Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson’s trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease. Inflammatory bowel disease (IBD) is a chronic, remitting and relapsing inflammatory disease of the gastrointestinal tract characterized by inflammation and mucosal tissue damage and is associated with significant morbidity. Ulcerative colitis and Crohn’s disease are the two most common forms of IBD. Ulcerative colitis and Crohn’s disease differ from each other in physiology, but show similar symptoms such as severe diarrhea, rectal bleeding, abdominal pain, fever, and weight loss ,2. CliniDextran sulfate sodium (DSS) is a water-soluble sulfated polysaccharide. Oral administration of DSS to trigger acute colitis has been widely used in experimental animal models for preclinical studies of IBD, because the pathophysiology resembles human Ulcerative colitis ,13,14. RIn this study, we investigated glomerular structural changes focusing on specific types of glomerular collagens and GFB-related proteins after DSS administration, to demonstrate the coexistence of glomerular structural changes and IBD in a DSS-induced colitis mouse model. This study should help establish an experimental animal model for further elucidation of the clinical pathological mechanisms of IBD-associated renal disease.p < 0.05)  A. DAI sc < 0.05) B. Colon  < 0.05) C. Histol < 0.05) D. These Human and mouse studies have indicated the involvement of non-intestinal organs in IBD ,6,7,8,9.Glomerular collagens including type IV collagen , type I collagen  and type V collagen  ,27 were The GFB comprises glomerular endothelial cells, the GBM and podocytes ,23. PodoWestern blot analysis of these four proteins in glomeruli showed declines in all proteins B, consisThese findings confirmed that DSS administration caused podocyte damage  and changes to endothelial adherens junctions in glomerular endothelium (reductions in VE-cadherin). These results are shown in Renal manifestations and complications in patients with IBD have been reported in several clinical and experimental studies from recent years ,10,11,28This study adopted the DSS-induced colitis mouse model and confirmed symptoms of DSS-induced colitis such as body weight loss, diarrhea, gross bleeding and colon architecture destruction , similarWe noticed that kidney size and weight were decreased in DSS-colitis mice after DSS administration , since kTo clarify the assumption that DSS induces renal structural change, we investigated the changes in specific types of collagens in the glomeruli. Our results showed that type IV collagen was decreased in the GBM, whereas type I and V collagens increased in the renal glomerular capillary loops and interstitium of DSS-colitis mice . Type IVRenal disease has been reported in human IBD patients, but GFB-related protein changes have not been closely investigated. The present study investigated four proteins  to clarify GFB damage, because these proteins have not been investigated in the kidneys of DSS-colitis mice, and the relevance of GBM damage in the DSS-induced colitis mouse model has not been reported yet. Our results showed declined expression of all four proteins in glomeruli after DSS administration . PodocytTaken together, our results imply that DSS administration could cause the glomerular collagen changes, including decreased type IV collagen as a supporting ECM of GBM structure and deposition of type I and type V collagens in renal interstitium. These collagen changes might lead to structural damage to podocytes such as a loss of polarity and detachment from the GBM, as well as loss of endothelial cell junctions, eventually causing renal disease. Loss of the podocyte cytoskeletal proteins synaptopodin and podocalyxin and the slit diaphragms protein nephrin, as well as the defective endothelial cells adherens junction protein (VE-cadherin) which may be associated with podocyte damage.Based on our findings, the lack or insufficiency of these GFB-related proteins in DSS-colitis mice might cause glomerular structural damage, and consequently lead to damage not only to podocytes, but also to adherens junctions in the vasculature, which might result in GFB damage.In conclusion, this study used the DSS-induced colitis mouse model, a very common experimental model of colitis, to clarify changes in glomerular collagens and GFB-related proteins after DSS administration. These findings on glomerular structural change in experimental mice with DSS-induced colitis should lead to novel uses of the animal model for further investigations into IBD-associated renal disease.Seven-week-old male Slc:ICR strain mice weighing 28–30 g  were housed in the Central Animal House at the University of Tsukuba under climate-controlled conditions . Cages were cleaned and sterilized every week. All mice were given free access to food and water. Animal experiments were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Science Council of Japan and Ministry of Education, Culture, Sports, Science and Technology of Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Tsukuba  based on the Institutional Animal Care and Use Committee (IACUC). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.w/v) DSS  dissolved in drinking water for eight days. Six mice per group were used in each experiment, and were not allowed to access to any other source of water. Mouse weights were monitored daily to quantify the systemic consequences of colitis. Mice were fed with normal water in place of DSS water on Day 8 and euthanized on Day 9. Colon length and kidney weight were measured for each mouse at harvest, then fixed in 10% neutral buffered formalin  or stored at −80 °C for further use. Mice receiving only distilled water were used as controls.As a widely used model of IBD, the DSS-induced colitis mouse model was used in this study to investigate changes to renal glomerular collagens and GFB-related proteins. This mouse model was established by inducing colitis with the administration of DSS in drinking water for eight days , followiWeight loss, stool consistency, and rectal bleeding were recorded daily to assess the severity of DSS-colitis. DAI was determined based on the methods described previously . BrieflyTo detect injury to the colon and renal tissues, prepared tissues were cut into 2-μm thick sections using a microtome  and stained with hematoxylin and eosin (HE) for histological investigations. To assess the GBM, periodic acid-Schiff (PAS) was used to estimate the glomerular deposition of matrix glycoprotein . Briefly® 488 or 568 ; double-stained with rhodamine-conjugated phalloidin  for F-actin; and finally submerged in fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) . Confocal imaging was performed according to the method described previously [® 488, and 568 signals were detected at laser excitation wavelengths of 488 nm and 543 nm, respectively.Immunofluorescence staining was performed by following the method described previously . Brieflyeviously  with a cKidney cortex tissue from each mouse was removed, glomeruli were isolated using a serial sieving method described previously , and the3VO4, 1 mM NaF, 20 mM Na4P2O7, 0.02% NaN3, 1% Triton X-100, 0.1% SDS and 1 mM PMSF) with 1% protease inhibitor cocktail  by sonication , followed by incubation on ice for 10 min and centrifugation at 10,000 rpm for 10 min. Supernatant was collected and protein concentration was quantitated using the Micro BCA Protein Assay kit  according to the instructions from the manufacturer. Thirty micrograms of total protein were loaded per lane and separated by 7.5% polyacrylamide gels, followed by transferring onto methanol-activated PVDF membrane . Protein-transferred membranes were incubated with primary antibodies against type I collagen, type IV collagen, type V collagen, synaptopodin, VE-cadherin, nephrin, podocalyxin and β-actin, respectively, at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were visualized with chemiluminescence substrate for 1 min and detected using a luminescent image analyzer . Band density was quantitated densitometrically with Image J software  by calculating the average optical density in each band. Relative and normalized protein expressions were calculated using the ratio of each protein density to β-actin density.Kidney cortices (~50 mg) or isolated glomeruli from kidney cortices of each mouse were homogenized in lysis buffer  in at least 3 independent experiments. The significance of differences between groups were analyzed using Student’s t-test. A probability level of p < 0.05 was considered significant.Statistical analyses were performed using Prism software . All data are expressed as mean ± standard error of the mean (SEM) from 6 replicates (


SUOX). Clinically, the disorder is classified as one of two forms: the late-onset mild form or the classic early-onset form. The latter is life-threatening and always leads to death during early childhood. Mild ISOD cases are rare and may benefit from dietary therapy. To date, no cases of ISOD have been reported to recover spontaneously. Here, we present three mild ISOD cases in one family, each with a stable clinical course and spontaneous recovery.Isolated sulfite oxidase deficiency (ISOD) is an autosomal recessive disorder caused by a deficiency of sulfite oxidase, which is encoded by the sulfite oxidase gene . The siblings included two males and one female with late ages of onset (12–16 months) and presented with specific neuroimaging abnormalities limited to the bilateral globus pallidus and substantia nigra. The three patients had decreased plasma homocysteine levels. They exhibited a monophasic clinical course continuing up to 8.5 years even without dietary therapy.All three siblings had two novel compound heterozygous mutations in the This is the first report of mild ISOD cases with a stable clinical course and spontaneous recovery without dietary therapy. Our study provides an expansion for the clinical spectrum of ISOD. Furthermore, we highlight the importance of including ISOD in the differential diagnosis for patients presenting with late-onset symptoms, bilaterally symmetric regions of abnormal intensities in the basal ganglia, and decreased plasma homocysteine levels. SUOX) [SUOX gene result in a decrease in SUOX activity, leading to an accumulation of sulfur-containing amino acids, inducing mitochondrial impairment and energy failure, and resulting in severe neurological damage [Isolated sulfite oxidase deficiency  is an inborn error of metabolism characterized by severe neurological impairment including dystonia severe psychomotor delay, refractory seizures, and lens dislocation. Clinically, the disorder is classified as one of two forms: mild or severe. Severe ISOD often presents within the first 72 h of life and leads to death during early childhood in most cases , 2. ISODSUOX) . Mutatiol damage , but clil damage . Therefo. This study was approved by the Ethics committee of the Affiliated Hospital of Zunyi Medical University, China.The three patients in this report were children of a non-consanguineous Chinese couple Fig. a who wer1Before age 1, psychomotor development in the firstborn male patient was normal, and he could walk independently. This patient exhibited rapid regression of acquired motor skills and cognition after a 2-day episode of mild diarrhea at age 1. Seizures were not observed. On examination, his growth was found to be in the 90th percentile, and his alertness and eye movement were both normal. He was not able to sit. No dysmorphic features, tremor, ataxia, or involuntary movements were observed. Cranial nerve examination findings were normal. He had generalized hypotonia with normal deep tendon reflexes. His blood lactate level was slightly increased, but uric acid, plasma glucose, and cerebrospinal fluid (CSF) test results were unremarkable. Brain magnetic resonance imaging (MRI) using T2-weighted (T2WI) and fluid-attenuated inversion recovery (FLAIR) sequencing demonstrated high signal in lesions limited to the bilateral globus pallidus and substantia nigra . Whole-exome sequencing showed that all three siblings harbored two novel compound heterozygous mutations  in the SUOX is associated with ISOD, and therefore, the levels of sulfite in the urine and plasma homocysteine were measured for each of the three children. This indicated that sulfite concentrations were higher than normal and that homocysteine levels were lower than normal in all three children (Table en Table . The thrVery few patients present with the mild form of ISOD. Previously, only seven patients with mild ISOD have been reported in the literature –11, 13. SUOX gene have a milder clinical presentation than those with null mutations [SUOX gene only lead to decreased biosynthesis of SUOX, whereas null mutations of the SUOX gene lead to complete abolishment of SUOX biosynthesis [SUOX gene, further supporting the hypothesis that missense mutations of the SUOX gene contribute to a milder presentation.Mechanisms for milder presentations of ISOD have not been fully elucidated. It has been reported that patients with missense mutations in the utations , since mynthesis . The genOur case series and 2 cases of late-onset mild ISOD reported by others show decreased homocysteine levels. Sulfite reacts readily with free thiol groups to form sulfocysteine. It can also conjugate with homocysteine to form sulfocysteine, thus depleting plasma homocysteine . The sliMore than 90% of patients with classic early-onset ISOD show severe cerebral and cerebellar atrophy and/or cystic white matter changes , 14, 15.Restricting dietary intake of methionine, cysteine, and taurine has been reported to be effective in patients with mild ISOD –11. TouaIn conclusion, to our knowledge, this is the first literature review to summarize the characteristics of late-onset mild ISOD and the first report of late-onset mild ISOD cases with a stable clinical course and spontaneous recovery without dietary therapy. We propose that when patients present with late-onset symptoms, a monophasic clinical course, neuroimaging indicating a bilateral globus pallidus lesion, and decreased homocysteine levels, ISOD should be considered. Furthermore, we can reasonably predict a good prognosis for children with late-onset mild ISOD based on their monophasic clinical course and reversible neuroimaging features.Additional file 1: Video S1. Video for case 1 at the age of 9.5 years showing the movement ability. The boy had unsteady gait, no choreiform movements were observed.Additional file 2: Video S2. Video for case 2 at the age of 5.5 years showing the movement ability The girl could walk several steps without support, with an unsteady gait and choreiform movements.Additional file 3: Video S3. Video for case 2 at the age of 5.5 years showing the language ability. Her vocabulary was normal, she could read smoothly.Additional file 4: Video S4. Video for case 3 at the age of 4.5 years showing the movement ability. He could not sit until age 4.5 years because of generalized hypertonia. He had some mild choreiform movements.


