Fragaria × ananassa) is a genetically complex allo-octoploid crop with 28 pairs of chromosomes (2n = 8x = 56) for which a genome sequence is not yet available. The diploid Fragaria vesca is considered the donor species of one of the octoploid sub-genomes and its available genome sequence can be used as a reference for genomic studies. A wide number of strawberry cultivars are stored in ex situ germplasm collections world-wide but a number of previous studies have addressed the genetic diversity present within a limited number of these collections. Here, we report the development and application of two platforms based on the implementation of Diversity Array Technology (DArT) markers for high-throughput genotyping in strawberry. The first DArT microarray was used to evaluate the genetic diversity of 62 strawberry cultivars that represent a wide range of variation based on phenotype, geographical and temporal origin and pedigrees. A total of 603 DArT markers were used to evaluate the diversity and structure of the population and their cluster analyses revealed that these markers were highly efficient in classifying the accessions in groups based on historical, geographical and pedigree-based cues. The second DArTseq platform took benefit of the complexity reduction method optimized for strawberry and the development of next generation sequencing technologies. The strawberry DArTseq was used to generate a total of 9,386 SNP markers in the previously developed ‘232’ × ‘1392’ mapping population, of which, 4,242 high quality markers were further selected to saturate this map after several filtering steps. The high-throughput platforms here developed for genotyping strawberry will facilitate genome-wide characterizations of large accessions sets and complement other available options.Cultivated strawberry ( Furthermore, DArT has been applied successfully to species with large genomes such as barley [Efforts of crop improvement in polyploid species are hampered by the complexity of the genome and the difficulties to develop high-throughput genotyping platforms. Diversity Arrays Technology (DArT) offers an inexpensive and high throughput whole-genome genotyping technique as initially shown for rice . The effs barley  and withs barley , hexaplos barley ,5 or thes barley . The DArs barley . Contrars barley  and mores barley ,9. DArTss barley –13. SimiFragaria, which encompasses all soft-fruited strawberry species, belongs to the Rosaceae family, which comprises many economically important species such as apple, peach, and plum. F. × ananassa (2n = 8x = 56), the cultivated octoploid strawberry, is the most economically relevant soft berry, with a total harvested area of 361,662 ha and a production of 7,739,622 t in 2013 . In addition, strawberry is considered as a model species for the study of non-climacteric ripening in fleshy fruits and as so it is the subject of numerous studies [F. virginiana and the South American domesticated F. chiloensis [The genus  studies ,15. Thisiloensis ,17. Systiloensis . As a reiloensis ,19. Althiloensis ,22.In spite of its narrow genetic variation, strawberry shows a large diversity in many traits such as biotic and abiotic stress tolerance –25, fruiBrassica napus, wheat, sugarcane and cultivated strawberry [F. vesca [® array, comprising more than 90K SNPs derived from short-read sequences from a panel of 19 octoploid accessions [F. vesca reference genome displays high macrosynteny with the octoploid strawberries genomes [® array for the genetic characterization of strawberry has already been shown [F. vesca reference genome for the SNP discovery process has resulted in a bias towards markers in the F. vesca-derived sub-genome in comparison to the other 3 sub-genomes. An additional problem of the strawberry SNP array arises from interpretation of the complex signal dosages arising from the combination of alleles from the different sub-genomes [Strawberry accessions have been genotyped using several methods such as Random Amplified Polymorphic DNA (RAPDs) ,33, amplrawberry –53. The F. vesca  allowed cessions . The dip genomes , and par genomes ,57. The en shown ,56. Howe-genomes .http://www.diversityarrays.com), the second one taking benefit from the development of NGS. Our main objective was to prove DArT in a genetically complex species where several possible alleles were expected. The first DArT microarray platform was obtained from genomic representations derived from 62 widely diverse accessions that cover a wide range of variation based on phenotype, and geographical and temporal origin. Using this platform, we obtained a clear picture of the genetic diversity and structure of an octoploid strawberry collection. The second platform, DArTseqTM, thanks to NGS technologies, provided a much larger number of SNP markers compared to the DArT microarray and was successfully used to develop a high-density genetic map of strawberry using the ‘232’ × ‘1392’ population [To provide alternative high-throughput genotyping techniques useful for