Doricha eliza), an endangered hummingbird, is endemic to Mexico where two populations have a disjunct distribution. One population is distributed along the northern tip of the Yucatan Peninsula whereas the other is mostly restricted to central Veracruz. Despite their disjunct distribution, previous work has failed to detect morphological or behavioral differences between these populations. Here we use variation in morphology, mtDNA and nuDNA sequences to determine the degree of morphological and molecular divergence between populations, their divergence time, and historical demography. We use species distribution modeling and niche divergence tests to infer the relative roles of vicariance and dispersal in driving divergence in the genus. Our Bayesian and maximum likelihood phylogenetic analyses revealed that Doricha eliza populations form a monophyletic clade and support their sister relationship with D. enicura. We found marked genetic differentiation, with reciprocal monophyly of haplotypes and highly restricted gene flow, supporting a history of isolation over the last 120,000 years. Genetic divergence between populations is consistent with the lack of overlap in environmental space and slight morphological differences between males. Our findings indicate that the divergence of the Veracruz and Yucatan populations is best explained by a combination of a short period of isolation exacerbated by subsequent divergence in climate conditions, and that rather than vicariance, the two isolated ranges of D. eliza are the product of recent colonization and divergence in isolation.The Mexican Sheartail ( Doricha eliza) is an endemic to Mexico, and globally is a near threatened species according to the IUCN Red List TrochilusCalliphlox, Calothorax, Myrtis, Thaumastura, Rhodopis, and Doricha; D. eliza in the bees group D. enicura), and multiple loci are needed to fully resolve the phylogenetic position of the Mexican Sheartail within the Mellisugini The Mexican Sheartail Hummingbird  What is the level of genetic differentiation between the Veracruz and Yucatan populations? (3) Are disjunct populations currently connected by gene flow? (4) When did the Veracruz and Yucatan populations split? And (5) was the divergence between the two disjunct populations caused by vicariance or dispersal? To answer these questions, we conducted Bayesian and maximum likelihood phylogenetic analyses of mitochondrial and nuclear DNA markers and time estimates of intraspecific genetic divergence. We also used morphological data, genetic diversity and historical demographic indices, modeling ancestral distribution, and use of niche divergence tests to infer the history of the Veracruz and Yucatan populations and the relative roles of dispersal and vicariance in driving divergence in the genus.In this study, we ask the following questions: (1) what is the phylogenetic position of We obtained the collecting permit to conduct this work from Mexico’s Secretaría de Medio Ambiente y Recursos Naturales, Instituto Nacional de Ecología, Dirección General de Vida Silvestre (permit number: INE SGPA/DGVS/07701/11) for the field study described. This collecting permit specifically allowed for the collection of tail feathers from the birds. Manipulation of birds in the field was minimal. Birds were captured with mist nets, measured, and their two outermost tail feathers were removed for genetic analyses before the birds were released. All procedures with birds were carried out in accordance with the Guidelines for the Use of Wild Birds in Research proposed by the North American Ornithological Council and the ethics of experimental procedures were revised and authorized by the Animal Care and Use Committee under the Graduate Studies Committee  of the Instituto de Ecología, A.C. (INECOL). While the field studies involve an endangered and protected species, no specific permits are required for field studies such as the one described here.D. eliza during the 2011 and 2012 breeding seasons. Ten hummingbirds were collected in central Veracruz at the following locations: Xalapa, El Lencero, Miradores and Chavarrillo. Feather samples were collected from 15 individuals of the Yucatan population at Rio Lagartos and Chicxulub (Table S1). We sequenced the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 2 gene (ND2) and the complete ATP synthase 6 and ATP synthase 8 coding region (ATPase), and the nuclear 20454 locus from tail feathers of 25 D. eliza, and sequenced or downloaded sequence data from GenBank for the sister species D. enicura and for the outgroups, the bee hummingbirds Calothorax lucifer, C. pulcher, Selasphorus rufus, S. sasin, S. platycercus, S. calliope, Atthis heloisa, Archilochus colubris and Tilmatura dupontii, and the emerald Amazilia cyanocephala (Table S2). We also obtained ND2 sequences from GenBank for an additional 17 species of the bee hummingbird group , 11 representative taxa of the mountain gems group and 12 species of the emeralds group to be used for sequence alignment and as outgroups (Table S2).Feather samples were collected from a total of 25 Taq polymerase , 4.0 mM MgCl2, and 0.29 µM of each primer. PCR reactions were performed in a 2720 thermal cycler  or in an Eppendorf Mastercycler thermal cycler . For amplification of the ND2, cycling parameters consisted of initial denaturation at 94°C for 3 min, followed by 40 cycles at 94°C for 45 sec, annealing at 47–48°C for 45 sec, 72°C for 30 sec, and a final step at 72°C for 5 min. The protocol for amplifying ATPase 68 was an initial denaturation at 95°C for 2 min, followed by 40 cycles at 92°C for 40 sec, annealing at 47–50°C for 1 min, 73°C for 2 min, and a final step at 73°C for 3 min. Amplification of the 20454 locus included initial denaturation at 94°C for 1.30 min, followed by 40 cycles at 94°C for 30 sec, annealing at 50–52°C for 30 sec, 72°C for 45 sec, and a final step at 72°C for 10 min. PCR products were purified with QIAquick (Qiagen Inc.) and sequenced in both directions to check the validity of sequence data using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). The products were read on a 310 automated DNA sequencer (Applied Biosystems) at the INECOL’s sequencing facility. Finally, sequences were assembled using Sequencher v4.9  and then manually aligned using SE-AL v2.0a11 . All sequences are deposited under the following GenBank accession numbers: KJ710519–KJ710624 (Table S2).Total genomic DNA was extracted using the Qiagen DNeasy blood and tissue extraction kit , following the manufacturer’s protocol. Using polymerase chain reaction (PCR), we amplified fragments from three mitochondrial DNA (mtDNA) coding genes: ND2  and maximum likelihood (ML) methods. The BI analyses were run in MrBayes v3.12 Doricha eliza and available sequences for North American and South American members of the Mellisugini clade D. eliza and outgroups, and the third with the nuclear 20454 locus data set. All DNA markers used and their accession numbers are listed in Table S2. We used jModeltest v1.1 http://tree.bio.ed.ac.uk/software/figtree/). The BI analyses included two additional sets of analyses using the combined data set (ND2+ATPase+20454): the first used a single model for the entire combined loci data set , and the second set employed partition-specific DNA evolution models of each gene. For each data set, two parallel Markov chain Monte Carlo (MCMC) analyses were executed simultaneously, and each was run for 50 million generations, sampling every 1000 generations. We computed Bayes factors with the harmonic means Phylogenetic relationships among mtDNA sequences of ML analyses were performed using default values and the same evolution models as in the Bayesian analyses. Node support for the ML tree was estimated with 1000 bootstrap replicates, and nodes were considered highly supported when bootstrap values were ≥70% D. eliza, we used ND2, ATPase and 20454 sequences for all D. eliza samples under the multispecies coalescent method of *BEAST Doricha enicura, Calothorax lucifer and C. pulcher were the outgroups. We employed a relaxed molecular clock model with branch rates drawn independently from a lognormal distribution and the Yule process as a tree prior under a continuous population size model. The models of molecular evolution that best fit our sequence data under the Akaike information criterion  and the convergence of runs was confirmed by effective sample sizes (ESS) >200 for all parameters and by visual inspection of traces within and between replicates using Tracer v1.5 http://beast.bio.ed.ac.uk/TreeAnnotator). Nucleotide substitution models selected with jModeltest v1.1 –2 substitutions/site/lineage/million years (s/s/l/My) for ND2, 2.2×10–2 s/s/l/My for ATPase, and 1×10–3 s/s/l/My for 20454 based on rates obtained for Hawaiian honeycreepers Doricha species To estimate relationships between populations of ion AIC;  were HKYD. eliza. We ran two individual analyses to estimate TMRCA, one with all the ND2 sequences of bee hummingbirds available from previous studies D. eliza and the other hummingbird species listed above. All sequences used and those retrieved from GenBank are listed with their accession numbers in Table S2. The best-fit model of evolution, GTR+I+G for the ND2 and GTR+I for the ND2+ATPase data set, was estimated from the data sets using jModeltest and an uncorrelated lognormal relaxed model selected in BEAST as the clock model. A coalescent model assuming constant population size was used to model the tree prior. The coalescent tree prior used in these analyses appears to fit better when mixed data sets are predominantly intraspecific data Table S2). All of the samples of D. eliza were used in both analyses, rather than just the unique haplotypes, to avoid overestimating evolutionary time scales BEAST v1.7.4 FST values and analysis of molecular variance  using DnaSP v5.1 Hd) and nucleotide diversity (π) using Arlequin To infer genealogical relationships among haplotypes, the ND2, ATPase and phased 20454 sequence data sets were separately analyzed using the statistical parsimony algorithm, implemented in TCS v2.1 ce AMOVA . The AMOD. eliza were inferred by means of neutrality tests and mismatch distributions carried out in Arlequin v3.1 DFsNe) over time in BEAST. This analysis was performed for each genetic group separately and for the two groups combined (concatenated). Concatenated analysis has been proposed to satisfy the assumption of lineages interbreeding in scenarios where divergence is recent and there is low genetic structure –2 substitutions/site/lineage/million years (s/s/l/My) for ND2 and 2.2×10–2 s/s/l/My for ATPase based on rates for Hawaiian honeycreepers The demographic histories of the Veracruz and Yucatan populations of t) between the Veracruz and Yucatan populations of D. eliza, the effective number of migrants per generation (mV to Y and mY to V), and the effective population size of the ancestral (qA) and descendant populations (qV and qY). We used mitochondrial and nuclear phased haplotypes to produce maximum-likelihood estimates and confidence intervals for splitting times, effective population sizes, and gene flow –2 substitutions/site/lineage/million years (s/s/l/My) for ND2, 2.2×10–2 s/s/l/My for ATPase and 1×10–3 s/s/l/My for 20454 obtained for Hawaiian honeycreepers a+[s/(1–s)] a is the time to maturity and s is the adult annual survival rate. Based on this, the estimate for T was 3.04 years. To convert the time since divergence parameter of IMa to years, t, we divided the time parameter (B) by the mutation rate per year (U) converted to per locus rate by multiplying by the fragment length in base pairs, and calculated for the rates described above.We used the ‘isolation-with-migration’ coalescent model as implemented in the program IMa D. eliza resided during the Last Glacial Maximum  and Last Interglacial . We assembled a data set of 121 unique records (51 for Veracruz and 76 for the Yucatan) from georeferenced museum , and analyzed the data with the maximum entropy algorithm in MaxEnt c. 1 km2; D. eliza’s occurrence