Mullerornis and Aepyornis, and represent small-bodied and medium-bodied aepyornithids, respectively. Aepyornis contains two distinct morphometric subgroups, which are identified as the largely allopatric species A. hildebrandti and A. maximus. The third morphotype, which has not previously been recognized as a distinct genus, is described as the novel taxon Vorombe titan. Vorombe represents the largest-bodied aepyornithid and is the world's largest bird, with a mean body mass of almost 650 kg. This new taxonomic framework for the Aepyornithidae provides an important new baseline for future studies of avian evolution and the Quaternary ecology of Madagascar.Madagascar's now-extinct radiation of large-bodied ratites, the elephant birds (Aepyornithidae), has been subject to little modern research compared to the island's mammalian megafauna and other Late Quaternary giant birds. The family's convoluted and conflicting taxonomic history has hindered accurate interpretation of morphological diversity and has restricted modern research into their evolutionary history, biogeography and ecology. We present a new quantitative analysis of patterns of morphological diversity of aepyornithid skeletal elements, including material from all major global collections of aepyornithid skeletal remains, and constituting the first taxonomic reassessment of the family for over 50 years. Linear morphometric data collected from appendicular limb elements, and including nearly all type specimens, were examined using multivariate cluster analysis and the Bayesian information criterion, and with estimation of missing data using multiple imputation and expectation maximization algorithms. These analyses recover three distinct skeletal morphotypes within the Aepyornithidae. Two of these morphotypes are associated with the type specimens of the existing genera  Then someone turned up another thigh-bone four feet six or more, and that they called Aepyornis titan … if they get any more Aepyornises, he reckons some scientific swell will go and burst a blood-vessel.”Aepyornis Island [H. G. Wells, s Island An accurate understanding of taxonomy and diversity in recently extinct groups is necessary in order to understand evolutionary processes that have contributed to the functioning of past ecosystems, patterns of regional biogeography and ecological disruption caused by humans in prehistory ,3. HowevThe Quaternary faunal record of Madagascar contains a unique and extraordinary megafauna, including giant lemurs, hippopotami, giant tortoises and the world's largest birds, the elephant birds. These taxa all survived into the Late Holocene and became extinct following the arrival of prehistoric human settlers, with available radiometric data suggesting that elephant birds persisted until around 1000 years ago . Studies2.Following the presentation and description of the world's largest egg and enormous avian skeletal remains from Madagascar in 1851 , the eleRichard Owen investigated diversity within another extinct insular radiation of giant island-endemic ratites, the moa (Aves: Dinornithiformes) of New Zealand, through a series of linear measurements of leg bones  that allowed separation and diagnosis of moa taxa  ,16. ThesAepyornis Geoffroy Saint-Hilaire, 1851, type species Aepyornis maximus Geoffroy Saint-Hilaire, 1851 [Mullerornis Milne-Edwards and Grandidier, 1894 [Mullerornis betsilei [Flacourtia Andrews, 1895, type species Mullerornis rudis [Aepyornis (approx. 400 kg) and Mullerornis (approx. 100 kg). Differentiation of species was based largely on linear measurements of the limited remains then available for study in respective national collections and via inter-museum loans of casts. Most of these taxa were erected between 1893 and 1895, and authors attempted to demonstrate their authority by synonymizing ‘competing’ taxa, often focusing on laying claim to the largest birds  was summarized by Brodkorb [M. grandis Lamberton, 1934 [Aepyornis recognized geographical co-occurrence of A. maximus and A. medius in both the central west coast region and the extreme south of Madagascar, with A. hildebrandti Burckhardt, 1893 [Mullerornis was also considered to contain two geographically co-occurring species, M. agilis Milne-Edwards and Grandidier, 1894 [M. rudis Milne-Edwards and Grandidier, 1894 [M. betsilei Milne-Edwards and Grandidier, 1894 [In the early twentieth century, further attempts to clarify the taxonomic diversity of the Aepyornithidae were made by Monnier , LambrecBrodkorb . Althougon, 1934 , based oon, 1934 ,29–31  and Mullerornis (approx. 2 mm thick) [The elephant birds have been the focus of remarkably little study during the late twentieth and early twenty-first centuries in comparison to moa and many other Quaternary megafaunal vertebrates. Following the recent development of methods of evolutionary and ecological analysis using ancient biomolecules, elephant bird material has been studied in efforts to reconstruct their evolutionary history and phylogenetic relationships , dietary maximus ,30. Howefication , combinefication  or eggshfication . Aepyorn3.Multivariate analysis of morphometric data derived from skeletal elements constitutes a significantly more powerful diagnostic tool for delimiting taxa than the univariate and bivariate methods used in historical aepyornithid systematic studies. However, multivariate methods require data frames with no missing values. As aepyornithid remains are rarely found completely intact, attempts to quantify multivariate morphometric data inclusive of all available specimens must compensate for these data gaps –37.Omission of characters and specimens from analysis is a common method for addressing the problem of missing data ,37,38. HThe alternative to omitting data is to estimate missing values while preserving natural variation of characters within taxa. One approach, imputation based on the means of observed variables, can create conservative models that can underrepresent natural variation within the morphological range for a given taxon  and may Many studies that aim to test the validity of a given taxonomic hypothesis using morphometric data are supported by a well-delimited higher-order nomenclature and good geographical provenance of specimens ,41,42. CTo clarify the confused state of elephant bird taxonomy, and to assess how many taxonomic units represented by distinct morphological clusters can be identified within a rigorously determined quantitative framework, we performed a series of morphometric analyses on linear measurement data from almost all of the specimens of aepyornithid appendicular limb elements available for study in global museum collections. We used an iterative modelling approach to permit comparison between models alternately assigning specimens to a varying number of clusters . This st4.4.1.n = 97), tibiotarsi (n = 162) and tarsometatarsi (n = 87) of adult individuals (defined on the basis of full fusion of epiphyses) were studied from the following collections: American Museum of Natural History, USA (AMNH), Centre ValBio, Madagascar (CVB), Museum für Naturkunde, Germany (MfN), Museum National d'Histoire Naturelle, France (MNHN), Natural History Museum, UK (NHMUK), Naturhistorisches Museum, Austria (NHMW), Oxford University Museum, UK (OUMNH), Université d'Antananarivo, Madagascar (UA), Natural History Museum, University of Oslo, Norway (UIO), United States National Museum, USA (USNM) and Zoologiska Museum, Uppsala Universitet, Sweden (ZIUU) . A standard series of 20 femoral, 20 tibiotarsal and 44 tarsometatarsal measurements were taken where possible  were completely intact and undamaged . As some taxa might only be represented by broken specimens, proportions of missing linear measurements from broken specimens were examined in 5% stepped increments. Selection of first-round data frames was defined by the inclusion of elements with less than 25% of linear measurements missing (approx. 50% of available specimens) to minimize imputation and maximize potential taxonomic inclusion. Skeletal elements with more than 25% of linear measurements missing were omitted from the first round of imputation calculations and taxonomic assessments. The first-round data frames included 48 femora (49% of specimens and 11.6% imputed data), 73 tibiotarsi (45% of specimens and 7.8% imputed data) and 46 tarsometatarsi .k-fold cross-validation as a regulation mechanism to remove noise and improve prediction quality. The ‘tuned parameters’ were determined by fivefold cross-validation to find the PCA loadings that produced the smallest mean square error of predictions, using the ‘estim_ncpPCA’ function of the MissMDA package [All statistical analysis was performed in R v. 3.1.3 . MI meth package . Linear 4.3.PCA was conducted on observed and imputed data derived from the first round of EM imputation, to investigate whether morphometric measurement data are able to identify discrete clusters of elephant bird specimens that are likely to correspond with taxonomically distinct groups. This approach extracts and summarizes the major features of morphometric shape variation and reduces high dimensionality to examine the distribution of different taxonomic groups in shape-space, without making any prior assumptions about the pattern of clustering of specimens.The package ‘MClust’  was usedA second dataset was generated to include all data with more than 25% missing specimens, any available type specimens that had not yet been included in cluster models, and all specimens with location data. Missing data point imputation, clustering, and removal of specimens demonstrating high levels of classification uncertainty were then performed. Clustering was performed using the same method as above, but the number of clusters was limited to the number observed from the first round of analysis.The large amount of missing data included in this second phase of imputation means that cluster assignment of these poorer-quality specimens must be interpreted with caution; however, this represents the only quantitative framework for identifying distinct morphological forms from incomplete remains of elephant birds. Where accession data were available for specimens, their cluster and geographical location was recorded to examine any potential pattern of spatial distributions. This second dataset included 64 femora (26.9% missing datapoints), 95 tibiotarsi (22.8% missing datapoints) and 70 tarsometatarsi (27.0% missing datapoints) .All PCA analyses were re-run using log-transformed data, to further reduce the potential confounding influence of variation in size alone .4.4.ANOVAs were performed on individual measurements for each morphological cluster of femora, tibiotarsi and tarsometatarsi, in order to describe the measurement parameters of each cluster and therefore define the taxonomic groupings represented by each cluster. Mass estimations were calculated using the Campbell and Marcus algorithm for estimating body mass in birds from femoral least-shaft circumference (LogM = 2.411 × LogLCF − 0.065) . Mean ma4.5.14C dating at the Oxford Radiocarbon Accelerator Unit, Oxford, UK and calibrated using ShCal13 [Bone samples from elephant bird specimens assigned to different morphometric clusters were submitted for accelerator mass spectrometer (AMS)  ShCal13  implemen ShCal13 .5.5.1.From our sample, 41 femora, 83 tibiotarsi and 41 tarsometatarsi were excluded from the first round of analysis due to exceeding the more than 25% missing marker criterion for taxonomic assessment. The percentage of total imputed data generated in this round was 11.6% for femora, 9.1% for tibiotarsi