genetic analysis of diverse strawberry populations, here we report on the development of two DArT platforms for octoploid strawberry  were used for DArT marker development in this study, including the parental lines of the ‘232’ × ‘1392’ mapping population and 4 progenies. They were obtained from the IFAPA strawberry germplasm collection (ESP138) located at Centro IFAPA Churriana Málaga Spain or from the CIREF strawberry germplasm collection (FRA207) located at Douville France. Cultivar names, their year of release, pedigree and geographical origin are shown in https://www.bordeaux.inra.fr/genberry/).A total of 62 accessions of strawberry . The combination of PstI and TaqI produced most promising results and this complexity reduction method was applied to construct libraries of 7,680 genomic clones in total from 62 strawberry accessions  as descrhttp://www.diversityarrays.com/software.html). DArTsoft was also used to convert signal intensities to presence/absence (binary) scores used in the downstream analysis. To determine marker quality (reproducibility of markers), 32 accessions were genotyped in technical replication (two independent libraries and marker extraction) and consistency of allele calling was used to determine reproducibility statistics and to select high-quality markers. In a polyploid like strawberry some of the missing data is due to a number of reasons such as copy number differences, presence of heterozygotes/hemizygotes or null alleles. The informativeness of the DArT markers was determined by calculating the polymorphism information content (PIC) within the 62 diverse strawberry cultivars [Each sample (the 62 diverse genotypes) was assayed using methods described above for library construction. Genomic representations were labeled with fluorescent dyes (Cy3 and Cy5). Labeled targets were then hybridized to printed DArT arrays for 16 hours at 62°C in a water bath. Slides were processed as described in  and scanultivars . The maxSimilarly to the DArT microarray, the DArTseq technology was optimized for strawberry by selecting the most appropriate complexity reduction method (both the size of the representation and the fraction of a genome selected for assays). Four methods of complexity reduction were tested in strawberry (data not presented) and the PstI-MseI method was selected. DNA samples are processed in digestion/ligation reactions principally as per  but replAfter PCR, equimolar amounts of amplification products from each sample were bulked and applied to c-Bot (Illumina) bridge PCR, followed by sequencing on Illumina GAIIx. The sequencing (single read) was run for 77 cycles in two lanes. Sequences generated were processed using proprietary DArT analytical pipelines. In the primary pipeline the fastq files are first processed to filter away poor quality sequences, applying more stringent selection criteria to the barcode region compared to the rest of the sequence. In that way the assignments of the sequences to specific samples carried in the “barcode split” step are very reliable. Approximately 600,000 (+/- 7%) sequences per barcode/sample were used in marker calling. Finally, identical sequences are collapsed into FASTQCOL. The propriety software package DArTsoft14 is used for marker discovery and scoring from FASTQCOL files. The FASTQCOL files from the samples of ‘232’ × ‘1392’ population were analyzed using DArTsoft14 to output candidate SNP and silicoDArT markers which are polymorphic within the set of samples (SilicoDArT markers are sequences with presence/absence variation in the DArTseq genomic representation). All unique sequences from the set of FASTQCOL files are identified, and clustered by sequence similarity at a distance threshold of 3 base variations. The sequence clusters are then parsed into SNP and silicoDArT markers utilizing a range of metadata parameters derived from the quantity and distribution of each sequence across all samples in the analysis.Similarly to DArT microarray, a high level of technical replication is included in the DArTseq genotyping process, which enables reproducibility scores to be calculated for each candidate marker. The candidate markers output by DArTsoft14 are further filtered on the basis of the reproducibility values, average count for each sequence or row sum (sequencing depth), the balance of average counts for each SNP allele, and the call rate (proportion of samples for which the marker is scored).F. × ananassa and Comarum palustre, formerly Potentilla palustris). Clade strength was tested by 1,000 bootstrap analyses performed with the SEQBOOT program [DArTs were scored as 0/1 and they were used as different inputs for the RESTDIST and NEIGHBOR programs of the PHYLIP 3.6 software package to construct Neighbor-Join phylograms, based on Felsenstein’s modification of the Nei and Li restriction fragment distance . Phylogr program .The genetic structure of the germplasm collection was analyzed performing Principal Coordinate Analysis (PCoA) implemented in the program GenAlex 6.41  and