points, and then ran a correlation analysis to eliminate correlated environmental variables using the program PAST v2.12 We constructed a species distribution model SDM  to prediWe employed a multivariate method To examine differences in morphological variation between the mist-netted Veracruz and Yucatan adult hummingbirds used in the genetic analyses, six measures were taken using a dial calliper with a precision of 0.1 mm and a wing ruler: total body length ; exposed culmen ; bill width at the base ; and wing chord  for both males and females, and tail length  for females, and the length of the outermost rectrices (r5), from the base of the uropygial gland to the tip of the longest rectrix (left and right) for males. All measurements were taken by YLV. Measurements for two juvenile males from Yucatan were discarded from the analysis. To examine morphological differences between populations, for males and females we conducted a one-way non-parametric Kruskal-Wallis test using genetic group as fixed factors and morphological measures as dependent variables. These analyses were performed using SPSS v17 for Mac .D. eliza and other species in the bee hummingbird group were reconstructed using Bayesian inference (BI) and Maximum Likelihood (ML). The BI and ML analyses yielded the same general topologies with minor differences in the position of some terminal branches. Only BI trees are shown. Both the BI and ML trees of the ND2 sequence data set confirmed that D. eliza and D. enicura form a highly supported monophyletic clade . A highly supported relationship between Doricha and Calothorax species was retrieved , yet the relationships within these genera of sheartails and those between other members of the Mellisugini are not fully resolved . The level of polymorphism found in the Mellisugini of the nuclear 20454 locus was low and several haplotypes were shared among species. Most interspecific relationships in the Mellisugini were not resolved when only using 20454 . In contrast, the interspecific phylogenetic relationships among species in the Mellisugini were more fully resolved when using the combined ND2+ATPase+20454 data set , and monophyly of sheartails  is also retrieved with high support . Individuals of D. eliza are retrieved as a monophyletic group , with a split separating the Veracruz and Yucatan populations  in BEAST was estimated to be 1.03 Ma (95% HPD 1.608-0.309 Ma) and 1.46 Ma (95% HPD 2.104-0.852 Ma) between Calothorax species  when using the ND2 data set of Mellisugini representatives and 222,000 years ago  when using the ND2+ATPase data set. Estimates of the TMRCA for Doricha species and for the sheartails  indicate that the splits occurred at 1.04 Ma  and 2.06 Ma  when using the ND2 data set, and at 0.7 Ma  and 1.21 Ma  when using the ND2+ATPase data set.Relationships between n *BEAST Figure 2D. eliza (S2) produced 10 haplotypes for ND2, and 8 haplotypes for ATPase, resulting in 10 haplotypes for the concatenated sequence (1077 bp). Phylogenetic analysis of the ND2, ATPase, and concatenated ND2+ATPase haplotypes revealed genetic divergence between the Veracruz and Yucatan populations with no shared haplotypes (h) and nucleotide diversity (π) were moderate for both the Veracruz  and Yucatan  populations. When samples were combined, overall h was 0.796, and overall π was 0.0020 indicating relatively high levels of genetic diversity in the Mexican Sheartail hummingbird. The AMOVA results revealed strong population structure  when samples were grouped by geographic area. Mitochondrial divergence between the Veracruz and Yucatan populations was low (Dxy = 0.35%), whereas genetic divergence between populations of D. eliza and the other members of the sheartails ranged from 0.46% to 3.47%  when the Veracruz and Yucatan populations were pooled  was estimated to be 5,380  and the sizes of the two descendant populations were NVERACRUZ = 1,830  and NYUCATAN = 1,410 . Migration rates between genetic groups (mYUCATAN→VERACRUZ and m VERACRUZ→YUCATAN) were 1.28  and 1.02 , respectively, and the divergence time (t) between genetic groups was estimated to be 22,100 years ago .IMa results are summarized in D. eliza , and Guatemala.The current distribution predicted by MAXENT Figure 4D. eliza (Veracruz and Yucatan records). The first niche axis (38.8% of variation) was associated with isothermality (BIO3) and precipitation of driest month (BIO14). The second niche axis (34.2%) was associated with annual precipitation (BIO12) and annual mean temperature (BIO1), and the third axis (15.7%) was associated with mean annual range (BIO2) and temperature seasonality (BIO4). Tests of niche divergence and conservatism on these three niche axes showed evidence for niche conservatism on niche axis 1 and niche divergence on niche axis 2 and 3 , yet males from the Yucatan population had significantly smaller left outermost rectrices  than those from Veracruz .Morphological analysis detected no significant differences in the mean values of most traits between the Veracruz and Yucatan populations  sequences and the available ND2 (1041 bp) sequences were downloaded from GenBank, which led to a high percentage of missing characters. Although some studies found that incomplete data could bias the ML and BI analysis Archilochus, Atthis, Calothorax, Calliphlox, Calypte, Chaetocercus, Doricha, Eulidia, Microstilbon, Myrmia, Myrtis, Rhodopis, Selasphorus (incl. Stellula), Thaumastura and Tilmatura, are recovered within two groups (sheartails and “Selasphorus” + woodstars). A previous study Doricha, Calothorax, Atthis and Tilmatura in the Mellisugini as suggested by McGuire et al. Tilmatura dupontii as the only representative of the woodstars and sister to all other bees in that study, whereas our phylogenetic analyses place T. dupontii closer to South American woodstars.A molecular phylogeny combining the available ND2 sequences to resolve the relationships within the Mellisugini (bees) clade was not available until now. Here we used a ND2 data set from four taxa assigned to the genera D. eliza) is strongly supported as the sister group to D. enicura, and together they appear as the sister to Calothorax species forming the group of sheartails with strong support. The relationship between sheartails and woodstars, however, received moderate support, and Archilochus colubris and A. alexandri cluster with the woodstars. In a recent study surveying Mellisugini relationships using nuclear and mtDNA sequences Archilochus species appeared in a clade with Calliphlox evelynae and Mellisuga minima but a sister relationship between this clade and sheartails  was not supported. More data is necessary, including that of Mellisuga helenae, Chaetocercus heliodor, Ch. astreans, Ch. berlepschi and Ch. jourdanii, to verify this position and to corroborate the monophyly of woodstars. Based on the BI and ML analyses of the combined data set (ND2+ATPase+20454), we propose that D. eliza is sister to D. enicura and both form a monophyletic clade with Calothorax species.According to our phylogenetic analyses of the combined data set (ND2+ATPase+20454), the Mexican Sheartail hummingbird  is required for genetic and morphological variation to arise in natural populations. For Mexican Sheartails, the monophyly of D. eliza, the question of how their isolation occurred remains unanswered. It is likely that the Veracruz population represents a relatively recent colonization event, though it is difficulty to directly observe immigration events in nature Mellisugini are a recently diverged lineage D. eliza but reciprocal monophyly of haplotypes, supporting the hypothesis of a short history of isolation. Moderate levels of haplotype and nucleotide diversity of populations suggest relatively small population sizes and founder effects. Significant genetic differentiation between populations and limited gene flow resulting from barriers to dispersal have been found for montane hummingbird species in particular, such as Speckled Hummingbird (Adelomyia melanogenys) Campylopterus curvipennis) Amazilia cyanocephala) Selasphorus platycercus) Lampornis amethystinus) . Limited differentiation has been found in other species with a lowland distribution, such as the Rufous-tailed Hummingbird (Amazilia tzacatl) Phaethornis longirostris) A. t. handleyi), endemic to the Caribbean island Escudo de Veraguas in western Panama, initially described as a distinct species on the basis of its considerably larger size and darker plumage, is slightly differentiated  from the mainland A. tzacatl c. 10 km away Our results reveal moderate mtDNA divergence between the Veracruz and Yucatan populations of D. eliza separated by the Gulf of Mexico would have been exposed and eventually adapted to the different environmental conditions. Populations of the Mexican Sheartail separated by 780 km (and by the Gulf) are distributed in a unique environmental space, implying that the different environmental conditions in the Yucatan Peninsula and in Veracruz would have reduced gene flow, as shown by the IMa results, and this would have reinforced the divergence of the two mtDNA haplogroups following the initial spatial separation. This scenario is supported by our tests of niche divergence and conservatism that compared the amount of climatic divergence to the null expectation of background climatic divergence and that showed evidence for niche divergence between the D. eliza records of Veracruz and the Yucatan on two axes of environmental space related to annual precipitation and mean diurnal temperature range (PC2 and PC3). These findings support the hypothesis that climatic niche dissimilarity between D. eliza populations separated by the Gulf seems to have reduced gene flow. Our analyses of D. eliza, combining a phylogeographic and species distribution modeling approach, suggest that the observed patterns of genetic variation and divergence between the Veracruz and Yucatan populations are best explained by a combination of isolation exacerbated by subsequent climate differentiation between regions. Although the latter may be true for species that disperse poorly or that are reluctant to cross areas of less hospitable habitat for physiological reasons, niche divergence for other species with poor dispersal may mean enhanced opportunities for isolation and reduced gene flow, thereby increasing the likelihood of speciation.Overall, population differentiation in the Mexican Sheartail seems primarily enhanced by isolation, which is reasonable for populations separated by a long distance. The Veracruz colonization hypothesis is consistent with the lower migration rate of the Veracruz population to Yucatan than was found for the opposite direction, and with the results of the tests of niche conservatism that suggest that the Veracruz colonization with gene flow was facilitated by niche similarity (PC1). Consequently, following geographic isolation, the populations of D. eliza hummingbirds from Veracruz are similar in most trait mean values to individuals from the Yucatan population , and thus increased sexual selection in the smaller population of Veracruz.Our morphological analysis confirmed that see also . PatternD. eliza are genetically differentiated, and that the outermost rectrices of male hummingbirds from Veracruz are longer than those of the males from Yucatan. The Mexican Sheartail Hummingbird is globally near threatened and both the Veracruz and Yucatan populations are locally endangered with population declines in Veracruz resulting from severe habitat degradation caused by livestock grazing, sugarcane cultivation and residential development, while the Yucatan population is under pressure mainly from the development of its coastal dune habitat for tourism D. eliza constitute distinct genetic lineages, and that the importance of these populations as reservoirs of endemic genetic diversity require different management approaches and merit targeted conservation efforts to preserve the unique genetic pools of both populations and their habitats.Our results reveal that the Veracruz and Yucatan populations of Figure S1Stills from video recording, showing moments of a rocking pendulum flight displayed by a Doricha eliza male to a female at the nest. (A) Photograph shows a male D. eliza from the Veracruz population. Photograph by Gerardo Sánchez Vigil. (B) Photograph shows a female D. eliza from the Veracruz population. Photograph by Yuyini Licona Vera. (C–J) The male begins the courtship display doing a pendulum flight (from left to right) in front of the female. During the display, the male extends his throat feathers and fully displays tail rectrices, while approaching the female repeatedly. The entire time, the female at the nest follows the male’s movements (red arrows). This pendulum flight is done repeatedly around the female (from right to left and from left to right) and is finished with an upward flight (not shown in the video). The video is available as supplementary material – (TIF)Click here for additional data file.Figure S2Bayesian posterior probabilities and bootstrap support for MrBayes and Maximum Likelihood analyses. Illustration of tree topology based on ND2 sequences for North American and South American members of the Mellisugini clade. Values above branches denote posterior probabilities (PP) and those below branches denote bootstrap values.