and 5.5% for tarsometatarsi. Five femora, 49 tibiotarsi and one tarsometatarsus were excluded from taxonomic classification through clustering due to high uncertainty in classification (greater than 5% uncertainty). Four femora, 43 tibiotarsi and 12 tarsometatarsi were excluded from subsequent biogeographical analysis, again due to greater than 5% uncertainty of classification. For the biogeographical assessment dataset, 26.9% of femoral markers, 23.0% of tibiotarsal markers and 27.0% of tarsometatarsal markers were not observed and so were imputed.Cluster analysis performed separately on PCA weightings created from each specimen's linear measurements from all three limb bones revealed that the comprehensive sample of elephant bird specimens analysed in this study fall into multiple distinct morphometric groups, defined as a stable result by BIC differentiation between cluster models of greater than 2 . Femora  and tibiIn all taxonomic PCA clusters, PC1 was highly correlated (greater than 0.75) with almost all measurements from each skeletal element , indicating a primary separation of clusters based on overall size . However, clusters overlap in size ranges and can also be differentiated by other patterns of distinctive morphotype variation, with clear autocorrelation between size and differing morphology.In cluster analysis of log-transformed data, femoral data were more stable than tarsometatarsal data. Four stable groups were recovered in unsupervised clustering of femoral data: clusters 1, 2 and 3 were separated along PC1, and cluster 2 was further subdivided into two subclusters on PC2. When supervised clustering based on these four groups was then applied to tarsometatarsal data, this produced the same classification of all tarsometatarsal specimens within the same clusters as in non-transformed cluster analysis .5.2.Tarsometatarsal data provide the best-resolved assessment of morphological diversity within aepyornithids, as the four clusters based on data for this limb bone resolve well and group membership is the most stable . FemoralAepyornis Geoffroy Saint-Hilaire, 1851; Mullerornis Milne-Edwards and Grandidier, 1894 and Flacourtia Andrews, 1895. The type specimens or type series of 10 of the 15 species that have been assigned to these genera can still be located in museum collections, and were included within the clustering analysis. The taxonomic identity of each morphometric cluster was established by determining which type specimens were included within which clusters, and which of these type specimens represented the oldest available taxonomic name , Mullerornis agilis Milne-Edwards and Grandidier, 1894 (part of syntype series: tibiotarsus) and Mullerornis rudis Milne-Edwards and Grandidier, 1894 (part of syntype series: tibiotarsus). Cluster 2 contains the intermediate-sized specimens across all skeletal element datasets and contains well-predicted type material of Aepyornis hildebrandti Burckhardt, 1893 (part of syntype series: tarsometatarsus), Aepyornis medius Milne-Edwards and Grandidier, 1869 (holotype: femur), Aepyornis cursor Milne-Edwards and Grandidier, 1894 (holotype: tarsometatarsus), Aepyornis lentus Milne-Edwards and Grandidier, 1894 (holotype: tarsometatarsus) and Aepyornis gracilis Monnier, 1913 (holotype: femur). When subdivided by supervised cluster classification (four groups), cluster 2a contains the type material of Aepyornis hildebrandti (part of syntype series: tarsometatarsus), Aepyornis lentus and Aepyornis gracilis. The syntype femur of Aepyornis hildebrandti was also assigned to cluster 2a, but this specimen is incomplete and cluster assignment was poorly predicted due to high uncertainty, so taxonomic assignment of the name Aepyornis hildebrandti was based solely on the tarsometatarsus. Cluster 2b contained the holotypes of Aepyornis medius and Aepyornis cursor. All tibiotarsi that fell within cluster 2 demonstrated high uncertainty of cluster classification and were therefore not used for taxonomic assessment. Cluster 3 contains the largest specimens of all skeletal elements and contains the syntype material of Aepyornis titan Andrews, 1894  and Aepyornis ingens Milne-Edwards and Grandidier, 1894 (tibiotarsus and tarsometatarsus).Cluster 1 represents the smallest specimens across all skeletal element datasets and contains the type material of Aepyornis maximus by Geoffroy Saint-Hilaire [A. maximus as reported by Owen is 127 mm, indicating that the type series of this species falls within the range of cluster 2b.Owing to the vague description of skeletal measurements, only eggshell dimensions can be compared accurately from the original description of -Hilaire . However-Hilaire  publishe-Hilaire , but are-Hilaire  that canMullerornis betsilei type series by Milne-Edwards & Grandidier [M. betsilei (70 mm) falls within the range of cluster 1.The original published description of the andidier  includesMullerornis grandis. Here, we use the minimum femoral shaft circumference, which shows discrete measurement values between clusters for Mullerornis . The minimum shaft circumference range reported for M. grandis (125–145 mm) falls within the upper range of cluster 1.Lamberton  includedAepyornis mulleri was described on the basis of a skull, mandible, vertebrae, ribs, sternum, part of pelvis, ‘the leg bones’ and phalanges [A. mulleri was previously considered to be a subjective synonym of A. hildebrandti by Monnier [halanges . No publ Monnier , but is Aepyornis Geoffroy Saint-Hilaire, 1851 was first used to describe Aepyornis maximus, which our data demonstrate can be assigned to cluster 2b, and this name can therefore be interpreted as the senior synonym for all of cluster 2. The genus Mullerornis Milne-Edwards and Grandidier, 1894 was first used to describe Mullerornis betsilei, which was subsequently designated as the type species by Richmond [Aepyornis in 1894, represents a further distinct morphotype which on this basis also needs to be recognized as distinct at the genus level. A third aepyornithid genus name, Flacourtia Andrews, 1895, is also available, but the holotype tibiotarsus of the type species Mullerornis rudis clusters reliably within cluster 1, and so the name Flacourtia represents a junior synonym of Mullerornis and cannot be used to describe cluster 3. There is therefore no available genus name that can be applied to cluster 3.The genus Richmond , and whiRichmond ,18, thisMullerornis), and so we apply the oldest species name for this cluster, Aepyornis modestus, to name the single species that can be recognized in this genus. Cluster 2 (Aepyornis) can be separated into two distinct morphological groups on the basis of both tarsometatarsal and femoral data, and we interpret these groups as representing separate species within the same genus: the oldest available species names within each cluster are Aepyornis hildebrandti (cluster 2a) and Aepyornis maximus (cluster 2b). No morphological differentiation can be demonstrated within cluster 3 (unnamed genus). Within this cluster, the two species names Aepyornis titan and Aepyornis ingens were both published in 1894, but titan (published January 1894) predates ingens (published February 1894) by one month, so that the oldest available species name for this group is Aepyornis titan. Body mass estimates for these four recognized aepyornithid taxa are given in Our analysis does not distinguish distinct morphotypes within cluster 1 . This specimen is the type specimen for Aepyornis lentus and is missing more than 25% of measurement data, leading to potential unreliability of cluster assignment. New AMS dates for specimens assigned to Aepyornis hildebrandti and Vorombe titan are given in Owing to the poorly resolved clustering of tibiotarsal data, we selected only femoral and tarsometatarsal geographical location data to reconstruct distributions of newly defined elephant bird taxa. Specimens with high uncertainty were also removed from the pooled location dataset. Locality data associated with well-resolved specimens in our analysis  are plotted by species in ighlands . Almost 6.Order Struthioniformes Latham, 1790 [am, 1790 Family Aepyornithidae Bonaparte, 1853 [te, 1853 Revised diagnosis:Femur: Trochanter femoris extremely large, expanded medio-laterally and medio-distally. Crista trochanteris rounded and convex, expanded cranio-caudally and medially, oriented slightly caudally; muscle scars present on lateral facies. Facies articularis antitrochanterica concave. Shaft flattened caudally. Linea intermuscularis caudalis leads to medial margin of shaft. Linea intermuscularis cranialis leads to distal margin of shaft. Impression of m. gastrocnemialis lateralis presents as large, deep pit on cranio-lateral margin, just proximal to very wide trochlea fibularis. Sulcus patellaris broad, deep u-shape in distal aspect, laterally directed.Tibiotarsus: Distally flattened, much wider medio-laterally than cranio-caudally deep. Pronounced crista cnemialis lateralis, expanded proximally. Pronounced crista cnemialis cranialis, expanded medially. Both cnemialis crista oriented medially. Incisura tibialis wide, deep u-shape in proximal aspect, separating facies articularis lateralis and crista cnemialis lateralis. Facies articularis prominent and rounded, longer proximo-distally than latero-medially. Shaft narrows from proximal end, with linea intermuscularis cranialis terminating on lateral margin. Canalis extensoris proximal to condylus lateralis at distal end. Condylus lateralis with greater caudo-cranial expansion than condylus medialis. Pons supratendineus present. Distal end much wider medio-laterally than cranio-caudally.Tarsometatarsus: Proportionately long, with triangular facies dorsalis. Proximal end and trochleae expanded medio-laterally; lateral margin expanded proximally, medial margin enlarged distally. Shaft flattened dorsally. Single high and long hypotarsal ridge. In anterior view, foramina closely spaced within sulcus extensorius formed by flattened-triangular orientation of metatarsi. In posterior view, foramina widely separated by hypotarsal ridge oriented away from midline of broad, long shaft. Distal end with large intertrochlear notches. Three trochleae; trochlea III always terminating furthest distally and marginally forward of shaft and larger than trochleae II and IV, which are nearly equal in size.Aepyornis Geoffroy Saint-Hilaire, 1851Genus  [re, 1851 Aepiornis Geoffroy Saint-Hilaire, 1851, p. 52 [1, p. 52 Epiornis Muller and Baldamus, 1851, p. 48 [1, p. 48 Epyornis Bonaparte, 1853, p. 139 [, p. 139 Type species:Aepyornis maximus Geoffroy Saint-Hilaire, 1851 (by monotypy) [onotypy) .Recognized species:Aepyornis maximus Geoffroy Saint-Hilaire, 1851 [Aepyornis hildebrandti Burckhardt, 1893 [re, 1851 ; Aepyorndt, 1893 .Revised diagnosis:Femur: Proportionately broader and more robust than Mullerornis, and slightly more robust than Vorombe. Facies articularis antitrochanterica is shallow concave surface between trochanter femoris and caput femoris, which are oriented at shallower angles proximally than distally. Significantly larger than Mullerornis in following measurements: F1–F4, F6, F10–F12, F16 . Significantly smaller than Vorombe in following measurements: F1–F14, F16–F17, F19–F20 .Tibiotarsus: Shaft broader in proportion to overall size in comparison to other genera. Smaller tibiotarsi (A. hildebrandti) of similar length to Mullerornis but considerably more robust, with more rounded cnemial crista. Proximal end expanded, particularly medio-laterally. Margin between crista cnemialis cranialis and crista cnemialis lateralis flatter than other genera.Tarsometatarsus: Smaller tarsometatarsi (A. hildebrandti) of similar length to Mullerornis but medio-laterally broader and with much shallower triangular cross-section. Trochlea IV distally larger and longer than trochlea II. Significantly larger than Mullerornis in following measurements: Tmt2–Tmt6, Tmt9–Tmt11, Tmt13–Tmt25, Tmt27–Tmt31, Tmt33–Tmt39 . Significantly smaller than Vorombe in following measurements: Tmt1, Tmt3–Tmt6, Tmt10–Tmt11, Tmt13–Tmt22, Tmt27–Tmt28, Tmt31, Tmt33, Tmt35–Tmt36, Tmt38–Tmt41, Tmt43–Tmt44 .Revised description:Femur: (In addition to diagnostic features above) Crista trochanteris large, rounded and convex. Medio-distal margin of caput femoris with broad curvature, transitioning into medial margin of shaft. Shaft narrows from proximal end, with straight middle section, and expanding distally into condylus medialis. Condylus medialis expanded proximo-distally. Trochlea fibularis with acute angle to lateral margin of shaft and trochanter femoris. Fossa poplitea with pronounced, proximally arched margin; very large, positioned above lateral portion of condylus medialis and sulcus patellaris.Tibiotarsus: (In addition to diagnostic features above) Crista cnemialis cranialis directed proximo-medially, extending proximally to crista cnemialis lateralis. Rounded, proximally expanded crista cnemialis lateralis extends into ridge, leading into prominent, straight linea intermuscularis cranialis that terminates approximately 50% along length of shaft on lateral margin. Proximal margin of sulcus intercnemialis is very shallow concave curve between the two crista in cranial view. Shaft medially straight and laterally curved, expanding into distal condyles. Distal articular surface broad and shallow.Tarsometatarsus: (In addition to diagnostic features above) Very robust . Medio-laterally broad at proximal end, with rounded lateral portion; expanded plantar-dorsally, transitioning into shaft. Tuberositas m. tibialis cranialis small, rounded, slightly larger medio-laterally than proximo-distally. Shaft very broad, narrowing slightly in medial section, with both medial and lateral margins having continuous broad concave curvatures.Aepyornis maximus Geoffroy Saint-Hilaire, 1851 [re, 1851 Aepyornis maximus Geoffroy Saint-Hilaire, 1851, p. 104 [Aepyornis medius Milne-Edwards and Grandidier, 1869, p. 97 [9, p. 97 Aepyornis cursor Milne-Edwards and Grandidier, 1894, p. 124 [Syntype series: Tarsometatarsus of adult individual and two eggs, from ‘the south coast’ of Madagascar, purchased from Merchant Captain M. Abadie. Original tarsometatarsus now cannot be located, and eggs cannot be distinguished from other collections in MNHN.Lectotype: Tarsometatarsus from original syntype series , designated herein.Revised diagnosis:Femur: Compared to A. hildebrandti, has similar length and width of proximal and distal ends, but markedly more robust; trochanter femoris larger in proportion to total femur size and more expanded proximally and dorsoventrally; caput femoris slightly shorter medially; shaft with greater circumference and more clearly defined linea intermuscularis cranialis. Significantly larger than A. hildebrandti in F15 .Tibiotarsus: Compared to A. hildebrandti, crista cnemialis lateralis more expanded proximally and more laterally oriented, and with shallower angle of transition into pronounced linea intermuscularis cranialis; fascia gastrocnemialis proportionally larger; shaft proportionally wider, and similarly expanded at proximal and distal ends; distal condyles more expanded medio-laterally and protrude equally distally. See Tarsometatarsus: Compared to A. hildebrandti, proximal end flatter, with less proximal expansion on lateral metatarsal and more expanded medio-laterally; expansion of medial margin continues more distally; trochleae proportionately less expanded. Significantly larger than A. hildebrandti in following measurements: Tmt9, Tmt14, Tmt19, Tmt33, Tmt35–Tmt36, Tmt40, Tmt43 . See Revised description:Femur: (In addition to descriptions and diagnostic features above) Comparatively long and very stout . Condylus medialis expanded medially and with flatter distal surface leading into trochlea fibularis; fossa poplitea with pronounced, proximally arched margin with slight orientation towards medial shaft margin.Tibiotarsus: (In addition to descriptions and diagnostic features above) Long and very robust . Shaft with distinct curvature on lateral margin.Tarsometatarsus: (In addition to descriptions and diagnostic features above) Long and stout . Foramina within shallow fossa infracotylaris dorsalis, which has slight concave curvature from proximal margin of articular surface. Proximal end more medio-laterally enlarged and with slightly greater lateral length than distal end.Proportions of limb elements: 1 : 1.6 : 0.9 (tarsometatarsus : tibiotarsus : femur). Data in tables Measurements of type material (mm) reported by Owen [: Extreme breadth across trochlear condyles, 127 mm (5 inches); transverse diameter of shaft 6 inches above lower end, 74 mm (2.9 inches); antero-posterior diameter of shaft 6 inches above lower end, 33 mm (1.3 inches). by Owen : ExtremeAepyornis hildebrandti Burckhardt, 1893 [dt, 1893 Aepyornis hildebrandti Burckhardt, 1893, p. 127 [, p. 127 Aepyornis lentus Milne-Edwards and Grandidier, 1894, p. 124 [Aepyornis gracilis Monnier, 1913, p. 15 [3, p. 15 Syntype series: Femur (MfN MB.AV.73), tibiotarsus (MfN MB.AV.70), tarsometatarsus (MfN MB.AV.67), from Antsirabe, Madagascar.Lectotype: Tarsometatarsus (MfN MB.AV.67), designated by Brodkorb [Brodkorb .Revised diagnosis:Femur: Compared to A. maximus, trochanter femoris proportionally smaller and less expanded proximally and dorsoventrally; caput femoris longer medially, with more continuous curvature of distal margin; shaft much more slender, with less well-defined linea intermuscularis cranialis. Significantly smaller than A. maximus in F15 .Tibiotarsus: Compared to A. maximus, crista cnemialis lateralis less expanded proximally and more medially oriented, and with more acute angle of transition into weak linea intermuscularis cranialis; concavity from crista cnemialis cranialis to shaft less acute; fascia gastrocnemialis smaller; shaft proportionally narrower, and more expanded at proximal end than at distal end; distal condyles more expanded proximo-distally; condylus lateralis protrudes distally to condylus medialis.Tarsometatarsus: Compared to A. maximus, metatarsal IV more expanded proximally, creating angled proximal articular surface; medial margin less expanded distally; trochleae more expanded medio-laterally. Significantly smaller than A. maximus in following measurements: Tmt9, Tmt14, Tmt19, Tmt33, Tmt35–Tmt36, Tmt40, Tmt43 .Revised description:Femur: (In addition to descriptions and diagnostic features above) Comparatively short but robust . Condylus medialis expanded proximo-distally; concave fascia leading into trochlea fibularis; fossa poplitea with pronounced, proximally arched margin in centre of shaft.Tibiotarsus: (In addition to descriptions and diagnostic features above) Long and robust . Shaft with distinct curvature on lateral margin.Tarsometatarsus: (In addition to descriptions and diagnostic features above) Small and stout . Foramina within fossa infracotylaris dorsalis, which has concave curvature from proximal margin of articular surface. Distal end more medio-laterally enlarged than proximal end; lateral length greater than medial length.Proportions of limb elements: 1 : 1.5 : 1 (tarsometatarsus : tibiotarsus : femur). Data in tables Measurements of type material (mm). MfN MB.AV.73: F2 = 43.24; F3 = 158; F4 = 52.7; F16 = 100.4; F20 = 11.2. MfN MB.AV.70: Tt1 = 473; Tt2 = 39.32; Tt3 = 110; Tt4 = 26.76; Tt5 = 90.1; Tt6 = 59.34; Tt7 = 72.7; Tt8 = 128.8; Tt9 = 85.54; Tt10 = 93.92; Tt11 = 30.4; Tt12 = 65.6; Tt13 = 59.16; Tt14 = 70.88; Tt15 = 53.48; Tt16 = 28.02; Tt17 = 18.62; Tt18 = 43.32; Tt19 = 71.6; Tt20 = 153. MfN MB.AV.67: Tmt1 = 266; Tmt2 = 21.04; Tmt3 = 42.98; Tmt4 = 41.42; Tmt5 = 96.02; Tmt8 = 51.88; Tmt9 = 97.12; Tmt10 = 68.86; Tmt11 = 96.86; Tmt13 = 40.7; Tmt14 = 32.76; Tmt15 = 31.82; Tmt16 = 36.02; Tmt17 = 44.94; Tmt18 = 40.48; Tmt19 = 45.6; Tmt20 = 36.18; Tmt21 = 31.02; Tmt22 = 33.8; Tmt23 = 42.34; Tmt24 = 40.24; Tmt25 = 37.44; Tmt26 = 42.84; Tmt27 = 45.7; Tmt28 = 45.14; Tmt29 = 34.16; Tmt30 = 41.5; Tmt31 = 36.8; Tmt32 = 13.28; Tmt33 = 80.28; Tmt34 = 19.16; Tmt35 = 52.8; Tmt36 = 38.38; Tmt41 = 252; Tmt42 = 237; Tmt43 = 261; Tmt44 = 256.Mullerornis Milne-Edwards and Grandidier, 1894Genus  [Flacourtia Andrews, 1895, p 23 [95, p 23 Type species:Mullerornis betsilei Milne-Edwards and Grandidier, 1894 [er, 1894 .Recognized species:Mullerornis modestus  [r, 1869) .Revised diagnosis:Femur: Smaller, proportionately narrower and less robust than Aepyornis or Vorombe. Facies articularis antitrochanterica and caput femoris form smooth concave surface, oriented proximo-distally at shallower angle proximally than distally. Distal end medio-laterally expanded. Significantly smaller than Aepyornis in following measurements: F1–F4, F6, F10–F12, F16 . Significantly smaller than Vorombe in following measurements: F1–F14, F16–F17, F19–F20 .Tibiotarsus: Similar in total length to Aepyornis hildebrandti, but with more slender shaft and well-defined, protruding cnemial crista. Proximal end expanded laterally, but with reduced medial expansion compared to other genera. Crista cnemialis lateralis prominent, projecting proximally, forming distinct curved and laterally positioned ridge. Crista cnemialis cranialis more prominent than in other genera, expanded markedly medially to form extremely pronounced curve into shaft. Margin between crista cnemialis cranialis and crista cnemialis lateralis sharply concave.Tarsometatarsus: Similar in length to Aepyornis hildebrandti, but markedly narrower. Shaft with acute triangular cross-section. Trochleae with reduced lateral expansion and minimal medial expansion. Trochlea IV protrudes distal to trochlea II; trochlea III protrudes distal to trochleae II and IV. Significantly smaller than Aepyornis in following measurements: Tmt2–Tmt6, Tmt9–Tmt11, Tmt13–Tmt25, Tmt27–Tmt31, Tmt33–Tmt39 . Significantly smaller than Vorombe in following measurements: Tmt1–Tmt6, Tmt9–Tmt36, Tmt38–Tmt41, Tmt43–Tmt44 .Revised description:Femur: (In addition to descriptions and diagnostic features above) Short and slender . Crista trochanterica large, rounded and convex at proximal end. Distal margin of caput femoris with reduced concave curvature. Shaft narrows in middle, curved medially and laterally, expanding into broad condylus medialis; with reduced concave curvature on distal fascia. Medio-distal condyle much less expanded than latero-distal condyle, and protrudes proximally. Fossa poplitea very large in proportion to size of femur, with poorly defined proximal margin, positioned above sulcus patellaris. Trochlea fibularis very large in proportion to size of femur; oriented disto-laterally, pointing away from trochanter femoris.Tibiotarsus: (In addition to descriptions and diagnostic features above) Long and slender . Crista cnemialis cranialis extends markedly past crista cnemialis lateralis, directed proximo-medially. Crista cnemialis lateralis protrudes markedly medially, transitioning sharply into clear linea intermuscularis that approaches lateral margin approximately 50% along shaft length, then runs parallel and becomes undefined above condylus lateralis. Proximal margin of sulcus intercnemialis is sharply concave curve between the two crista in cranial view. Shaft relatively straight, narrowing markedly on medial margin but with only shallow curvature