by uF. vesca reference map, followed by 1–4 (following the same order as in the previously published ‘232’ × ‘1392’ maps) for each of the 4 homeologous linkage groups. Linkage maps were drawn using MapChart 2.2 for Windows [Selected SNP markers derived from the DArTseq platform were used in combination with previously mapped SSR, SSCP and AFLP  for map  Windows .F. vesca pseudo-chromosome assembly [Physical map positions of DArT-derived SNPs and microsatellites used in this study were obtained by aligning the DArT sequences  and the model-based Bayesian clustering method implemented in STRUCTURE. The most likely number of clusters (K) was evaluated considering the ΔK criterion , that gaGenetic divergence among samples was also studied using DArT markers and the PCoA approach based on a genetic distance matrix with data standardization and it was largely consistent with the STRUCTURE results . The fir2 test; p = 0.01) and an additional 693 alleles (6.6%) did not fit the simplex ratios  and were regarded as distorted and also excluded. Among the remaining 6,770 simplex markers, 3,370 (49.8%) were in pseudo-test cross configurations  and 1,531 (22.6%) heterozygous in the female and male, respectively). The remaining 3,400 (50.2%) simplex markers were present in both parents and fitted a 3:1 ratio. The high number of 3:1 markers suggests a close relationship between the two parents, as previously reported [A total of 9,386 SNP markers were produced by the DArT platform and provided as 18,772 binary SNP allele scorings for the presence/absence (0/1) of the reference versus SNP allele scores. Due to the polyploidy of strawberry, DArTseq SNPs were filtered as alleles to avoid confusion between sub-genomes. A total of 6,744 35.9%) of the SNP alleles was monomorphic in the progeny and were removed. Markers with missing values in more than 10% individuals (more than nine progeny lines) or in any of the two parental lines, or with 0 scores in both parents were excluded . The remaining markers  were tested for closeness to the various segregation ratios present in an octoploid species [.9% of threported , and shoThe final number of selected SNPs was 4,242 . Among them, 1,839 (43.3%) were ‘232’-derived markers, 1,531 (36.1%) were derived from ‘1392’ and 872 (20.6%) had an inter-cross configuration. The 4,242 SNPs were used for mapping, in combination with 408 SSR and gene specific markers previously mapped . Only 19The final number of markers positioned in the consensus ‘232’ × ‘1392’ linkage map was 2,089 that provided high coverage of the genome as the 7 homoeology groups (HGs) were represented and the smallest LG was 30.3 cM long Figs . A totalF. vesca genome [F. × ananassa LGs belonging to HG I, an inversion in a segment at the top of pseudochromosome 2 in comparison to three F. × ananassa LGs of HG II or another in F. vesca pseudochromosome 3 and three homoeologous LGs in F. × ananassa HG III. In other instances, rearrangements were detected in only one homoeologous LG compared to F. vesca or the rest of the sub-genomes, as one large inversion involving more than half of LG II-2  were mapped on a different chromosome to that expected based on the latest assembly of a genome  . In molecular breeding, this advantage is important for developing new markers for marker-assisted selection based in the identified DArT marker sequences. The DArT clones used to analyze diversity in strawberry could be sequenced for future works or for comparison to the mapped DArTseqTM markers. However, the choice of complexity reduction method was optimized to generate the optimal restriction fragment size for each platform and would result in a very small overlap of markers between them. Furthermore, the higher cost-effectiveness and larger number of markers generated by the DArTseqTM platform makes this technology more useful for future studies.Compared to the DArT microarray platform, which is based on genome complexity reduction using restriction enzymes followed by hybridization to microarrays , the DArplatform ,77 combiF. vesca  . However, high throughput platforms offer the advantage of cost and time efficient whole genome coverage. After this work, two complementary platforms are now available for high throughput genotyping of the octoploid strawberry: the DArTseq here developed and the 90K Axiom® SNP array [TM and other genotyping by sequencing approaches, practically all fixed arrays suffer from ascertainment bias, especially when developed using not very representative reference genome and fairly small sampling of diversity for marker discovery. In the particular case of the 90K Axiom® SNP array developed for strawberry, it was based on the F. vesca reference genome and, when used for mapping in the octoploid strawberry, suffers from a bias to one of the sub-genomes, as shown in the ‘Holiday’ × ‘Korona’ and DA × MO linkage maps [TM pipeline can be used as an useful alternative to fixed sequence approaches for molecular diversity analyses and to generate extremely dense linkage maps