(TIF)Click here for additional data file.Figure S3Bayesian posterior probabilities and bootstrap support for MrBayes and Maximum Likelihood analyses. Illustration of tree topology based on the nuDNA locus 20454 unphased sequences from D. eliza and outgroups. Values above branches denote posterior probabilities (PP) and those below branches denote bootstrap values.(TIF)Click here for additional data file.Figure S4Morphological differences between the Veracruz and Yucatan populations of D. eliza males. Data are means and 95% confidence intervals for total body length (A), exposed culmen (B), base bill-width (C), wing chord (D), length of right outermost rectrix (E), and length of left outermost rectrix (F). Measurements are in mm.(TIF)Click here for additional data file.Figure S5Morphological differences between the Veracruz and Yucatan populations of D. eliza females. Data are means and 95% confidence intervals for total body length (A), exposed culmen (B), base bill-width (C), wing chord (D), and tail length (E). Measurements are in mm.(TIF)Click here for additional data file.Table S1Code for identification (ID), sex, state and locality of origin, geographic coordinates and elevation of sampled individuals of Doricha eliza.(DOC)Click here for additional data file.Table S2Species names, sequence data and GenBank accession numbers for Doricha eliza (25) and outgroups (51) used in this study.(DOC)Click here for additional data file.Table S3Code for identification (ID), sex, morphological data and GenBank accession numbers for Doricha eliza individuals used in this study.(DOC)Click here for additional data file.Video S1Rocking pendulum flight as displayed by male Mexican Sheartails. The video was recorded at Miradores, Veracruz during the breeding season (07 September 2012).(MP4)Click here for additional data file.


Extended esophageal endoscopic submucosal dissection (ESD) is highly responsible for esophageal stricture. We conducted a comparative study in a porcine model to evaluate the effectiveness of adipose tissue-derived stromal cell (ADSC) double cell sheet transplantation.Twelve female pigs were treated with 5 cm long hemi-circumferential ESD and randomized in two groups. ADSC group (n = 6) received 4 double cell sheets of allogenic ADSC on a paper support membrane and control group (n = 6) received 4 paper support membranes. ADSC were labelled with PKH-67 fluorophore to allow probe-based confocal laser endomicroscopie (pCLE) monitoring. After 28 days follow-up, animals were sacrificed. At days 3, 14 and 28, endoscopic evaluation with pCLE and esophagography were performed.P = 0.08) and higher gain weight on day 28. pCLE demonstrated a compatible cell signal in 4 animals of the ADSC group at day 3. In ADSC group, endoscopy showed that 1 out of 6(17%) animals developed a severe esophageal stricture comparatively to 100% (5/5) in the control group; P = 0.015. Esophagography demonstrated a decreased degree of stricture in the ADSC group on day 14  and day 28 . Histological analysis showed a decreased fibrosis development in the ADSC group, in terms of surface  and maximal depth .One animal from the control group was excluded (anesthetic complication). Animals from ADSC group showed less frequent alimentary trouble (17% vs 80%; In this model, transplantation of allogenic ADSC organized in double cell sheets after extended esophegeal ESD is strongly associated with a lower esophageal stricture’s rate. Specific measurements were performed on the most pathological slide of each animal as follows: maximal thickness of the slide (extending from digestive lumen to serous layer), maximal thickness of re-epithelialization, maximal thickness of fibrosis, maximal surface of fibrosis, and maximal length of defect of muscularis propriae (characterized by the distance between the banks of muscularis propriae on each side). Fibrotic invasion of the inner and outer muscular layer was defined as the presence of fibrosis tissue continually from digestive lumen to muscular layers. Finally, inflammation analysis was performed semi quantitatively based on the detection of inflammatory cells through the different layers.After sacrifice, esophagi were extracted and strictures areas were isolated for macroscopic evaluation and measurements. Esophagi pieces were then fixed in 10% buffered formalin, embedded in paraffin and processed into 4 μm-thick sections. Hematoxylin Eosin and Saffron (HES) was used to stain slides. Slides were digitalized for computerized analysis . Categorical data were expressed as percentages and compared with the Fischer exact test. Agostino and Pearson omnibus was used to confirm non-normality of our continuous data. We compared them with the Mann-Whitney test and expressed them as median values and range or mean ± standard deviation. A P>0.05). Transplantation was successful for each animal, defined as the application of all four grafts in the esophageal bed scars. We harvested all resected specimen and size was comparable between the two groups. Average specimen height and width was respectively 2.6 cm and 1.3 cm; P>0.05  in the ADSC group vs 40% (2/5) in the control group; P = 0.017) on day 14 and 46% vs. 90% (P = 0.03) on day 28, respectively for ADSC group and control group . [First, comparatively to other stem cell lineage, ADSC are easy to isolate in a large number and easy to expand tivity). ,39Secondly, we assumed that local application of ADSC would decrease abnormal fibrosis and assist mucosal normal regeneration. We used a double-sheet construction in order to improve the effectiveness of transplantation. Two ADSC sheets instead of one sheet were chosen for the following reasons: 1/ an ADSC 3D construct made of two ADSC sheets layered up upon each other (with their preserved extracellular matrix) allow a better cell survival into the ulcer bed because it protects the cells from aggressive external environment; 2/ neoangiogenesis could be stimulated by the superposition of two ADSC sheets promoting proangiogenic factors diffusion, which enables engraftment; and 3/ a double ADSC sheet is more easy to manipulate during the endoscopic procedure. Two ADSC sheets instead of three or more sheets were chosen because the higher the number of sheets, the higher will be the risk of necrosis and cell death inside the 3D construct. Comparatively Okhi et al. applied a different type of cell sheet. They transplanted mature cell sheets approaching the native esophagus mucosal histological structure, composed of a stratified autologous oral mucosal sheet consisted of 3–5 layers. These sheets were obtained after 14 days of cell incubation using a temperature culture cell dish. Finally, based on previous studies we decided to transplant four double cell sheets per animal, with the objective to cover approximatively half of the wounded surface.in vivo.Our first hypothesis to explain the antifibrotic effect of ADSC sheet transplantation is the consequence of multiple factors released by the ADSC. This paracrine effect of ADSC seems to happen early, just after transplantation. Mechanisms of fibrosis are complex. After external aggression, as endoscopic resection, inflammatory response could be severe and uncontrolled resulting in abnormal healing with fibrosis development. ADSC probably influence this initial inflammatory response, particularly by producing TGFβ. ,41 MoreoDespite the success of the transplantation procedure, one animal from the ADSC group developed a severe stricture with feeding difficulties. Confirmation of severe stricture was obtained on day 14 after morphological evaluation. Interestingly, endoscopic and radiological evaluation showed a proximal stringy stricture, but of very small dimension compared to strictures observed in the control group. Three hypothesis might explain this unfavorable evolution: 1/ a technical early failure of the cell grafts; 2/ a poor ADSC survival despite a positive fluorescent signal at day 3; and 3/ the cells destruction after day 3 that could be exacerbated by a difficult and longer pCLE procedure. Okhi et al  also repOne limitation of our study was the small number of animals included. Ideally, additional experiments could strengthen our results, especially for the clinical evaluation which was not different between groups. Even if clinical evaluation was very important, it was more difficult and less objective than endoscopic and radiological evaluation, in particular in terms of regurgitations and vomiting score in pigs. Concerning the weight gain, difference was not significant between the control group and the ADSC group. However, because of rapid growth in young pigs (6 month-old pigs), to reveal a significant weight gain difference seems to be challenging and would likely require a high number of supplementary animals.In conclusion, double cell sheet transplantation of allogenic ADSC appears to be an effective preventive treatment of esophageal stricture after large endoscopic submucosal dissection. Moreover, pCLE seems to be a useful tool in order to monitor cells transplantation. Further studies are needed to confirm these results and explore the role of ADSC-sheets on the wound healing process. These preclinical results are very promising and encourage us to undertake the clinical evaluation of ADSC sheets, as a preventive treatment of esophageal stricture formation in patients treated by large ESD.