on lateral margin and only minor expansion into distal condyles. Distal condyles protrude equally at distal end.Tarsometatarsus: (In addition to descriptions and diagnostic features above) Small and slender . Proximal end with small amount of lateral expansion and marginal medial expansion. Hypotarsal ridge very broad and deep in proximal aspect. Proximal fascia relatively flat, with minimal proximo-distal expansion. Foramina within fossa infracotylaris dorsalis that has concave curvature from proximal margin of articular surface. Tuberositas m. tibialis cranialis centrally positioned, rounded and slightly larger medio-laterally than proximo-distally. Shaft narrow with lateral margin reducing towards distal end, and relatively straight medial margin.Mullerornis modestus  [r, 1869) Aepyornis modestus Milne-Edwards and Grandidier, 1869, p. 314 [, p. 314 Mullerornis agilis Milne-Edwards and Grandidier, 1894, p. 125 [Mullerornis betsilei Milne-Edwards and Grandidier, 1894, p. 125 [Mullerornis rudis Milne-Edwards and Grandidier, 1894, p. 125 [Holotype: Femur (MNHN 1908-5), from Ambolisatra, Madagascar.Revised diagnosis: As for genus.Revised description: As for genus.Proportions of limb elements: 1 : 1.5 : 0.9 (tarsometatarsus : tibiotarsus : femur). Data are presented in tables Measurements of type material (mm). MNHN 1908-5: F1 = 255; F2 = 29.9; F3 = 121; F4 = 41.84; F5 = 112; F6 = 34.9; F7 = 34.54; F8 = 68.54; F9 = 90.66; F10 = 55.98; F11 = 63.66; F12 = 223; F14 = 240; F16 = 96.52; F17 = 75.88; F18 = 95.24; F19 = 23.36; F20 = 11.66.Vorombe gen. nov.Genus Etymology: From the Malagasy for ‘big bird’ (neuter).Type species:Aepyornis titan Andrews, 1894 [ws, 1894 Recognized species:Vorombe titan  [s, 1894) Diagnosis:Femur: Extremely large and robust in comparison to other genera, with enlarged proximal and distal ends. Medio-distal margin of caput femoris with more acute curvature than in other genera. Facies antitrochanterica and caput femoris form smooth concave surface. Caput femoris oriented at equal angles perpendicular to shaft proximo-distally. Marked crista supracondylaris medialis present (absent in other genera). Condylus medialis expanded medially and flatter than in Aepyornis. Significantly larger than both Aepyornis and Mullerornis in all measurements .Tibiotarsus: Extremely large in comparison to other genera. Proximal and distal ends enlarged, particularly medio-laterally, with proximal articular surface marginally more concave than Aepyornis but much less than Mullerornis, and with more pronounced narrowing transition into shaft; shaft narrower in proportion to total length compared to Aepyornis. Lateral condyle markedly more expanded distally and laterally than in other genera, terminating distal to condylus medialis.Tarsometatarsus: Considerably larger and markedly more expanded medio-laterally than other genera, particularly at proximal and distal ends. Lateral portion of proximal articular surface protrudes proximally to medial portion, creating markedly angled proximal articular surface similar to A. hildebrandti. Trochlea II protrudes marginally proximal to trochlea IV. Trochleae II and IV more equal in size than in other genera; expanded similarly both medio-laterally and dorsoventrally. Significantly larger than Mullerornis in all measurements . Significantly larger than Aepyornis in following measurements: Tmt1, Tmt3–Tmt6, Tmt10–Tmt11, Tmt13–Tmt22, Tmt27–Tmt28, Tmt31, Tmt33, Tmt35–Tmt36, Tmt38–Tmt41, Tmt43–Tmt44 .Description:Femur: (In addition to descriptions and diagnostic features above) Robust . Crista trochanterica large, rounded and convex. Medio-distal margin of caput femoris transitions into medial margin of narrowing, medially straight shaft, which then expands into condylus medialis. Condylus lateralis expanded proximally. Trochlea fibularis very large, shallow and broad; parallel to shaft and trochanter femoris. Fossa poplitea with poorly defined proximal margin; transitions smoothly into shaft, positioned above lateral portion of condylus medialis and sulcus patellaris.Tibiotarsus: (In addition to descriptions and diagnostic features above) Very long . Crista cnemialis cranialis extends past crista cnemialis lateralis, directed proximo-medially. Crista cnemialis lateralis rounded, medially and marginally proximally expanded; transitions via smooth curve into medial surface of shaft, extending into prominent, straight and well-defined linea intermuscularis terminating on lateral margin just proximal to distal condyles. Proximal margin of sulcus intercnemialis very shallow concave curve between the two crista in cranial view. Shaft narrowing near proximal end on medial margin, but with only shallow curvature on lateral margin, becoming very straight and parallel at midshaft before expanding markedly into distal condyles.Tarsometatarsus: (In addition to descriptions and diagnostic features above) Robust  and long. Extremely medio-laterally broad at proximal ends; lateral portion rounded and expanded plantar-dorsally. Hypotarsal ridge very broad and deep in proximal aspect. Foramina within shallow fossa infracotylaris dorsalis that has slight concave curvature from proximal margin of articular surface. Tuberositas m. tibialis cranialis small, rounded, slightly larger medio-laterally than proximo-distally. Shaft highly tapered and broad; medial margin becoming straight, lateral margin retains continuous broad concave curvature.Vorombe titan (Andrews 1894) [ws 1894) Aepyornis titan Andrews 1894, p. 18 [4, p. 18 Aepyornis ingens Milne-Edwards and Grandidier, 1894, p. 124 [Syntype series: Femur (NHMUK A439), tibiotarsus (NHMUK A437), from Itampolo (Itampulu Vé), Madagascar ; newly designated (signated .Diagnosis: As for genus.Description: As for genus.Proportions of limb elements: 1 : 1.8 : 1 (tarsometatarsus : tibiotarsus : femur). Data are summarized in tables Measurements of type material (mm). NHMUK A439: F2 = 71; F3 = 271; F4 = 91.5; F5 = 232; F6 = 68.6; F7 = 74; F9 = 203; F11 = 141; F14 = 414; F15 = 346; F19 = 32; F20 = 24. NHMUK A437: Tt2 = 75.8; Tt3 = 206; Tt4 = 44.3; Tt5 = 162; Tt6 = 112.5; Tt7 = 134.5; Tt20 = 263.7.Mullerornis samples) represents specimens that are already uncontroversially recognized as being distinct at the genus level from the other clusters, the comparably morphometrically distinct largest-bodied cluster (cluster 3) must therefore also be recognized as taxonomically distinct at the genus level. Further morphometric subdivision within cluster 2 is interpreted as representing species-level differentiation. We therefore identify three valid elephant bird genera, two of which are monotypic, and one of which contains two species.Our study provides the first rigorous quantitative analysis of morphometric variation within elephant birds, using data from almost all of the specimens available for study in global museum collections, and employing multivariate analyses of morphometric data with methods for estimating missing values that are robust to potential sources of error. This exhaustive analysis fundamentally revises the taxonomic framework for understanding diversity and variation within elephant birds, compared to historical taxonomic reviews that were based largely on qualitative assessment of much smaller sample sizes of specimens. We demonstrate that three main morphometric clusters can be identified within measurement data for elephant bird appendicular elements, with one cluster further divisible into two separate subclusters. As one of these clusters  [Aepyornis spp. and three Mullerornis spp.) [Our new data-driven taxonomic revision recognizes both different numbers and different identities of elephant bird taxa compared to previous assessments. Our taxonomic framework recognizes only four elephant bird species, substantially reducing the number of valid species recognized by earlier authors, who variously identified 15 different putative species tables ; for exaepyornis , Lambertis spp.)  and Brodis spp.) . As our is spp.) .Mullerornis has generally been interpreted in recent decades as comprising three species, M. agilis, M. betsilei and M. rudis. However, not only do we synonymize these three taxa as representing a single species on the basis of morphometric analysis, but we also identify the name M. modestus as the senior synonym for all three taxa; this name was previously considered to be a junior synonym of Aepyornis maximus [Aepyornis maximus has commonly been interpreted as the largest elephant bird, both in older taxonomic reviews and also in popular culture, but the type material of this first elephant bird to be described has rarely been considered since its original description, with the species concept of A. maximus instead becoming associated with later collections of very large elephant bird bones that have been erroneously assigned to the taxon. Our analysis demonstrates that the name Aepyornis is in fact not associated with the largest known elephant bird material, but instead represents the medium-sized genus-level cluster in our morphometric analysis, with this genus containing only two diagnosable species (A. hildebrandti and A. maximus) compared with previous assumptions of four or more congeners  exceeds estimates based on comparable data for other extinct Quaternary giant birds such as Dinornis (Dinornithiformes: range = 61–275 kg) and Dromornis  [Vorombe on the basis of size; this specimen had a least-shaft circumference of 308 mm and a corresponding mass estimate of 860 kg, making this the largest known bird individual ever recorded. This body mass estimate is comparable to or greater than available estimates for the smallest sauropod dinosaurs  [Aepyornis maximus based on broad, qualitative size ranges assumed for this ‘wastebasket taxon’, leading to underestimation of the true size of the largest elephant birds by previous authors [As the name urements  demonstr60.0 kg) , giving  authors .Aepyornis maximus  [Aepyornis hildebrandti and Vorombe titan demonstrate that specimens assigned to these clusters are also Holocene in age  . New dire in age . We can Dinornis, which was formerly interpreted as representing taxonomic variation, has been shown instead to constitute extreme reversed sexual size dimorphism [Aepyornis maximus and A. medius, two formerly recognized species that were considered to be distinguishable only by size, could represent male and female morphs of the same species [. Indeed, although we do not exclude the possibility that elephant birds exhibited sexual size dimorphism, our morphometric clusters are scaled and therefore independent of size, and are differentiated by more complex patterns of variation across a large series of characters that would not be expected from sexual size dimorphism. Any sexual size dimorphism is therefore likely to be captured as within-cluster variation in our analysis, and our clusters are better interpreted as representing distinct taxonomic units. Recent quantitative analysis has similarly failed to detect any reliable morphometric differentiation of sexual dimorphs in non-avian dinosaurs [Morphological variation in the giant moa morphism ,67, and morphism . Several species . Our quainosaurs . Howeverinosaurs ,67,70.M. modestus, A. maximus and V. titan in the south and southwest of Madagascar and into the central highlands. The substantial disparity in size between these different taxa suggests that these birds were able to coexist by exploiting distinct dietary niches and floral interactions [Aepyornis lentus’ is excluded from biogeographical consideration due to potential unreliability of cluster assignment, all of the specimens assigned to A. hildebrandti in our analysis are restricted to the highest elevations of the central highlands at Antsirabe and Masinandreina. This biogeographical pattern suggests that, whereas different elephant bird genera were morphologically and ecologically distinct enough to be able to coexist in the same landscapes, different species within the same genus (Aepyornis) displayed largely allopatric differentiation between different ecoregions. This spatial pattern is also shown in many other vertebrate taxa across Madagascar today [Dinornis that exhibited size differences across altitudinal gradients and habitat types have been shown to be conspecific through ancient DNA analysis [Pachyornis on New Zealand's South Island, with P. elephantopus occurring in lowland habitats and P. australis restricted to subalpine shrublands and fellfields during the Holocene [Aepyornis therefore provides further support for our interpretation of clusters 2a and 2b as representing taxonomic variation rather than sexual dimorphism.Locality data associated with elephant bird specimens included in distinct morphometric clusters demonstrate the sympatric co-occurrence of ractions ,71. Howear today , and simar today . Althouganalysis , comparaHolocene . The allPrevious assumptions of elephant bird species richness (15 putative proposed species variously accepted by different authors; We encourage further investigation of elephant bird systematics and taxonomy, employing complementary data and methods to those presented in this study. In particular, the suggested bimodality in thickness of elephant bird eggshell  was cons