suitable for QTL detection and genome-wide association studies (GWAS).SSRs have been the preferred marker for genetic diversity as well as for QTL mapping in strawberry ,37–46. TNP array . The firNP array ,78,79. Fage maps ,56. TherF. virginiana) as pollen source, and the ‘Frutilla’ or Chilean strawberry (F. chiloensis) occurred accidentally [The analysis of genetic diversity and population structure here reported highlights the history of the two first centuries of the cultivated strawberry breeding programs, which have been conducted in the past mainly in USA and Europe. Breeding of the cultivated strawberry begun shortly after its origin in the 1760s, when a cross between the Scarlet strawberry Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Spatial variations in flow rates and temporal changes in water content show that date palms utilize stem water for daily transpiration and nightly xylem reservoir recharge. in vivo accounting of the date palm diurnal and seasonal water mass balance. By incorporating these methods it was possible to determine that date palms substantially rely on the exploitation and recharge of the stem reservoir in their water budget. The stem of mature date palms can hold up to 1 m3 of water and supply 25% of daily transpiration (i.e. 5000 l of water in 100 d of summer). The internal stem water reservoir is consistently recharged by over 50 l per night which allows for successive daytime reuse throughout the entire growing season. More broadly, these findings suggest that internal water allocation and night-time soil-water availability could provide useful information for improving date palm irrigation practices.During the summer, evaporative demand at midday often exceeds the transport capacity of most desert plants. However, date palms maintain their ecological dominance with sustained and uniquely high rates of transpiration. This high rate of flow cannot be attributed to soil water supply alone. In order to quantify intra-plant water allocation in irrigated date palms, three water-sensing techniques have been incorporated: heat dissipation, gravimetric sampling, and time domain reflectrometry. Each of these methods has known limitations but their integration resulted in a quantitative  Espeletia) to transpire when soil water is frozen  cannot reveal variations in water storage as they only detect inter-flows (the flow between a system and its environment). Investigating the intra-system flow requires the evaluation of water status  or waterPhoenix dactylifera L. Arecaceae) originated in the desert oases of Iraq and adapted to desert conditions and prolonged drought periods. Palms are monocotyledonous with no vascular cambium and, therefore, are permanently dependent on the primary xylem  to achieve optimal plant growth and yield  investigating temporal variations in the water storage of field-cultivated date palms; (ii) quantifying the internal water availability of date palms; and (iii) describing seasonal changes in internal water use in date palms.The function of water storage in forest palms has previously been addressed , b. YePhoenix dactylifera L., cv. Hayany) were selected for the research. The trees were 35 years old, 15 m high, and 0.4 m in diameter and were calculated to hold over 1 m3 of water [volume multiplied by 60% water content (WC)]. The plantation was irrigated through a drip irrigation system  following the local field-service extension recommendations (commonly used by growers): multiplying the reference evapotranspiration (ET0) by crop and season factors.The research was conducted in a date palm plantation located in the northern Jordan valley , Israel, between June and December, 2012. The Jordan valley region is semi-arid with an annual precipitation of 383mm (December–March). Minimum and maximum daily temperatures are 8.5 °C (January) and 37.7 °C (July), respectively, and the mean daily relative humidity (RH) varies between 75% (January) and 53% (July). The experimental site has mineral alluvial soil and is located at 200 m below sea level. Five date palm trees , wind speed , air dry temperature , and air relative humidity . Air water vapour pressure deficit  was calculated from the daily and hourly averages of temperatures and relative humidity. The reference evapotranspiration  was computed according to the Penman–Montieth (PM) equation  is the vapour pressure curve slope, Rn (MJ m–2 d–1) is the net radiation  was calculated by a second order quadratic equation  and 2mm d–1 during the winter  also decreased from c. 250 l d–1 tree–1 during the summer to c. 120 l d–1 tree–1 during the winter. In late autumn, ET0 and SF varied dramatically across consecutive days due to unstable climatic conditions. However, SF in the lower stem (2 m) was limited to 150 l d–1 tree–1 and was nearly constant throughout both the summer and autumn. SF values at 2 m and 10 m were equal only in winter , and decreased between 1500h and 2000h. Lower-stem SF on the other hand increased as early as 0100h, peaked at 1000h (c. 14 