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Autologous skin graft is frequently used in the field of plastic, and reconstructive surgery. The engraftment is dependent upon revascularization and angiogenesis, which can be regulated by different factors. In addition to its hematopoietic effects, erythropoietin is shown to positively affect the wound healing process.We studied effects of human erythropoietin on revascularization of full thickness skin grafts in rat.Forty adult Albino male rats were selected for this study. Full thickness skin graft was performed for them, and the effects of systemic, and localized administration of erythropoietin on vascularization of the graft area were evaluated in four groups as following: inverse group underwent full thickness skin graft; in normal saline group normal saline was injected under the fascia of grafted area for seven days; systemic EPO group received systemic erythropoietin for seven days after the surgery; and in graft EPO group, erythropoietin was injected under the fascia of grafted area after full thickness skin grafting for seven days.Forty adult Albino male rats (n = 40), with weights ranging from 356 to 469 g (mean 391.5 ± 29.6 g) were included. The vascular densities of central margins were significantly different between inverse group and graft EPO groups , and vascular density of central margins of normal saline group and graft EPO groups were significantly different too .EPO can stimulate angiogenesis which has an important role in wound healing. So, local administration of EPO seems to be beneficial in engraftment. Autologous skin graft is frequently used in the field of plastic, and reconstructive surgery , which iThe presence of EPO receptors on epithelial cells raised the hypothesis that EPO may directly affect the complex network of cytokines and growth factors involved in both the maturation of erythrocytes and the proliferation of endothelial cells , 9. EPO Because of all of the interesting characteristics of EPO, we studied effects of human erythropoietin on neovascularization of full thickness skin grafts in rat.Forty adult Albino male rats (n = 40), with weights ranging from 356 to 469 g (mean 391.5 ± 29.6 g), were selected for this study. During the experiments, animals were housed one per cage, maintained under controlled environmental conditions , and were provided with standard laboratory food and water ad libitum. The experiments were conducted in accordance with the Declaration of Helsinki and with the Guide for the care and use of laboratory animals. After induction of general anesthesia with ketamine hydrochloride (30 mg/kg intramuscularly) and xylazine (12 mg/kg intramuscularly), the surgical site was shaved and prepped with povidone iodine and chlorhexidine solution. Animals were covered with sterile surgical drapes with only the operative site exposed. A 2 × 3 cm full thickness skin graft was taken from the area between the two scapula bones on the dorsum of each rat . The skiThe animals were randomly allocated into the following groups:Group 1 : Underwent full thickness skin graft.Group 2 : After full thickness skin grafting, normal saline was injected under fascia of the grafted area for seven days.Group 3 : After performing surgery, the rats received systemic EPO (300 Unit/kg) for seven days.Group 4 : After full thickness skin grafting, EPO (300 Unit/kg) was injected under the fascia of grafted area for seven days.On the 7th day, rats were sacrificed and biopsies were taken from the grafted area. After fixation, staining of the samples with immunohistochemistry methods for CD31 and anti F-VШ, as accepted endothelial markers  was doneData were collected, analyzed and reported as mean and standard deviation (mean ± SD). ANOVA test and post hoc comparison test were used for comparison of differences between four groups. Statistical analysis was carried out by using SPSS 19.0 software. P ≤ 0.05 was considered as statistically significant.Forty adult Albino male rats (n = 40), with weights ranging from 356 to 469 g (mean 391.5 ± 29.6 g) were included. ANOVA test showed significant difference in vascular density of central margins  .For analyzing the differences of central margins among the groups post hoc test was used. Vascular density of central margins was significantly different between inverse group and graft EPO group, , and vascular density of central margins of normal saline group and Graft EPO group showed statistical difference, too .The angiogenic cascade is known to result from a concerted interaction between the extracellular matrix, cells, and growth factors . After tEnhancement of neovascularization by EPO has been attributed to direct effect of EPO on VEGF production and on endothelial cell mitosis , 11. The


Granulomatosis with polyangiitis (GPA), formerly known as Wegener’s granulomatosis (WG), belongs to the group of ANCA-associated necrotizing vasculitides. This study describes the clinical picture of the disease in a large cohort of GPA paediatric patients.Children with age at diagnosis ≤ 18 years, fulfilling the EULAR/PRINTO/PRES GPA/WG classification criteria were extracted from the PRINTO vasculitis database. The clinical signs/symptoms and laboratory features were analysed before or at the time of diagnosis and at least 3 months thereafter and compared with other paediatric and adult case series (>50 patients) derived from the literature.The 56 children with GPA/WG were predominantly females (68%) and Caucasians (82%) with a median age at disease onset of 11.7 years, and a median delay in diagnosis of 4.2 months. The most frequent organ systems involved before/at the time of diagnosis were ears, nose, throat (91%), constitutional  (89%), respiratory (79%), mucosa and skin (64%), musculoskeletal (59%), and eye (35%), 67% were ANCA-PR3 positive, while haematuria/proteinuria was present in > 50% of the children. In adult series, the frequency of female involvement ranged from 29% to 50% with lower frequencies of constitutional , ears, nose, throat , respiratory , laboratory involvement and higher frequency of conductive hearing loss than in this paediatric series.Paediatric patients compared to adults with GPA/WG have similar pattern of clinical manifestations but different frequencies of organ involvement. Granulomatosis with polyangiitis (GPA), previously known as Wegener’s granulomatosis (WG),  is a necThe estimated annual incidence of the disease in adults is 1:100,000, and 90% of the patients are Caucasians . In chilThe clinical and laboratory picture of GPA/WG was described in several large cohorts of predominantly adult patients -12 but tThe aim of this project was to describe the clinical and laboratory features at presentation of childhood GPA/WG in a large international cohort of paediatric patients collected by PRINTO, and compare this series with other paediatric series and with adult patients with WG/GPA derived from the literature.The PRINTO database contains data on 1398 patients with childhood vasculitides, with age at diagnosis ≤ 18 years, vasculitis diagnosis after year 2000, as previously described ,17. The For the purposes of this analysis, we extracted all patients fulfilling the c-GPA/WG EULAR/PRINTO/PRES classification criteria -17. PatiThe study was approved by the ethics committee of the Gaslini Hospital  and by the ethics committees of all remaining participating centres and informed consent was obtained from parent(s) as required by the national law in each participating country.http://www.ncbi.nlm.nih.gov/pubmed/) search was used to retrieve studies, which included patients with GPA/WG (both children and adults), and included both prospective and retrospective cases describing the clinical picture of at least 50 patients. Search terms were “(((Granulomatosis AND (Wegener OR Wegener’s OR polyangiitis)) OR (necrotizing AND vasculitis))) AND (group OR cohort OR patients) AND (prospective OR prospectively OR retrospective OR retrospectively) AND demographic AND age AND clinical AND study” in English.A PubMed (st and 3rd quartile); the categorical data were reported as absolute frequencies and as percentage frequencies of measured subjects.The quantitative demographic, clinical and laboratory data were reported as median .st and 3rd quartile 8.5-14.5 years) and with median delay in diagnosis of 4.2 (1.0-9.6) months.A total of 56/67 patients fulfilling the EULAR/PRINTO/PRES childhood GPA/WG classification criteria were identified; the remaining 11 patients were excluded for the following reasons: 1 age at onset > 18 years, 1 for the presence of other co-morbid condition, 7 excluded by consensus evaluation, 4 not fulfilling the EULAR/PRINTO/PRES as previously described ,17. Eigh, pulmonary , and eye  involvement which remained substantially unchanged (data not shown).The Table When evaluated for ANCA positivity, at the time of diagnosis, 67% children were ELISA PR3 and 26% ELISA MPO ANCA positive which decreased to 39% and 19.5% respectively at the subsequent measurement at least 3 months later; ANCA immunofluorescence staining was positive in 82.7% at diagnosis and in 47.6% 3 months thereafter. Haematuria/proteinuria was present in > 50% cases at the time of diagnosis (with necrotizing pauci-immune glomerulonephritis in 23/28 [82.1%] biopsies available) and in >30% at least 3 months later.The Table When compared to adult series, adult patients showed lower frequencies of constitutional , ENT , respiratory , and renal (haematuria or red blood cells casts) involvement, and a higher frequency of conductive hearing loss.In this study, we have analysed the clinical features at the time of diagnosis of a large sample of children with GPA/WG whose data were retrieved from the PRINTO database and were collected on the occasion of the project that led to the validation of the EULAR/PRINTO/PRES criteria for the classification of childhood vasculitis -17.Granulomatosis with polyangiits is very rare in children and, although it appears to be the same disease observed in adults, it is not known if childhood presentations are characterized by differences in the frequency of some clinical features well described in adults. Indeed, in other connective tissue diseases, such as systemic lupus erythematosus or juvenile dermatomyositis, the frequency of several clinical manifestations differed from those observed in adults ,19.We have therefore investigated the clinical and laboratory features present at the time of diagnosis in our series of 56 children and compared them with those of the only other large series of paediatric patients (N = 65) reported in the literature as well as with 3 large (N > 50) reported series of adult patients with GPA/WG.In agreement with previous studies in children, we have found a gender disproportion, as 68% of our patients were females, ,8,10.In the present paediatric series, the disease was characterised by high frequency of constitutional symptoms, upper and lower respiratory tract, mucosa, skin and musculoskeletal involvement. The frequencies of symptoms typically decreased in the first few months after diagnosis, presumably due to the effect of treatment although data on treatment modalities were not collected in the database. Our series confirms the previous observation by Cabral et al that patients with time to diagnosis longer than 12 months have a higher frequency of ear, nose and throat and cutaneous symptoms and less renal symptoms in comparison with earlier diagnosed patients . This liComparing the paediatric and adult cohorts, while organ involvement, signs and symptoms were similar, there were differences in their frequencies at disease presentation. Adults had a lower frequency of constitutional, ENT and respiratory involvement; while hearing loss was more frequent in the adult population.The current work is limited by the lack of information on treatment and disease outcomes. In addition, comparison of clinical and laboratory findings with literature data might be hampered by the lack of standardised definition and by differences in the time of evaluation in the different series. Moreover our study was likely hampered by inconsistencies in laboratory techniques (in particular in relation to ANCA testing), and by the exclusion of cases in this series who did not fulfil the new paediatric classification criteria for c-GPA. Indeed, we recognise that patients may present with limited forms of the disease, without initially fulfilling all criteria for classification and clinicians must remain vigilant in relation to this presentation in children.In conclusion, paediatric patients compared to adults with GPA/WG have a similar pattern of clinical manifestations but present different frequencies of organ involvement.Children with GPA/WG are predominantly females/Caucasians with a median onset age of 11.7 years.Adult showed lower frequencies of female, constitutional, ears/nose/throat, respiratory, laboratory involvement and higher frequency of hearing loss.Paediatric patients compared to adults have similar clinical manifestations but different frequencies of organ involvement.The authors declare that they have no competing interest.We confirm that all the named co-authors have participated in the: conception and design, or analysis and interpretation of data drafting the article or revising it critically for important intellectual content, final approval of the version to be published. All authors read and approved the final manuscript.