Ventilatory parameters have been investigated in several species of Testudines, but few species have had their ventilatory pattern fully characterized by presenting all variables necessary to understand changes in breathing pattern seen under varying environmental conditions.Trachemys scripta and the terrestrial tortoise Chelonoidis carbonarius under normoxia, hypoxia, and hypercarbia and furthermore compiled respiratory data of testudine species from the literature to analyze the relative changes in each variable.We measured ventilation and gas exchange at 25 °C in the semi-aquatic turtle NVP) were three to four times longer in T. scripta than in C. carbonarius. Hypoxia and hypercarbia significantly increased ventilation in both species and decreased TNVP and oxygen consumption in T. scripta but not in C. carbonarius.During normoxia both species studied showed an episodic breathing pattern with two to three breaths per episode, but the non-ventilatory periods (TC. carbonarius did show considerable non-ventilatory periods with more than one breath per breathing episode, and the breathing pattern in T. scripta was found to diverge significantly from predictions based on mechanical analyses of the respiratory system. A quantitative analysis of the literature showed that relative changes in the ventilatory patterns of chelonians in response to hypoxia and hyperbarbia were qualitatively similar among species, although there were variations in the magnitude of change.Contrary to expectations, the breathing pattern in  The order Testudines differs from the other groups of reptiles by the presence of a rigid shell, impeding lung ventilation through movement of the ribs . To overTestudines can be divided into two suborders. The 100 species of Pleurodira are characterized by a retraction of the neck in the horizontal plane, whereas the 250 species of Cryptodira retract their neck in the vertical plane . All PleTrachemys scripta and Chrysemys picta have been used in numerous respiratory studies , the semi-aquatic dies see . Althougdies see . While tdies see . Furtherta e.g., . The totta e.g., . Fong, ZTestudo pardalis employs regular single breaths separated by short breath-holds. A regular singlet breathing behavior has also been shown by Testudo graeca and by Testudo horsfieldi. The semi-aquatic Pelomedusa subrufa, on the other hand, uses breathing episodes containing several ventilations interspaced by longer breath-holds  and the South American red-footed tortoise Chelonoidis carbonarius (Testudinidae). Trachemys scripta, the model species for cardiorespiratory studies, was investigated because no previous study reported all ventilatory variables obtained from the same animals and experimental protocols, both under hypoxic and hypercarbic conditions, whereas C. carbonarius was chosen because it is a widespread South American tortoise that has not had its respiratory physiology investigated previously. Furthermore, the present data were compiled together with available data from the literature to characterize the general response of Testudines to hypoxia and hypercarbia and to verify if terrestrial species show a significantly different ventilatory pattern compared to semi-aquatic species.Given these variations in the breathing pattern during normoxia, hypoxia, and hypercarbia among Testudines, and considering the very few ventilatory data available for terrestrial species, the aim of the present study was to analyze the ventilatory response to different gas mixtures in two cryptodirans, the red-eared slider T. scripta  and C. carbonarius  living under natural conditions were obtained from the Jacarezário, Univeridade Estadual Paulista “Júlio de Mesquita Filho”, Rio Claro, SP, Brazil, transported to the laboratory at the University of São Paulo in Ribeirão Preto, SP, and maintained for at least three months before experimentation to acclimate to laboratory conditions. Experiments were performed between November 2014 and February 2015 following approval by the Instituto Chico Mendes de Conservação da Biodiversidade  and Comissão de Ética no Uso de Animais . Animals were maintained under a 12 h light/dark photoperiod cycle, in a temperature-controlled room at 25 ± 2 °C and received a mixed diet supplemented with amino acids, vitamins and minerals  three times a week. T. scripta were housed in a box with a water reservoir for diving whereas C. carbonarius were housed in boxes whose bottom was covered with wooden chips.Adults of both sexes of T. scripta were placed in an aquarium with a single access to an inverted funnel, and each individual only needed to extend its neck and protrude its nostrils into the chamber for air breathing. C. carbonarius were placed in a plastic box and a mask was fitted to the head of each animal for respirometry and a collar was fixed to the neck to prevent head retraction. The dead space of the funnel or the mask was never larger than 40 ml. The exit of the funnel and the frontal tip of the mask were equipped with a pneumotach (Fleisch tube), which was connected to a spirometer . The gas inside the funnel or mask was sampled at 180  ml min−1, dried, and pulled to a gas analyzer . Data were recorded and analyzed using PowerLab 8/35 and LabChart 7.0 .Animals were submitted to open respirometry following 2 were used to calibrate the gas exchange measurements. In all cases, calibrations resulted in linear regressions with R2 >0.95.Both the funnel and the mask were calibrated by injections of known volumes, using an Inspira ventilator , and concentrations of gas, supplied by a Pegas 4000MF gas mixer . Air was used for the spirometer calibration with volumes ranging from 1 to 60 ml and different volumes and concentrations of O2) or progressively increasing hypercarbic  exposures, and after that animals were exposed again to normoxia. The exposure time of each gas mixture, as well as normoxic conditions, was 2 h. Animals were first exposed to one randomly chosen progressive gas exposure and the following day to the other one.Experimental temperature and photoperiod were the same as during maintenance, and all animals were fasted for three to seven days before experimentation to avoid the confounding effects of digestion on metabolism. The animals were weighed one day before the beginning of each experimental treatment. Before any measurements, animals were placed into the experimental setup or equipped with a mask at least 12 h before initiation of experiments. Experimentation started around 08:00, and ventilation and gas exchange were measured first under normoxic conditions, followed by progressively decreasing hypoxic , frequency of breathing episodes , duration of non-ventilatory period , duration of inspiration , duration of expiration , total duration of one ventilatory cycle , tidal volume , breathing frequency , and oxygen consumption  (−1), the relative duration of expiration (TEXP∕TTOT), the relation between inspiration and expiration (TINSP∕TEXP), the expiratory flow rate , minute ventilation (−1 min−1), oxygen consumption (2 kg−1 min−1), and air convection requirement (The last hour of each exposure was used to extract the following data: breathing frequency during breathing episodes (fO2 kg−1) . During P < 0.05 were considered significant.Data were analyzed using GraphPad Prism 6.0 and applying Repeated Measures ANOVA followed by a Tukey’s multiple comparison test. Values of To compare the results of the present study with previously published data, we searched for relevant publications using Pubmed, Web of Science, and Google Scholar databases using keywords such as ‘turtle’, ‘Testudines’, ‘hypoxia’, ‘hypercarbia’, ‘hypercapnia’, ‘ventilation’, ‘gas exchange’, etc. Values of respiratory variables of Testudines obtained under exposure to environmental hypoxia or hypercarbia (but not anoxia or hypoxic-hypercarbia) measured at temperatures between 20 and 30 °C were included . Data frTNV P was, on average, three to four times longer in T. scripta than in C. carbonarius. In T. scripta, hypoxia significantly increased fE, VT, Repi, TNV P and C. carbonarius significantly increased fE, TINSP, VT, fR, fR in C. carbonarius, which was significantly greater when compared to the pre-hypoxic value. Exposure to CO2 increased T. scripta, whereas in C. carbonarius TINSP, TTOT, VT, fR, NV P and f′ significantly decreased  were not significantly affected by either hypoxia nor hypercarbia, just as expiratory flow rate (VT/TEXP), but the latter did show a tendency to increase in both species with increasing levels of hypoxia and hypercarbia , whereas hypoxia caused a greater increase in fE, but slightly decreased fRepi (exception C. carbonarius). Neither hypoxia nor hypercarbia drastically altered TINSP, TEXP, TTOT, and f′ , in C. picta   . In the Testudo graeca and T. pardalis, respectively, but in both species breathing pattern consisted of just one ventilatory cycle interspersed by short and regular non-ventilatory periods. In the present study, both, T. scripta and, unexpectedly, C. carbonarius, showed more than one ventilatory cycle per breathing episode, but the mean duration of the non-ventilatory periods was lower in C. carbonarius when compared to T. scripta. aO2 and pH and increasing PaCO2 , possibly explaining the episodic breathing seen in this terrestrial species, but currently a physiological explication for this behavior is lacking. Interestingly, other ectothermic terrestrial species such as varanid  are similar to ours. E as in the present study (from 8.4 ± 1.6 in normoxia during winter up to 37.1 ± 2.3 episodes.h−1 in summer), but found considerable variation in fRepi through different seasons, ranging from 3.6 ± 0.4 breaths.episode−1 in normoxia during winter up to 26.1 ± 5.4 breaths.episode−1 in hypoxic-hypercarbia during autumn, thereby demonstrating considerable seasonal variation in breathing pattern in T. scripta. Repi and fE during normoxia and hypoxia, and Repi during normoxia. Repi during both normoxia and hypercarbia, and Repi at 10–12% CO2 