l h–1 tree–1), remained constant throughout the afternoon, and approximated the 10 m SF only at 1800h  and late afternoon (1800h) resulted in a daily depletion of 50 l of stored water from a single tree (WC was similar at both heights. Throughout the growing season (June–October) the average WC in the stem was 0.62 m3 m–3 . However, WC at 10 m increased (by 4%) 14 d after the WC at 2 m stabilized and it continued to fluctuate according to changes in ET0.Instantaneous h height : it was d midday . Continu2 m3 m–3  and it secreased , and theET0 is well met by daytime SF in the upper stem of date palms  available for intra-stem allocation under reduced water potentials  and ceases when the two flows are back in equilibrium (1700h; Stem xylem vessels have low specific water capacitance . Hence, le water . This cheenhouse  and Dougm 1700h; . Night-tWC in red maple trees, peaking in the summer of a wet year and decreasing towards winter. In date palms, the high summer day-time evaporative demand imposes the major use of stem water and differentiates between lower and upper stem flows (WC increases. At this time of the year the lower stem is nearly saturated as water-storage depletion is limited to the upper stem which is still affected by the evaporative demand.The hourly variations in water sources for transpiration are seasonal and depend on soil water availability and evaporative demand. em flows . Yet, stem flows . TowardsUnder the dry summer conditions of a Mediterranean climate, the date palm is largely dependent on its stem water reservoir to meet hourly transpiration requirements and maintain diurnal and seasonal homeostasis in water uptake. Due to high intensity irrigation, cultivated date palms do not encounter prolonged water stress. Nevertheless, it was found that the midday evaporative demand during summer exceeds the water supplied by roots and stem water storage is depleted. The internal reservoir contributes 25% of the daily transpiration and is recharged at night for subsequent daytime use. Diurnal intra-water allocation is crucial to the photosynthetic performance of date palms as it enables optimum vegetative and reproductive growth in hot and dry desert summers. This makes the desert-adapted date palm a highly sustainable, productive, and economic crop in arid Mediterranean regions.


Carcharodon carcharias, of various lengths (1.5–3.9 m), sampled at two geographically separate areas off southern and eastern Australia. Muscle tissue was low in total lipid content  and was dominated by phospholipids  and polyunsaturated fatty acids . In contrast, liver was high in total lipid which varied between 51–81% wm and was dominated by triacylglycerols (>93%) and monounsaturated fatty acids (36±12%). With knowledge of total lipid and dry tissue mass, we estimated the energy density of muscle (18.4±0.1 kJ g−1 dm) and liver (34.1±3.2 kJ g−1 dm), demonstrating that white sharks have very high energetic requirements. High among-individual variation in these biochemical parameters and related trophic markers were observed, but were not related to any one biological or environmental factor. Signature fatty acid profiles suggest that white sharks over the size range examined are generalist predators with fish, elasmobranchs and mammalian blubber all contributing to the diet. The ecological applications and physiological influences of lipids in white sharks are discussed along with recommendations for future research, including the use of non-lethal sampling to examine the nutritional condition, energetics and dietary relationships among and between individuals. Such knowledge is fundamental to better understand the implications of environmental perturbations on this iconic and threatened species.Lipids are major sources of metabolic energy in sharks and are closely linked to environmental conditions and biological cycles, such as those related to diet, reproduction and migration. In this study, we report for the first time, the total lipid content, lipid class composition and fatty acid profiles of muscle and liver tissue of white sharks,  Carcharodon carcharias, is considered the most voracious apex predator in temperate marine ecosystems worldwide in situ observations of predation at aggregation sites  or scavenging events on whale carcasses The white shark, Rapid advances in technology, data treatment and other numerical techniques, and our understanding of their tissue biochemistry, are allowing ecologists to gain a more complete understanding of white shark biology. Tracking studies have shown that white sharks engage in broad-scale inshore and offshore migrations The aim of this study was to report the lipid content, lipid class composition and fatty acid profiles of the muscle and liver of white sharks sampled opportunistically from South Australia and New South Wales . As tissue differences in lipids are well known in other species of shark All procedures were ratified by independent Animal Ethics Committees, including the New South Wales Department of