Predicting molecular responses in human by extrapolating results from model organisms requires a precise understanding of the architecture and regulation of biological mechanisms across species.Here, we present a large-scale comparative analysis of organ and tissue transcriptomes involving the three mammalian species human, mouse and rat. To this end, we created a unique, highly standardized compendium of tissue expression. Representative tissue specific datasets were aggregated from more than 33,900 Affymetrix expression microarrays. For each organism, we created two expression datasets covering over 55 distinct tissue types with curated data from two independent microarray platforms. Principal component analysis (PCA) revealed that the tissue-specific architecture of transcriptomes is highly conserved between human, mouse and rat. Moreover, tissues with related biological function clustered tightly together, even if the underlying data originated from different labs and experimental settings. Overall, the expression variance caused by tissue type was approximately 10 times higher than the variance caused by perturbations or diseases, except for a subset of cancers and chemicals. Pairs of gene orthologs exhibited higher expression correlation between mouse and rat than with human. Finally, we show evidence that tissue expression profiles, if combined with sequence similarity, can improve the correct assignment of functionally related homologs across species.The results demonstrate that tissue-specific regulation is the main determinant of transcriptome composition and is highly conserved across mammalian species. Mouse and rat are frequent mammalian models in biomedical research to learn more about a disease, its diagnosis and its treatment. Comparing results obtained from mammalian models with those from human samples is essential to estimate conservation of molecular mechanisms across species and to refine prediction models. Cross-species comparisons can be carried out at several levels such as genomic or protein sequences, molecular abundances, or phenotypes. On the sequence level, the conservation of protein sequence between species has been extensively studied. A more recent development has been the study of genomic alterations, some of which appear to be highly relevant in translational efforts from model organisms to human. For exaIn contrast to tissue expression studies, the comparison of responses to perturbations has been much more difficult because available data consists of experiments carried out under conditions that are not easily comparable. In fact, from all public data currently available from gene expression repositories, very few experiments can be considered equivalent between human and mouse, such as cell cultures from the same tissue of origin and treated with the same chemical at identical concentration ranges and in a similar experimental setup. Therefore, comparisons have remained primarily descriptive, for example on the level of correlation network structures between species, assuminRecent comparative genomics efforts have led to a better understanding of conservation of gene expression between human and rodents. Nevertheless, there is still much debate about which regulatory aspects are conserved between gene homologs of different species. Earlier studies comparing the expression patterns of orthologous gene pairs in different tissues showed conflicting results. For example, some studies suggested that orthologous genes have dissimilar expression patterns,4,9-11, In this work, we overcome these limitations by combining three mammalian species, a large set of tissue types, two independent datasets per organism, and a data preparation methodology that delivers highly representative and robust expression values from a very large set of original data. Specifically, we integrated high quality human, mouse and rat data from more than 33,900 Affymetrix expression arrays across six microarray platforms (3 species × 2 platforms per species) and across at least 55 overlapping tissue types between each set of platforms. The original data for this study was obtained from Genevestigator, a databThe study was carried out on two distinct microarray platforms per species in order to assess the validity of the results across platforms and between independent experimental compositions. We grouped the platforms into two platform sets, SET 1 including arrays with lower transcript coverage (Affymetrix HG-U133A (20k), MG-U74Av2 (12k), and RG-U34 (8k)) and SET 2 including more recent full genome arrays (Affymetrix Human133 Plus 2.0 (47k), Mouse430 2.0 (40k) and Rat230 2.0 (31k)). To further minimize biases that can occur in our comparison between species, we carried out the following: • Overlap of tissue types: for all three species, we created data matrices with identical composition of tissues within each set.• Single vector per tissue type: for each tissue type and microarray platform, we calculated a single representative expression vector based on all samples annotated with this tissue type.• Selection of orthologs: we used OMA, a state• Selection of probe sets: we excluded, wherever possible, probe sets targeting multiple transcripts, keeping only highly specific probe sets.• Data normalization: a global normalization was performed across all data from a given microarray platform (see Methods section). Additionally, each probe set was normalized across all tissue types to yield a standardized representation of tissue specificity.This resulted in highly robust datasets representing tissue-specific expression for human, mouse and rat. These highly standardized and quality tested datasets allowed us to conveniently address the following hypotheses: 1. Hypothesis 1: The global architecture of tissue expression is conserved between human and rodent species.2. Hypothesis 2: Orthologs of more closely related species have a higher tissue expression correlation.3. Hypothesis 3: Tissue expression profiling can improve the mapping of functional orthologs.In order to evaluate the global architecture of tissue expression between human, mouse and rat gene orthologs (hypothesis 1), we performed a Principal Component Analysis (PCA) on SET 1 and SET 2, each containing 2127 and 8954 orthologous gene clusters represented by 56 and 55 tissue types, respectively  tissues from all other body parts . This suggests that all tissues of neural origin possess a major subset of genes that are strongly differentially expressed relative to tissues of other origin. For each species, the other principal components segregated all other tissue types  into clusters of anatomical parts that have a common origin or physiology. For example, a variety of muscle tissues formed a distinct cluster which was located close to another cluster involving heart tissues . On the opposite side of this component, related tissue clusters from the digestive, respiratory and renal systems were located. Interestingly, in SET 2, liver appears to be more closely located to the remaining tissues for mouse and rat in comparison to human. For SET 1, this is observed when principal component 1 is plotted against component 2. This result confirms previous findings in a comparison of human and mouse,21. FinaA particular case is the 'cell culture’ category. It appears distinct from the tissues from which these cells originated. The results suggest that bringing cells into culture causes a major shift in the transcriptome population. To assess the nature of this transformation more in detail, we identified a set of 177 genes exhibiting high expression in cell culture but minimal expression in all other normal tissues from SET 2. Gene Ontology enrichment analysis of these genes revealed that most of these genes belonged to apoptosis-related processes  ∼ kγ-. In this study, γ ranged from 2.9 to 3.1 which is in the typical range for a scale free network topology as a distance measure and complete linkage clustering.Hierarchical clustering was performed with the For analyzing the homologous gene clusters having m:n:p relationships between species, the gene clusters having a maximum of four related genes in each species were considered. For all possible combinations of the homologous genes in Human-Mouse, Human-Rat and Mouse-Rat, expression matrices were created, which were normalized and the pairwise Pearson correlation coefficient was calculated. The correlation values for Human-Mouse, Human-Rat and Mouse-Rat genes present in homologous gene clusters were then ranked from highest to lowest and the results were visualized using a box plot.nk/n, where n is the number of nodes in a network and nk is the number of nodes having degree k. A degree distribution was calculated for each percent of correlation threshold, and an average of these values was calculated for each of the ranges 0.85-0.89, 0.90-0.94 and 0.95-0.99 for each species. The degree distribution per range is represented as a log-log graph of average degree distribution versus degree.Correlation networks were constructed based on pairwise correlations between all genes of a given dataset. An edge between two genes represented by nodes was defined for correlation coefficients beyond a chosen threshold, resulting in a discretized matrix with 1, if the correlation coefficient was greater than that threshold and 0 otherwise. The degree for each gene was calculated from the matrix as a sum of number of 1s present in each row. The degree distribution was calculated by using the formula P(k) = GOstats package in Bioconductor, with hypergeometric distribution testing. The complete list of genes served as the universal set. A p-value of 0.001 was considered significant for genes having positive correlation, while a p-value of 0.05 was considered significant for genes having negative correlation.Gene Ontology enrichment analysis was performed on orthologous gene clusters with a Pearson correlation coefficient above 0.7 for both SET 1 and SET 2 and below -0.2 for both datasets. GO enrichment analysis was performed using the The second analysis of cell cultures versus normal tissues was carried out using the GENE SEARCH Anatomy tool from Genevestigator. To alloThe authors declare that they have no competing interest.AP, SSK and PZ performed data analysis. CD, VJ, OL, SB and TH contributed in data preparation and data curation. PZ and WG supervised the research project. All authors contributed to writing the manuscript.Gene expression values for sets of 2127 gene orthologs of human, mouse and rat across 56 tissue type categories. Orthologous gene sets are represented by Affymetrix probe sets from the platforms HG-U133A, MG-U74Av2, and RG-U34.Click here for fileGene expression values for sets of 8954 gene orthologs of human, mouse and rat across 56 tissue type categories. Orthologous gene sets are represented by Affymetrix probe sets from the platforms Human133 Plus 2.0, Mouse430 2.0, and Rat230 2.0.Click here for fileGene Ontology enrichment analysis of genes specifically expressed in the cell culture category as compared to all other tissues of SET 2.Click here for fileOutput from Genevestigator using the Anatomy tool from the Gene Search toolset to identify genes specifically expressed in cell culture but minimally expressed in normal tissues. Cell lines and blood cells were excluded from this analysis.Click here for fileGene Ontology enrichment analysis of genes specifically expressed in cell culture, but minimally expressed in normal tissues, as identified using Genevestigator.Click here for fileVariance of expression of all gene orthologs within different perturbations, as obtained from Genevestigator for the platforms corresponding to SET1 and SET2.Click here for file