to be 5.6 ± 1.0 at 28 °C.Comparing our data with previous studies on the effect of hypoxia or hypercarbia on ventilation and gas exchange in T, fR, 2 kg−1 min−1 , but are somewhat similar to the values found by 2 . The general response of T. scripta to reducing oxygen concentrations can be described by a moderate, when compared to the response during hypercarbia, increase in minute ventilation, mainly caused by increasing VT, and a reduction in oxygen consumption, thereby increasing the air convection requirement. These changes are generally more pronounced below 5% O2. The response to hypercarbia also includes an increase in ventilation due to an increase in VT and fR. In T. scripta neither hypoxia nor hypercarbia caused significant changes in TINSP, TEXP, TTOT, f′, and fRepi, whereas fE and TNV P, increased and decreased significantly, respectively.The values for minute ventilation in C. carbonarius during hypoxic or hypercarbic exposures, only data on VT, fR, Terrapene carolina carolina, possibly indicating that animals in their study may not have been resting quietly during normoxia. However, their 2 is identical to the values from other studies at similar oxygen concentrations. VT in C. carbonarius is on the lower end of data available for terrestrial Testudines, which may have been influenced by the relative large amount of bone tissue present in adult individuals of this species . Breathing frequency and In respect to udinidae .Gopherus polyphemus and Terrapene carolina, found values of oxygen consumption very similar to those of C. carbonarius during both normoxia and hypoxia. Interestingly, G. polyphemus spends a significant amount of time in burrows that may show hypoxia as well as hypercarbia, and whose critical oxygen level  was similar to the lowest G. polyphemus (0.05 mlO2 kg−1 min−1) and T. carolina (0.08 mlO2 kg−1 min−1) at less than 1% O2 , meaning that for a minute pump ventilation of 200 ml min−1. Animals should therefore ventilate at a frequency of 40 breaths min−1 and a tidal volume of 5 ml to ventilate the respiratory system with the lowest mechanical work, but such a breathing pattern would result in severe alkalosis due to increased CO2 excretion (T. scripta reached the greatest level of minute ventilation (215.9 ml min−1 kg−1) at 6% CO2, using a tidal volume of 57.2 ml kg−1 and an instantaneous breathing frequency of 18.3 breaths min−1 (fR = 3.0 breaths min−1), values much different from mechanical predictions. The significance of this variation in breathing pattern versus the mechanical predictions of work of breathing needs to be investigated to better understand the mechanical work of breathing of the Testudines respiratory system, since mechanical work of breathing increases markedly with increasing tidal volume, e.g., from 57 to 272 ml cmH2O min−1 kg−1 at 6.2 ml kg−1 and 3.0 breaths min−1 in undisturbed T. scripta versus 34.2 and 0.8 breaths min−1 in vagotomized T. scripta, each at 4% O2  show much smaller tidal volumes during normoxia than other chelonians (mostly between 10 and 20 ml kg−1), resulting in relatively larger increases in VT during hypoxia and hypercarbia than the other species. Both species also showed relatively larger increases in R and VT under normoxic conditions.The relatively large increases seen in VP. geoffroanus under both hypoxia and hypercarbia can be explained by the very low oxygen consumption under normoxic conditions, which could be a consequence of significant extra-pulmonary gas exchange or hypometabolism in this species , when compared to the incomplete PPS of the emydid T. scripta  merianae, whose static breathing mechanics was significantly affected by the removal of their post-hepatic septum (T. scripta.The chelonian respiratory system shows significant variations in lung structure, as well as in associated structures such as the post-pulmonary septum PPS; . The PPS scripta . The prec septum . SimilarC. carbonarius and the semi-aquatic T. scripta during hypoxic and hypercarbic conditions. Contrary to most previous reports on breathing pattern in terrestrial Testudines, C. carbonarius did show considerable non-ventilatory periods with more than one breath per episode. While our data confirm previous data on the general response of T. scripta to hypoxia and hypercarbia, breathing pattern has been found to diverge significantly from predictions based on mechanical analyses of the respiratory system.This is the first study to present all the different variables necessary to fully characterize the breathing pattern in the terrestrial Our meta-analysis demonstrates general trends regarding ventilatory parameters of Testudines when exposed to hypoxia or hypercarbia, but a multivariate analysis of the taxons respiratory physiology will need a complete set of ventilatory parameters from a much larger number of species. To date it is not possible to associate the variations in the magnitude of different respiratory variables with phylogeny, habitat, behavior, and/or lung structure, which could provide important information regarding the evolution of cardiorespiratory physiology in chelonians. Cardiovascular data regarding intracardiac shunt, pulmonary and systemic perfusion, and blood gases during hypoxia and hypercarbia are particularly needed from more species to fully understand blood gas homeostasis in such an important group of intermittent breathers.10.7717/peerj.5137/supp-1Table S1Click here for additional data file.


TCR-independent activation of these MAIT cells from blood and gut was maximized by extending the panel of cytokines to include TNF-superfamily member TL1A. RNA-seq experiments revealed that TCR-dependent and TCR-independent signals drive MAIT cells to exert overlapping and specific effector functions, affecting both host defense and tissue homeostasis. Although TCR triggering alone is insufficient to drive sustained activation, TCR-triggered MAIT cells showed specific enrichment of tissue-repair functions at the gene and protein levels and in in vitro assays. Altogether, these data indicate the blend of TCR-dependent and TCR-independent signaling to CD8+ MAIT cells may play a role in controlling the balance between healthy and pathological processes of tissue inflammation and repair.MAIT cells are an unconventional T cell population that can be activated through both TCR-dependent and TCR-independent mechanisms. Here, we examined the impact of combinations of TCR-dependent and TCR-independent signals in human CD8  •Activation of human MAIT cells is TCR-dependent or TCR-independent and enhanced by TL1A•TCR-dependent and TCR-independent triggering induces distinct transcriptional responses•TCR-dependent triggering of MAIT cells induces a tissue-repair program•in vivo studies in mice indicates a shared transcriptomeData integration with  in vivo, consistent with a homeostatic role for these cells in epithelia.Leng et al. explore the consequences of activation of human MAIT cells via their TCR and/or cytokines, including the gut-associated TNF-superfamily member TL1A. TCR triggering reveals a transcriptional program linked to tissue-repair functions seen  Human innate and adaptive immune systems form a critical partnership in immune defense against microorganisms. Studies have revealed several types of unconventional T lymphocytes that sit at the bridge between innate and adaptive immunity, including mucosal-associated invariant T (MAIT) cells . MAIT cein vivo. Normal expansion is only seen if ligand is delivered with a toll-like receptor (TLR) stimulus  B  . In vitrt manner . Cytokine (Gr) B . We and e (Gr) B , with a   . The autby H2M3) .+ memory T cells expressing IL-18Rα and DR3 (+CD4+ T cells in the presence of anti-CD3 or IL-12+IL-18 (Tumor necrosis factor (TNF)-like protein 1A (TL1A)/TNF superfamily member 15 (TNFSF15) is a gut-associated proinflammatory cytokine originally characterized in a screen for TNF-α homologous molecules. It is expressed by activated T cells, dendritic cells, and monocytes and signals through death receptor-3 (DR3) . TL1A is and DR3 . More sp12+IL-18 . This ma12+IL-18 .in vitro blood- and gut-derived MAIT cells. We find that IL-12 and IL-18, in synergy with TCR triggering, promote the activation of MAIT cells and that additional TL1A signaling can optimize this response in a dose-dependent manner. Triggering with TCR alone or supported by cytokines drives a set of functions linked to a tissue-repair gene expression signature, accompanied by relevant protein expression and function. Overall, our data provide insight into the precise nature of TCR- and cytokine-mediated human MAIT cell activation, characterized by a range of effector functions including not only emergency host defense but also ongoing homeostasis maintenance. This feature may be relevant to other innate-like T cell subsets found at barrier sites in humans.Here, we addressed how TCR-dependent and TCR-independent signals synergize and drive the activation of + T cells  in combination with suboptimal doses of IL-12 and IL-18 in a dose-dependent manner A–1C. Exp+ and CD161− CD8+ T cells  and IL-18 (50 ng/mL) with or without TL1A and/or IL-15 in combination with 5-OP-RU  to characterize transcriptional profiles of MAIT cells under different treatments: TCR , cytokines , and a combination thereof (labeled here as TC). Transcriptional profiles of differentially stimulated MAIT cells were compared with those of untreated (UT) cells. TCR beads were used at a 1:1 bead-to-cell ratio, and cytokines were used at the concentrations optimized earlier. To confirm activation, MAIT cells from the same donors were examined for their release of IFN-γ, TNF-α, and expression of GrB in response to the same stimulations A–S4C.The mRNA levels of 132, 1,124, or 1,375 genes were significantly modulated  by TCR, cytokines, or combined TCR and cytokine stimulation, respectively. Venn diagrams highlight the overlapping and unique transcriptional signatures elicited by these 3 stimulations . We founAnalysis of the other genes unique to TCR and cytokines (TC) indicates these two signals induce diverse physiological functions in MAIT cells A. Among The 1,594 genes with significantly altered expression levels among conditions were then plotted in a heatmap according to their normalized expressions using average linkage hierarchical clustering D. We alsWe next analyzed volcano plots that show differentially expressed transcriptional profiles of stimulated MAIT cells compared with their unstimulated counterparts F–4H and IL-26, oncostatin M (OSM), and heparin binding early growth factor (HBEGF)  G and 4H— (HBEGF) E–S4G. InGiven the range of responses seen after TCR- and cytokine-mediated activation, we speculated that functions of MAIT cells extended beyond conventional antimicrobial responses. The discovery of a skin-homing Tc17 subset in mouse responsive to commensal ligands has shed light on a unique form of adaptive immunity in which antimicrobial functions and tissue repair are coupled within the same subset of unconventional T cells . These cFirst, we examined the volcano plots in Next, genes that were significantly differentially expressed compared with unstimulated MAIT cells were identified from TCR-, C-, and TC-stimulated MAIT cells and statistically compared in aggregate to the tissue-repair gene dataset . Gene set enrichment analysis demonstrated significant enrichment (p < 0.0002) of these tissue repair-related genes in MAIT cells stimulated by TCR with or without cytokines A and 5B,E. coli led to the production of TNF, Furin, and CCL3 in a TCR-dependent manner, because it could be blocked fully or partially by anti-MR1 