Primary Industries (NSW DPI) Animal Research Authority (ACEC 12/07) and the Tasmanian Department of Primary Industries, Parks, Water and Environment (ACE 22/2011–2012). In South Australia, field work at the Neptune Islands was carried out under a South Australian S115 ministerial exemption 9902123. In NSW, field work was carried out under a NSW DPI Scientific Collection Permit (P07/0099-3.0 & P07/0099-4).Twenty one deceased white sharks of various lengths (1.5 to 3.9 m) were sampled opportunistically between 2001 to 2012 in waters off South Australia (SA) and along the New South Wales (NSW) coast, Australia . Most sp3:MeOH:milliQ H2O solvent mixture (10∶20∶7.5 mL) which was left overnight. The following day, the solution was broken into two phases by adding 10 mL CHCl3 and 10 mL saline milliQ H2O (9 g NaCl L−1) to give a final solvent ratio of 1∶1∶0.9. The lower layer was drained into a 50 mL round bottom flask and concentrated using a rotary evaporator. The extract was transferred in CHCl3 into a pre-weighed glass 2 mL vial. The solvent was blown down under a constant stream of nitrogen gas. The round bottom flask was rinsed twice with CHCl3 into the vial. The total lipid extract (TLE) was dried in the vial to constant weight and 1.5 mL of CHCl3 added.Total lipid content was extracted by the modified Bligh and Dyer Lipid class composition of tissues was determined using an Iatroscan Mark V TH10 thin layer chromatograph coupled with a flame ionization detector (TLC-FID). For each sample, the TLE was spotted and developed in a polar solvent system . Samples were also run in a non-polar solvent system (96∶4 v/v hexane:ether) to resolve hydrocarbons  from wax esters and diacylglyceryl ethers from triacylglycerols. All samples were run in duplicate along with standard solutions, which contained known quantities of common lipid classes. Chromarods were oven-dried for 10 min at 100°C and analyzed immediately. Peaks were quantified using SIC-480 Scientific Software.Fatty acid proportions were determined using a direct methylation procedure dm = 1 - LFdm and PCFwm = 1 - LFwm - WF). Energy density  of wet mass and dry mass of muscle and liver was then calculated using proximate composition data and published calorific values of 39.3 kJ g−1 for lipid and 17.8 kJ g−1 for protein and carbohydrates Water fraction (WF) of tissue mass was determined by taking weights before and after freeze-drying at −82°C for 48 h. From this, and acquired values of the lipid fraction (LF), we determined the wet/dry mass ratio of each tissue and the proportion that consisted of protein and carbohydrate , a software package for analyzing multivariate ecological data, to assess the groupings and investigate relationships within the fatty acid data set. Specifically, we used parametric, principal component analysis (PCA) and non-parametric cluster analysis.To assist in assessing broad scale food web groupings, a number of established fatty acid markers, or trophic indices, were calculated. These included 16:1ω7/16:0 and 22:6ω3/20:5ω3 to separate diatom versus dinoflagellate based energy channels in situ caught school, gummy and broadnose sharks  sampled off New South Wales in 2000 Of the total of fifty fatty acids detected in white sharks tissues, twenty two  were used in multivariate analyses to compare white shark muscle and liver profiles to other oceanic and coastal shark taxa with known dietary differences. Specifically, the mean fatty acid profiles of each of white shark muscle and liver, grouped according to cluster analyses, were compared to profiles of −1 wm (−1 wm). In other shark species, similar total lipid content has been detected; typically accounting for less than 1% wm White shark muscle was high in water (75.4±2.4%) and had a wet to dry mass ratio of 4.1±0.4 . Muscle −1 wm . Phospho−1 wm (−1 wm) and related this to their migration and reproductive cycles. For most oceanic shark species analyzed, triacylglycerol seems to be the dominant class of lipid accounting for >90% total lipid Liver had a wet to dry ratio of 1.2±0.1  and was −1 wm . Total l−1 wm  and intr−1 and the buoyancy in sea water (0.0103 g−1) is +0.095 mL−1 lipid −1, respectively Storage of large amounts of lipids which are high in energy and low in density are likely to assist white shark movement patterns −1 wm and 27.3 kJ g−1 wm, respectively, −1 dm) and energy content (590 to 4550 kJ dm) were observed in the Atlantic sharpnose shark in the Gulf of Mexico −1 wm The estimated energy density of white shark muscle was noticeably lower than that of the liver  than liver (4.2–17.9 kJ g−1 wm) with benthopelagic species, primarily gadiforms, having significantly larger lipid-rich livers than benthic species. In a comparative study of the energy density of 39 pelagic forage fishes −1 wm fish) was reported with small, fast maturing species having higher relative values than those species maturing at a larger size. Energy density of muscle and liver of Antarctic fishes were respectively lower (21–25 kJ g−1 dm) and higher  