The genotype-by-environment interactions of five parental environments with seed and plant performance are mediated by distinct genetic and molecular pathways, and the selective pressures that have shaped their natural variation. Arabidopsis genotypes that have been reported to be affected in seed traits. As expected, the various environments during seed development resulted in changed plant and/or seed performances. Comparative analysis clearly indicated that, overall, temperature plays the most dominant role in both plant and seed performance, whereas light has a prominent impact on plant traits. In comparison to temperature and light, nitrate mildly affected some of the plant and seed traits while phosphate had even less influence on those traits. Moreover, clear genotype-by-environment interactions were identified. This was shown by the fact that individual genotypes responded differentially to the environmental conditions. Low temperature significantly increased seed dormancy and decreased seed longevity of NILDOG1 and cyp707a1-1, whereas low light intensity increased seed dormancy and decreased seed longevity of NILDOG3 and NILDOG6. This also indicates that different genetic and molecular pathways are involved in the plant and seed responses. By identifying environmental conditions that affect the dormancy vs longevity correlation in the same way as previously identified naturally occurring loci, we have identified selective forces that probably shaped evolution for these important seed traits.Seed performance after dispersal is highly dependent on parental environmental cues, especially during seed formation and maturation. Here we examine which environmental factors are the most dominant in this respect and whether their effects are dependent on the genotypes under investigation. We studied the influence of light intensity, photoperiod, temperature, nitrate, and phosphate during seed development on five plant attributes and thirteen seed attributes, using 12  Seed performance refers to the capacity of seeds to germinate under various environmental conditions and represents a critical component of the plant life cycle that is of eminent ecological and agronomic importance. It has been observed that a change of temperature, photoperiod, or nutrient or drought stress, during seed development, maturation, and after dispersal, may strongly affect seed performance   levels were reduced around 3-fold (Arabidopsis is inversely correlated with seed nitrate content. Higher nitrate concentrations (50mM) administered to the mother plant led to less dormant seeds than seeds produced under standard nitrate conditions (10mM). Nitrate probably affects seed dormancy by its effect on ABA synthesis and degradation, since Arabidopsis seeds.One of the characteristics that determines seed performance is seed dormancy. In natural environments dormancy of dry seeds can be released by storage of the seeds for several months at mild temperatures (after-ripening) or by cold stratification, which is a low-temperature treatment of imbibed seeds (a fatua) , wheat (DELAY OF GERMINATION (DOG) loci.In addition to an effect on seed dormancy, environmental cues during seed development can also affect other traits that contribute to seed performance, such as seed weight, seed yield, ability to germinate, and longevity (or ‘storability’). A recent study has shown that some of these traits are directly linked. Prevailing stress conditions, such as high salt, osmotic stress, high and low temperature, ABA treatment, and artificial ageing have a negative effect on germination, whereas seed size has a negative correlation with germination in the presence of ABA but a positive correlation with the rate of germination (cis-epoxycarotenoid dioxygenases (NCEDs) catalyse the first committed step in ABA biosynthesis and NCED genes are key elements in the control of ABA levels in seeds  gene family, which catalyse steps of the ABA catabolic pathway, also play a prominent role in regulating endogenous ABA levels during seed development and germination , Columbia  and other genotypes with the Ler and Col genetic backgrounds were used in this study. NILDOG1-Cvi (Cape Verde Islands), NILDOG2-Cvi, NILDOG3-Cvi, NILDOG6-Kas-2 (Kashmir), NILDOG22-An-1 (Antwerpen)   and 70% relative humidity. Plants were grown in a standard nutrient solution (Supplementary Table S1) and watered three times per week. Upon the start of flowering, plants were transferred to the various environmental conditions  under a 16-h photoperiod of artificial light  were planted 5 d later in order to synchronize the flowering. In case individual plants had already started flowering, those flowers and siliques were removed to make sure all the seeds developed under the specific environmental conditions. Due to space limitation in the growth compartments each environment was performed as an independent experiment containing the control condition, except for the second nitrate and temperature experiment as these were performed at the same time and therefore share the control. All experiments with each growth condition were executed twice (first and second growth) for a robust confirmation of the phenotypes.Plants that were known to flower earlier  using a Nikon D80 camera fixed to a repro stand with a 60mm macro objective. The camera was connected to a computer with Nikon Camera Control Pro software version 2.0. Clustering of seeds was prevented as much as possible. The photographs were analysed using ImageJ (http://rsbweb.nih.gov/ij/) by combining colour thresholds (Y100–255U0–85V0–255) with particle analysis.Plant height, number of siliques per plant, and number of seeds per silique were scored for all three replicates. To investigate the number of seeds per silique and seed size, flowers that had opened at day 10 after the start of flowering were tagged and corresponding seeds were harvested. When the majority of siliques had turned yellow, watering of the plants was stopped and 7 d later dry mature seeds were harvested . Single seed weight was determined by weighing around 5mg of seeds, divided by the number of the weighed seeds, and converted to a 1000-seed weight by multiplying by 1000.–2 s–1). Pictures were taken twice a day for a period of 6 d using the same camera and software as described for number of seeds.Germination experiments were performed as described previously . DSDS50 is the closest time point to where a horizontal line at y = 50 crosses the fitted curve.Germination was scored using the Germinator package  in a closed tank with circulation for 5 d ; osmotic stress ; salt stress ; and ABA stress . ABA was dissolved in 10mM MES buffer (Sigma-Aldrich) and the pH adjusted to 5.8. To measure seed longevity, an artificial ageing test was performed by incubating seeds above a saturated ZnSO for 5 d . In the  for 5 d  and in oGmax) values were extracted from the germination assay using the Germinator package , and germination at 10°C the Gmax of most genotypes reached 100%. Therefore, to better distinguish the small differences between genotypes, the rate of germination  was also used for data analysis.Maximum germination . After centrifuging for 2min at 13 000rpm, 200 µl of the supernatant was transferred to an HPLC-vial.To measure nitrate, phosphate, and phytate content, 5mg of seeds were boiled at 100°C for 15min in 0.5ml 0.5M HCl and 50mg l–1 throughout the run. Contaminating anions in the eluents were removed using an ion trap column (ATC), installed between the pump and the sample injection valve. Anions were determined by conductivity detection. Background conductivity was decreased using an ASRS suppressor, with water as a counterflow. Peaks were identified and quantified using known external standards. External standards of nitrate, phosphate, and phytate were NaNO3 (Merck), Na2HPO4.2H2O (Merck), and Na(12)-IP6 IP6 (Sigma-Aldrich), respectively.HPLC-analysis was performed on a Dionex ICS2500 system with an AS11-HC column and an AG11-HC guard column and eluted with NaOH. The elution procedure was: 0–15min linear gradient of 25–100mM NaOH, then 15–20min 500mM NaOH followed by 20–35min 5mM NaOH. Flow rates were 1ml minAll data (both first and second growth) analysis was done in the statistical programming environment R 3.0.0.All data were analysed together. For comparison between traits each trait data set was normalized to a scale of 0–100. Analysis of variance (ANOVA) using linear models was used to determine the significance of the different environmental variables one by one.P = 0.000526).The data set was split up by environmental factors as shown in All data were used for this investigation. Pearson correlation was calculated for all trait pairs and significance was determined by linear regression.Data were split by environmental factors as described in erecta (Ler) and Columbia (Col-0), five near-isogenic lines  that are known to affect seed dormancy (DOG1 gene (dog1-1 and dog1-3) and three mutants of ABA biosynthesis and catabolism genes . Here, seed performance refers to the capacity of seeds to germinate under varying environmental conditions. Several seed germination traits were determined, including seed dormancy (DSDS50), seed longevity, and germination under stress conditions . The seed performance phenotypes observed in control conditions confirmed those that had been reported previously   retards plant growth and, as a result, extends the reproductive period . At 15°CDifferent nitrate and phosphate concentrations had no effect on the reproductive period. In addition, the combination of phosphate and high temperature shortened the reproductive period as for high temperature alone. This confirmed that changes in phosphate level do not affect the length of the reproductive period.Overall we have phenotyped 12 different genotypes  for 18 traits in 13 different environments and all these experiments have been performed twice. In order to assess and compare the importance of the different environmental factors on the phenotypes investigated, we performed statistical analysis on all data generated.Supplementary Table S2). The plant performance traits plant height and number of siliques per plant showed a strong and highly significant positive correlation. Plant height was also strongly correlated with seed weight, and, to a lesser extent, with seed size. Seed weight and seed size were strongly correlated. In general stress germination traits correlated with each other, especially germination in mannitol and salt, probably because both confer osmotic stress.A correlation matrix was generated for all pairs of measured traits to investigate associations between the characterized traits (P < 1×10–200) (er or Col) of the genotypes (P < 1×10–113). Also the effect of the parental environment