default parameters. Population module was run requiring a minimum genotyping of 75% of individuals to score loci, along with a calling likelihood filtering threshold of −25.Library preparation and sequencing was performed at IGA Technology Services , to generate genome-wide data from ddRADseq. In silico analysis of the 53, first excluding individuals with more than 25% missing genotypes (--mind 0.25), next setting a threshold of 0.01 for minor allele frequency (--maf 0.01) and finally, removing SNPs with a missing genotype rate of more than 25% (--geno 0.25). Due to high individual missingness, 13 samples were removed from the dataset as well as 35,013 SNPs after filtering for minor allele frequency and missing genotype rate. To exclude any potentially related individuals, a symmetric identical by state matrix was created with PLINK with a cutoff value of 0.05; one sample from Jordan (JO434) and three from Nigeria  were removed from further analyses 25 or Bactrian camel (NCBI accession: GCF_000767855.1)23 genomes using BBSplit v. 38.79 (https://sourceforge.net/projects/bbmap/), with the following settings: minratio = 1.0, ambiguous = toss, ambiguous2 = toss. We preprocessed these two genome assemblies with dustmasker v. 1.054, and the percent Bactrian camel was estimated as the number of reads that unambiguously mapped to the Bactrian camel genome divided by the total number of unambiguously mapped reads to both dromedary and Bactrian camel multiplied by 100. We removed three individuals from Iran  and six from Kazakhstan   and observed (HO) heterozygosities, AR, and inbreeding coefficients (FIS) were calculated with the R package Hierfstat v0.04-2259. We have used a parametric Welch t test implemented in R v.3.5.1 using the t.test function to compare mean HE,HO, and FIS between African and Asian dromedaries. Pairwise FST and AMOVA59 were analyzed with the program Arlequin 3.5.2.260.SNPs were tested for Hardy–Weinberg Equilibrium and linkage disequilibrium using VCFTOOLS v0.1.1528 to identify FST outlier loci putatively under of selection using default settings with a FDR61 of 0.05. To understand if SNPs putatively detected under selection were linked to significant biological pathways, we screened the respective RAD sequences for genes using the annotation of the new CamDro2 assembly25 and assessed their protein function with Genecards (http://www.genecards.org). To consider also regions in potential linkage disequilibrium, we included genes 200 kbp upstream and downstream of the SNPs under selection in the analysis.We applied BayeScan 2.133 to assess ancestry and possible structure among dromedary populations , using the lowest fivefold cross-validation error to choose the best number of clusters (K), from K = 1 to K = 10. To understand the phylogenetic relationship among individuals, we applied the Neighbor-Net method62 implemented in SplitsTree463, which is a neighbor-joining algorithm for constructing phylogenetic networks from a genetic  distance matrix created in PLINK.Individual-based PCA was performed with Adegenet v2.1.1 using the s.class option to represent principal components of known groups. Furthermore we applied ADMIXTURE v1.334 to investigate effective migration patterns in the global dromedary population. Based on a stepping-stone model, and assuming that migration is symmetric, EEMS uses Markov Chain Monte Carlo to estimate the diversity and migration parameters and produces maps which represent the posterior mean of effective migration and effective diversity across space. We performed three distinct runs, each consisting of 10 million MCMC iterations, discarding the initial 5 million as burn-in and saving every 49,995 iterations for a grid with 500 demes. All runs reached convergence and results were similar across replicates. The habitat polygon was obtained using Google Maps API v3 Tool (http://www.birdtheme.org/useful/v3tool.html) and results were plotted using the R package rEEMSplots as suggested in Petkova et al.34.We used the software estimated effective migration surfaces (EEMS)64, setting the Coalescent EBS40 as a tree prior and following Huson and Bryant63. EBS analysis was conducted on the global population where we randomly selected 50 RAD loci containing at least four but not more than six SNPs across at least 75% of the individuals using a custom R script (https://github.com/jelber2/RAD-Scripts/blob/master/RAD_Haplotypes.R)65. We repeated the EBS analysis for African and Asian dromedaries separately, but due to slight population structuring, Kenya and Hadhana populations were excluded from this analysis. We ran EBS analyses three times per continent, with 50 random RAD loci used in each run. A RAD locus-specific clock rate (per generation) was estimated by calculating the average number of differences between dromedary and Bactrian camel sequences, dividing by the length of the RAD loci, taking the average among SNP classes (number of SNPs per RAD locus from 4 to 6), dividing by the split time between the Bactrian camel and dromedary of ~4.4 million ybp23, and using a dromedary generation time of 5 years1. Each EBS was run for 2,100,000,000 chains using the RAD locus-specific clock rate of 1.809442e-08 to calibrate the time scale.We assessed the demographic history of the species employing a coalescent-based multi-locus analysis with variable loci using BEAST2 v. 2.5.1t test implemented in R v.3.5.1 using the t.test function to compare mean HE, HO, and FIS, and data are expressed as mean and SD. Pairwise FST values were performed including a minimum of three individuals per countries and their significance levels are represented with “+”. AMOVA were analyzed with the program Arlequin. P values below 0.05 are considered as statistically significant for all statistical tests in this work. All analyses are reproducible with access to genetic data (see “Data availability”).Sample size is outlined in Supplementary Data Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting SummaryPeer Review File