at 20 or 72 h, respectively   G. This e24–36 h) F and 5G.in vivo and in vitro (in vitro (5-OP-RU stimulation) and in vivo , because these were shown to align with activated H2M3-restricted cells from publicly available data from in vitro-activated human T cells and those in the mouse. In this analysis, the closest neighbors of the maximally activated cells (TC and C) were the Tc17 cells activated in the mouse skin and the in vivo chronically or in vitro-activated MAIT cells from in vivo. Hence, despite the cross-species comparison, stronger stimulation creates an important and relevant shift in the position on the dendrogram. These data indicate, in an unsupervised analysis, that closely shared transcriptional patterns exist between our in vitro-stimulated cells and different subsets that are performing tissue-repair functions (as well as host defense functions) in vivo in mice.We performed a data integration analysis by fusing RNA-seq datasets containing mouse data from activation studies in vitro  with ourin vitro . First, in vivo. Overlap was seen between the different human datasets , indicating the comparability of the studies despite the different protocols and sites.Finally, we performed, using an extension of this bioinformatic approach, alignment among all 3 generated datasets on human and mouse MAIT cell activation to assess their comparability. All 3 have shown that under conditions in which the TCR is stimulated, there is a tissue-repair signature, but when tested in both our dataset and that of Given the relationship between MAIT cells and epithelial maintenance, we wished to assess how closely associated such cells are with the epithelial layer. Because only limited data are available on this (partly because of the availability of suitable antibodies and the need for antibody combinations to reliably identify MAIT cells), we developed a high content imaging protocol based on chip cytometry. By costaining for multiple relevant markers  in colonic tissue, we could observe apposition between MAIT cells and intact epithelium, suggesting two-way cross talk is possible under homeostatic conditions .Figure 7in vitro and in vivo   , yet fulin model   and matrix metalloproteinases. T cell-derived furin has been shown to be critical in tissue protection in a transfer colitis model and affects regulatory T cell (Treg) development . CCL3 M. Both of closure F and 5G.in vivo . Thus, a reasonable body of data has emerged in parallel among the three studies that suggests MAIT cells possess tissue-repair activity of relevance in barrier defense.A similar set of RNA-seq data, functional data, and conclusions has been obtained using parallel experiments in human MAIT cells ytokines . Severalytokines . Looking−6).Taking together the data here and those of + MAIT cells. Their triggering behavior reflects that of other MAIT subsets in which this has been studied, although some functional differences may exist , our model suggests that ongoing maintenance of the barrier is an integral part of the function of such unconventional cells concentrated in epithelia, which goes hand-in-hand with control of microbial invasion.paul.klenerman@medawar.ox.ac.uk). Please note that this study did not generate new unique reagents.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Paul Klenerman .Healthy PBMCs were isolated from leukocyte cones (NHS Blood Services). For long-term storage, PBMCs were kept in liquid nitrogen with freezing media . These samples are fully anonymized, so data on age and gender are not available for comparison. Colonic tissues were collected in the form of polyp biopsies or from the uninvolved mucosa of patients with colorectal cancer. Patient information is shown in the table below. All patients involved gave written consent. Colonic tissues were digested at 37°C for overnight with Collagenase A (Roche) and DNase I (Sigma-Aldrich). Colonic lymphocytes were then isolated from the cell suspension by a Percoll-  gradient: cells were resuspended in 4ml of a 40% Percoll solution that was carefully overlayed over 4ml of 80% Percoll. After centrifugation , lymphocytes were obtained from the interphase between the two Percoll layers. A detailed protocol has been described by Characteristics of the CRC patients.2. Caco2 cells  were cultured at a starting density of 4x105 cell/cm2 in T-175 cell-culture flasks, using GlutaMAX medium supplemented with 10% fetal bovine serum (FBS), 1% MEM Non-essential-amino acid solution (NEAA), 100ug/mL Penicillin-Streptomycin, 2mM L-glutamine (Sigma Aldrich). Cultures were maintained with media exchange every second day and routinely split every week when cells had reached approximately 70% confluency.Cell lines were cultured at 37°C in 5% CO5 to 106 cells/mL in T-175 cell-culture using RPMI-1640 medium (Sigma Aldrich) supplemented with FBS, Penicillin-Streptomycin and L-glutamine.THP1 cells  were cultured at a density between 2x10+ T cells were positively labeled with CD8 Microbeads , and enriched from PBMCs using MS or LS columns following the manufacturer’s instructions (Miltenyi Biotech). Colonic lymphocytes were used without prior enrichment by CD8 microbeads and were maintained in R10 supplemented with 25ng/mL amphotericin B (GIBCO), 40 μg/mL gentamicin (GIBCO), and 10 μg/mL ciprofloxacin (Sigma-Aldrich).PBMCs were thawed, washed and maintained in RPMI 1640 with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin (R10) . CD8For non-specific TCR triggering, PBMCs, enriched CD8 T cells, sorted cells or colonic lymphocytes were stimulated with plate bound anti-CD3/28 antibodies (Miltenyi), or anti-CD3/28 beads (Miltenyi) at 1:1 ratio. ELISA plates (Greiner) were coated with 5 μg/mL anti-CD3/28 with the final volume of 100 μL at 4°C for overnight. Antibody mix was washed off the next morning, and plates were used after 1-hour 37°C incubation with R10. Anti-CD3/28 beads were prepared following the manufacturer’s instructions.5 MACS-enriched CD8s with 1x105 THP1 cells which had been previously pulsed with 10nM 5-OP-RU (kindly provided by David Fairlie) for 2 hours. Unpulsed THP1s were used as controls.MAIT-specific TCR triggering was achieved by co-culturing of 2x10For cytokine triggering, cells were stimulated for 20 hours with IL-12 (Miltenyi) at 2ng/mL, IL-18 (MBL) at 50ng/ml, IL-15 (Miltenyi) at 25ng/ml, TL1A (R&D) at 100ng/ml, unless otherwise stated.E. coli  at a 25 bacteria per cell (BpC) ratio overnight. Bacterially loaded THP1s were washed and co-cultured with MACS-enriched CD8s at a 1:2 ratio. In order to block the TCR-dependent component of this activation, in some experiments an anti-MR1 blocking antibody  was added to the co-cultures.For activation of MAIT cells by bacteria-derived ligands, THP1 cells were loaded with PFA-fixed  Brefeldin A  was added into the cell cultures for the last 4 hours before intracellular staining.Cells were stained with the antibodies and dyes listed in the + T cells were enriched from PBMCs of three healthy individuals and were rested overnight prior to sorting. On the next day, MAIT (CD1612+ Vα7.2+) cells were sorted using a Beckman Coulter MoFlo XDP and stimulated with a range of conditions including anti-CD3/28, cytokines (IL-12/IL-18/IL-15/TL1A), or the combination of both, or left untreated in R10 media for 24 hours. RNA was then extracted from these 12 samples using an RNeasy Micro kit (QIAGEN). The quantity and quality of extracted RNA was first evaluated using both a nanodrop spectrophotometer and the Agilent 2100 bioanalyzer. All samples had RNA integrity (RIN) values greater than 9 and were free from contaminating protein and organic compounds. RNaseq was performed by Wellcome Trust Centre for Human Genetics (University of Oxford) on a HiSeq4000v platform. Gene lists that were differentially expressed  between various conditions and their normalized expression values, as well as the principle component analysis (PCA) plots, were generated with Partek® Flow®, an online analysis platform for Next Generation Sequencing data (http://www.partek.com/partek-flow/), following the user’s guide. Volcano plots were generated with Prism software. Heatmaps were generated using normalized counts with Heatmapper (http://www.heatmapper.ca/expression/) with the averaged linkage clustering method and Pearson distance measurement method. Venn diagrams were drawn with an online diagram drawing platform developed by Ghent University, Belgium (http://bioinformatics.psb.ugent.be/webtools/Venn/). Gene set enrichment analysis (GSEA) was performed using GSEA version 3.0  and CD161- Vα7.2- cells were sorted from pre-enriched blood CD8+ T cells. These cells were then stimulated with anti-CD3/28, cytokines (IL-12/IL-18/IL-15/TL1A), the combination of both or left untreated in R10 media for 20 hours. The total RNA of sorted T cells was extracted with an RNeasy Micro kit (QIAGEN) and reverse transcribed using reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen). For detection of IL-26 mRNA, a 20X IL-26 human TaqMan® probe was used (Hs00218189_m1) with 2X TaqMan® Fast Advanced Master Mix (both from ThermoFisher Scientific). OSM and HEBGF cDNA quantification was performed with Roche ® hydrolysis probes , with GAPDH as the internal control .CD161+ were co-cultured with THP1 cells loaded with fixed E. coli at 25 BpC in the presence or absence of 20 μg/mL LEAF anti-MR1 Antibody (Biolegend). Supernatants were collected at 72 hours. A total of 1.5x104 Caco2 cells were seeded per well in a 96-well clear flat bottom plate (Corning) and grown to confluency at 37°C for 5 days with media exchange every 2 days. Monolayers were scratched using a WoundMaker (Essen Bioscience), washed with serum-free medium and incubated with CD8+ 72h hour supernatants diluted 1:4 with fresh media. As a negative control, fresh media was used. Time lapse imaging was recorded every 4 hours using IncuCyte S3 Live Cell Analysis System (Essen Bioscience) for 36 hours at 37°C. GlutaMAX medium supplemented with 10% FBS, 1% NEAA, Penicillin-Streptomycin and L-glutamine was used throughout this experiment.Enriched CD8in situ at room temperature for 10 minutes using 4% paraformaldehyde solution, then washed with 10-20 mL of PBS. Non-specific binding was blocked by incubating in 5% normal goat serum (Thermo Fisher cat016201) in PBS for at least one hour at room temperature. All antibodies were directly conjugated to their fluorophore and were diluted for the staining step in PBS. Immunostaining was performed using an iterative approach where up to three colors could be applied simultaneously. Fluorophores were subsequently bleached and a new round of antibodies applied to build up the panel . Sections were fixed he panel . Images https://www.immgen.org/) samples (voom function in limma R package (http://www.informatics.jax.org/orthology.shtml). Batch effects were removed using ComBat function in ‘sva’ R package . Statistical significance was assessed using paired Student’s t test, or repeated-measures two-way analysis of variances, with Bonferroni’s correction for multiple comparison assays. For the analysis of the RNaseq dataset, Partek Flow was used. For the GSE129906.The accession number for the raw and pre-processed data from the RNaseq datasets reported in this paper is GEO: The members of Oxford IBD Investigators are Carolina Arancibia-Carcamo, Adam Bailey, Eillie Barnes, Beth Bird-Lieberman, Oliver Brain, Barbara Braden, Jane Collier, James East, Alessandra Geremia, Lucy Howarth, Satish Keshav, Paul Klenerman, Simon Leedham, Rebecca Palmer, Fiona Powrie, Astor Rodrigues, Alison Simmons, Peter B. Sullivan, Simon P.L. Travis, and Holm H. Uhlig.