was very significant (P < 1×10–157). Of the environmental factors, temperature had the most significant effect on the traits measured  are presented in We used linear models/ANOVA to investigate the overall variance caused by the different parental environments. This analysis revealed that in general the effect of the genotype was the most pronounced (×10–200) , with a Light intensity affected all the plant phenotypes significantly . In highBoth photoperiod and light intensity are important light signals for plant performance, but they play distinct roles. Short days decreased the number of seeds per silique  while coLow temperature (15°C) during seed maturation resulted in yield increases. Plants had more siliques  that conThe effect of both nitrate and phosphate and a combination of phosphate regimes and high temperature on plant and seed performance was studied. Plants grown in the higher nitrate regime (20mM) produced more siliques . With rePhosphate levels correlated positively with the number of siliques per plant  Zhao et, E at 20P < 0.001) genotype-by-environment interactions for the parental maturation environments are listed in Supplementary Figure S1. To explore this in more detail, we focus on genotype-specific effects in the following section.Maturation environments have a noteworthy influence on seed dormancy levels, as well as on other plant and seed performance traits. However, several highly significant genotype-by-environment interactions suggest that the phenotypic plasticity varied among the 12 genotypes tested. All the significant .Low light intensity increased seed dormancy of NIL NILDOG6 . Germina NILDOG6 . HoweverDOG1 . This can be explained by the functional DOG1 Ler and Col alleles that are present in these lines, which is supported by the lack of response in the dog1 mutants . Also for temperature we identified a negative correlation between seed dormancy and seed longevity . Thus, the loss-of-function mutation in CYP707A2 leads to a defective response to nitrate and therefore no increase in dormancy.Nitrate dosage, particularly low nitrate (0mM) during silique formation, increased the dormancy levels of NILp707a1-1 , but notSupplementary Figure S2). Germination behaviour in mannitol and salt were positively correlated as described above . The nced6 nced9 double mutant that produces less ABA in its seeds , high nitrate, and high phosphate resulted in higher germination percentage under stress . Ler and Col genotypes responded with a similar trend to the changes in the environment but Col plants are taller, produce more siliques per plant, and are generally more stress tolerant  (er and Col when combining photoperiod and temperature (The usefulness of studying different genotypes became apparent from the fact that clear genotype-specific effects were observed. Most obvious is the effect of the genetic background ( ageing)  and 4. F ageing) , Fig. 8.perature . ContrarDOG3 and NILDOG6, whereas temperature mainly affected NILDOG1 and cyp707a1-1 .Photoperiod, phosphate, and phosphate × temperature combinations did not have significant effects on seed dormancy and longevity while light intensity, temperature, and nitrate showed clear genotype-specific responses . Low ligp707a1-1  and 8. I and Col . The prede novo biosynthesis and catabolism  than cyp707a1-1 .ABA metabolism and signalling are responsive to many important developmental processes and environmental cues, which makes it a key regulator of growth in changing environments . These pThe responses discussed above are direct effects of the environment on seed development and maturation; however, there might also be indirect responses. The increased length of the reproductive period at low temperature  can be tArabidopsis (Arabidopsis [i.e. leafy cotyledon1 (lec1), abscisic acid intensitive3 (abi3), transparent testa (tt), aberrant testa shape (ats), delay of germination1 (dog1), and the green seed mutant  are best synchronized with dormancy cycling mechanisms. These mechanisms involve de- and rehydration cycles allowing, at intervals, activation of repair mechanisms to counteract aging-related damage, such as the repair of damaged DNA, proteins, and mobilization of proteins  are still rather low compared to that of sunlight   which did not have any significant effect on either plant or seed performance , whereasIn general, a combination of low seed dormancy and high longevity is a desirable trait for crop species but also for seed conservation, although a certain level of dormancy may be required to prevent pre-harvest sprouting. Our study implies that manipulation of the growth conditions may be used to culture seeds with the required seed performance. Alternatively, monitoring the growth conditions during seed maturation may help to predict performance of the mature seed.In conclusion, our comparative analyses confirm and extend the notion that variable environmental conditions during seed development may result in variable plant and seed performance. Of all five parental environments analysed, temperature changes during seed maturation played a dominant role in both plant and seed performance, whereas light signalling (light intensity and photoperiod) had more impact on plant traits. Nitrate and phosphate displayed relatively mild effects on plant and seed performance. The observation that the different genotypes responded differentially to the environmental conditions indicates that different genetic and molecular pathways are involved in these responses.JXB online, and includes the ABA extraction and detection method as well as the following:Supplementary data can be found at Supplementary Table S1. Element concentrations in the standard nutrient solutionSupplementary Table S2. Trait by trait correlation/significance of plant and seed performance.Supplementary Figure S1. Seed dormancy and longevity levels of plants grown in control conditions .Supplementary Figure S2. Plant and seed performances of each genotype in five environments .Supplementary Figure S3. ABA levels in freshly harvested seeds of Ler, NILDOG1, Col, and cyp707a1-1 matured in low temperature and low nitrate compared with the control conditions.


Callithrix jacchus) are frequently used for biomedical research but can be afflicted with diarrhea—a serious and potentially lethal health problem. Enteropathogenic Escherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis in common marmosets, but the actual incidence of the disease and the relationship between EPEC and hematochezia are unknown. This study investigated the prevalence of EPEC infection in common marmosets and the association between EPEC and hematochezia. A total of 230 stool or rectal swab samples were collected from 230 common marmosets  and tested by culture-based detection and PCR amplification of VT1, VT2, LT, ST, eae, and bfp genes. Healthy animals were divided into three groups (n = 4 each for high and low concentration groups and n = 2 as negative control), and those in the experimental groups were perorally inoculated with a 2-ml of suspension of EPEC R811 strain adjusted to 5 × 108 (high concentration) and 5 × 104 (low concentration) CFU/ ml. Two animals in each group were examined 3 and 14 days post-inoculation (DPI). EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%), with a significant difference detected between presence of EPEC and sample status (P < 0.01). Acute hematochezia was observed in all animals of the high-concentration group but not in other groups at 1 or 2 DPI. A histopathological examination revealed the attachment of gram-negative bacilli to epithelial apical membranes and desquamated epithelial cells in the cecum of animals in the high-concentration group at 3 DPI. These findings suggest that EPEC is a causative agent of hemorrhagic typhlocolitis in common marmosets.Common marmosets ( Callithrix jacchus) is a New World primate used for biomedical research [The common marmoset (research . DiarrheEscherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis [Several intestinal pathogens cause diarrhea in common marmosets ,3. Enterocolitis . EPEC isocolitis ,6 and caocolitis ,6.Only a few methods such as survey and necropsy have been used to investigate hemorrhagic typhlocolitis associated with EPEC in common marmosets , and theSalmonella spp., Shigella spp., and Yersinia pseudotuberculosis in yearly monitoring. There were 98 healthy stool or rectal samples , 85 diarrhea or rectal swab samples (diarrhea samples), and 47 bloody stool or rectal swab samples (bloody stool samples)  kept in the Central Institute for Experimental Animals (CIEA) between June 2011 and August 2012. The animals were obtained a commercial vendor  or were born at the CIEA. The animals used in the study ranged in age from 1 month to 14.2 years  were housed in a conventional indoor facility in accordance with the Regulations for Animal Experimentation of CIEA based on the Guidelines for Proper Conduct of Animal Experiments , and were negative for samples) . Fresh sE. coli (including EPEC) and Salmonella spp., while SS agar  was used for detection of Salmonella spp., Shigella spp., and Yersinia pseudotuberculosis. Inoculated DHL agar plates were incubated under aerobic conditions at 37°C for 24 h (DHL agar) or 48 h (SS agar). For all suspected colonies of each pathogen (with the exception of E. coli), Gram staining as well as catalase, oxidase, and biochemical tests were carried out using commercially available kits . Antimicrobial susceptibility was tested using a commercially available kit . E. coli isolates were examined after confirming the presence of the eae gene with a secondary test (see below). For EPEC detection, 10 typical E. coli colonies on DHL agar were selected and mixed with 200 μl sterilized phosphate-buffered saline (PBS) to obtain a mixed colony suspension as a sample for the screening test. A portion of this suspension was inoculated on Heart Infusion Agar  and incubated under aerobic conditions at 37°C for 24 h for the secondary test. The suspensions were also tested for the presence of the VT1, VT2, LT, ST, eae, and bfp genes by PCR in the screening test. VT1, VT2, LT, and ST genes were detected with a commercially available PCR kit , while eae and bfp were detected as previously described [eae gene, each isolated bacterial colony on the Heart Infusion Agar was examined by PCR in a secondary test.Samples were inoculated on DHL agar  for detection of Escherichia coli; Denka Seiken Co., Tokyo, Japan). O antigen genotyping was carried out for three selected isolates as previously described [All EPEC isolates were serotyped by an agglutination test using a commercially available antisera kit  and relative humidity (40%–60%) on a 12:12-h light/dark cycle. Therapeutic treatment with antibiotics and/or analgesics was not provided, because such a treatment would have affected the experimental results. All animals were clinically healthy and confirmed free from Salmonella spp., Shigella spp., EPEC, and Yersinia pseudotuberculosis by culture tests of fecal samples.Male common marmosets  were obtained from a commercial vendor . The animals weighed 276–320 g at the time of infection. Animals were housed individually in stainless steel cages (409 × 610 × 1578 mm). The cages were positioned facing each other at a distance of 1.7 m to allow the animals to communicate visually and vocally. Animals were reared individually in order to obtain exact results in terms of fecal condition and to prevent secondary infection from cage-mates. Animals were provided wooden perches and a resting place made of wood as environmental enrichment, and were fed a balanced commercial primate diet  supplemented with ascorbic acid , vitamins A, D3, and E , and honey  and tap water eae gene and negative for VT1, VT2, LT, ST, and bfp genes). Biochemical characteristics and antimicrobial susceptibility of the strain are shown in EPEC R811 used for the experimental infection study was isolated from the bloody stool sample of a common marmoset and identified as EPEC based on biochemical features and by PCR  and one negative control group  were used in this study. The number of animals was selected based on a previous experimental infection study of common marmosets and was determined according to the number of cages in the biosafety level (BSL) 2 experimental room at our institute .8 (high concentration) or 5 × 104 (low concentration) CFU/ ml with sterile saline. These concentrations were determined based on previous experimental EPEC infection study in rhesus monkeys [Animals in the experimental group were perorally inoculated with 2 ml bacterial suspension adjusted to 5 × 10 monkeys . AnimalsBacterial strains were isolated from blood, duodenum contents, the cecum at autopsy, and daily from feces. Samples (except those from blood) were streaked directly onto DHL agar; 100 μl of blood was inoculated on DHL agar. Bacterial isolates were identified as described above.Esophagus, heart, lungs, mesenteric lymph nodes, spleen, stomach, small and large intestines, liver, gallbladder, pancreas, kidney, duodenum, cecum, and rectum were examined histologically. Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE) and toluidine blue and subjected to Gram staining according to standard procedures.2 test was used to compare EPEC isolation rates among clinically healthy, diarrhea, and bloody stool samples. Fisher’s exact test with Holm’s correction for multiple tests [http://www.R-project.org). Significance was determined at P < 0.01.Pearson’s χle tests  was usedThis study was carried out in strict accordance with the Regulations for Animal Experimentation of the CIEA based on the Guidelines for Proper Conduct of Animal Experiments . The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) for Animal Experimentation of the CIEA (permit no. 14007A). All animals in the study were reared in a BSL2 room used exclusively for experimental infection studies had free access to food and water, and were checked at least twice daily. All efforts were made to minimize painful procedures; when these were necessary, an attending veterinarian was consulted. Following the development of clinical signs, animals were checked multiple times daily. When clinical observations and scores reached defined levels based on the approved IACUC protocol, animals were sacrificed under deep anesthesia to minimize pain and distress. When animals became moribund or at the end of study, they were humanely euthanized as described above in accordance with current American Veterinary Medical Association Guidelines for Euthanasia of Animals.Salmonella spp., Shigella spp., and Y. pseudotuberculosis were not detected in any of the samples.A total of 74 stool or rectal swab samples were positive for EPEC. EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%) . SalmoneE. coli using a commercially available biochemical test kit. The isolates were positive for the eae gene and negative for VT1, VT2, LT, ST, and bfp genes by PCR. The serotypes of all isolates were undeterminable by the antisera kit. Three selected isolates were determined to be of the O85 genotype.All 74 EPEC isolates were identified as Two animals in the high-concentration group showed slight lethargy at 1 DPI and hematochezia at 1 and 2 DPI , which lTwo animals in the high-concentration group exhibiting slight lethargy and two animals in the low-concentration group without clinical symptoms were autopsied at 3 DPI. Slight ascites, enlargement of mesenteric lymph nodes, swollen spleen, hyperemia and atony of large intestine, and petechia of colon mucosa were observed in both animals in the high-concentration group Figs . A histo2 test, the three groups differed in terms of EPEC isolation rate (P < 0.01). Using the Holm method, there were significant differences in the EPEC isolation rate between the bloody stool sample group and the other two groups (adjusted P < 0.01 in both cases by Fisher’s exact test). There was no difference in EPEC isolation rate between clinically healthy and diarrhea samples (adjusted P = 0.09 by Fisher’s exact test).Based on Pearson’s χThere was a significant difference between sample status and EPEC presence. The EPEC-positive rate was higher in bloody stool samples than in healthy or diarrhea samples. The pathologic potential of EPEC in nonhuman primates has been previously reported. In one study of 18 nonhuman primates including nine common marmosets, the isolation ratio of EPEC from diarrhea and/or enteritis 47%) was higher than from apparently healthy animals (27%) . In anot7% was hiE. coli serogroup O26 was the causative strain of a hemorrhagic typhlocolitis outbreak in common marmosets [Saguinus fuscicollis) [In a previous report, armosets  and was icollis) . The isoicollis)  and bacticollis) .The actual pathogenicity of EPEC in common marmosets is poorly documented. We therefore carried out experimental infection of common marmosets with EPEC. We observed acute hematochezia in all animals in the high-concentration group, although symptoms disappeared between 5 and 9 DPI in animals that were monitored until 14 DPI. On the other hand, no hematochezia was observed in the low-concentration group, which was monitored until 14 DPI. Two animals in this group had diarrhea twice from 8–12 DPI. These results indicate that the onset of hematochezia depends on the quantity of EPEC cells to which an animal is exposed.EPEC was recovered from all animals in the high-concentration group throughout the experimental period. On the other hand, EPEC was recovered from two animals at 3 DPI and two at 5 and 6 DPI in the low-concentration group; of the latter, EPEC disappeared in one animal after 13 DPI, suggesting that it can cause continuous infection in common marmosets.E. coli strains derived from hemorrhagic typhlocolitis in common marmosets has been proposed to explain the difference in clinical symptoms between the two species [rtxA1), hemolysin E (ClyA), and Shigella enterotoxin 2 (shET2) in the EPEC R811 strain used in the present experimental study [The histopathological examination revealed attachment of gram-negative bacilli to epithelial apical membranes and desquamation of cecal epithelial cells in animals in the high-concentration group at 3 DPI . This is species . Recentlal study . AlthougIn conclusion, we demonstrated a high prevalence of EPEC infection with hematochezia in common marmosets using a relatively large number of samples, and induced hemorrhagic typhlocolitis in marmosets. This is the first study that has reproduced this disease in these animals by experimental EPEC infection. Our findings provide insight into the causes of diarrhea in common marmosets as well as an experimental model for studying this disease.


We present herein two cases of hard metal lung disease (HMLD) with distinct pathological findings. Both cases showed gradual improvements in pulmonary function over a period of a few years  after the avoidance of dust exposure, while improvements on high-resolution computed tomography were modest. The increased lymphocytes and decreased CD4/CD8 ratio in BALF observed at initial diagnosis normalized after the avoidance of dust exposure in one case. To the best of our knowledge, this is the first report demonstrating continual follow-up of pulmonary function and radiographic findings, and a comparison of BALF findings before and after avoidance of hard metal dust exposure. Hard metal lung disease (HMLD) is a rare occupational lung disease that can occur in workers engaged in the manufacture, utilization, or maintenance of tools composed of hard metal, a synthetic compound composed of tungsten carbide and cobalt , 2. Alth1) , 95.4 %), total lung capacity (TLC) , 87.2 %), and diffusing capacity of the lung for carbon monoxide . BALF obtained from the right middle lobe (S5) showed an increased percentage of lymphocytes (55.0 %) and a decreased CD4/CD8 ratio (0.40)  of the chest showed diffuse centrilobular micronodular opacities and curved linear shadows in the dorsum Fig. . PulmonaAfter reaching this diagnosis, we recommended the patient quit her job or transfer to a different workplace to completely avoid exposure to hard metal dust. However, she continued working at the same place without using a dust protective mask for financial reasons, and nonproductive cough continued. In addition, pulmonary function continued to worsen. Finally, 18 months after diagnosis, she obtained a transfer to a different department where she was completely isolated from exposure to hard metal dust. Her cough improved and had completely disappeared by 2 months after ending exposure to hard metal dust. A series of pulmonary function tests revealed continuous improvements over the course of 30 months without exposure to hard metal dust Fig. , althoug1 , TLC , and DLCO . Although BALF obtained from the right middle lobe (S5) demonstrated normal cell counts, multinucleated giant cells were observed  exposure to dust. Dyspnea gradually improved after reducing the dust exposure. Although a series of pulmonary function tests revealed improvements over the course of a year Fig. , improveThe clinical features of HMLD are known to be heterogeneous in several respects, such as pathological findings, BALF findings, and clinical course. In the two cases we encountered, thoracoscopic lung biopsy revealed distinct pathological findings, with one case characterized by typical GIP, and the other by diffuse centrilobular fibrosis around the respiratory bronchioles without GIP. In a study of 100 cases of HMLD, Asghar et al.  describeAvoidance of exposure to hard metal dust is central to preventing deterioration of this disease in any case, and improvements of the symptoms and/or radiological findings after avoiding exposure have been reported in several previous studies , 8–10. HIn some previously reported cases, respiratory failure progressed to death despite suspending exposure to the dust , 12, whiAlthough previous studies have suggested that increased lymphocytes and decreased CD4/CD8 ratio are characteristic BALF findings in subjects with HMLD , 12, 13,We have presented two cases of HMLD with continuous follow-up of pulmonary function and radiological findings before and after avoidance of hard metal dust exposure. The clinical courses of the two cases were presented, together with a comparison of BALF findings before and after avoidance of dust exposures.Written informed consent was obtained from both patients for the publication of this case report and the accompanying images.