and mammals, white shark liver has a higher energy density, and therefore capacity to store energy. Drazen −1. Our study adds information on the static energy available in the muscle and liver of white sharks and gives additional information on their energy budget and allocation strategies. Such information is vital for the calibration and validation of bioenergetics models at individual, population and ecosystem scales. These models, which describe the mass balance relationship between energy acquired, transformed and allocated to organism productivity , are increasingly being used to assist fisheries and conservation management A recent study A total of fifty fatty acids were identified in the white shark tissues analyzed  and S2, 20) polyunsaturated fatty acids have also been reported in large concentrations in the liver of other large, oceanic shark species Muscle fatty acid profiles of white shark in South Africa In addition to the large differences occurring between the fatty acid profile of the muscle and liver, we detected large variations among individual sharks . In the Large intra-specific differences in fatty acid profiles of tissues have been experimentally shown to reflect an organism's diet at different temporal scales No clear separation among juvenile and adult sharks was found in the present study. This is likely related to the fact that individuals were caught over a long and variable sampling period  and the animals were from a large size range (1.5–3.9 m). This variation, in association with the fact that white sharks engage in broad-scale inshore and offshore migrations Inter-specific comparisons can assist in the interpretation of fatty acid data and making dietary inferences, particularly where sample size is low Cluster analyses of the liver fatty acid profiles showed a higher magnitude of dissimilarity between sharks than that in the muscle, with an 80% resemblance cut-off identifying six separate groups . White sThe close similarity between muscle profiles of white shark in the present study and broadnose shark is fitting; the latter is a dominant predator in coastal systems, commonly consuming mammalian blubber and small elasmobranchs including spurdogs, school and gummy sharks Of particular interest in understanding the diets of white sharks is the contribution of oceanic relative to coastal prey. In a study of pelagic fishes in oceanic waters off eastern Australia, mako shark had the highest nitrogen isotopic value indicating their position as top predator in the system In this study we used lipid analyses to directly extract energetic and feeding related information on white sharks. We found that while the muscle is a site of structural energy, the liver has an even higher energy density than whale blubber and serves as a storage unit to fuel white shark migrations, growth and reproduction. Among-individual variation in fatty acid profiles suggest individuality in foraging strategies with some sort of a gradient between dominant or partial consumption of fish and mammals. Such information, although currently very limited in elasmobrachs, is required for the development of bioenergetics-based models including dynamic energy budget models and ecosystem models increasingly used in fisheries and conservation management. Indeed, measuring total lipid energy is appealing because lipids represent a common currency, linking food quality and quantity to many aspects of fish nutrition, reproduction and population dynamics. Future studies should include targeted sampling and utilization of this novel approach on live specimens, along with ongoing telemetry studies, to explore the relative contributions of biological and environmental factors influencing intra-specific variation. Such data will allow us to better understand the ecology of white shark and predict how they might respond to environmental perturbations.Table S1Muscle full fatty acid profiles of individual white sharks and a single basking shark (BS1) analyzed in this study.(DOCX)Click here for additional data file.Table S2Liver full fatty acid profiles of individual white sharks and a single basking shark (BS1) analyzed in this study.(DOCX)Click here for additional data file.


In an effort to explore new, noninvasive treatment options for spinal cord injuries (SCI), this study investigated the effects of electroacupuncture (EA) for SCI rat models. SCI was induced by a modified Allen's weight-drop method. We investigated the response of EA at Dazhui (GV 14) and Mingmen (GV 4) acupoints to understand the effects and mechanisms of EA in neuroprotection and neuronal function recovery after SCI. BBB testing was used to detect motor function of rats' hind limbs among groups, and EA was shown to promote the recovery of SCI rats' motor function. Nissl staining showed a restored neural morphology and an increase in the quantity of neurons after EA. Also, the antiapoptosis role was exposed by TUNEL staining. Western blotting analysis was used to determine the protein expression of neurotrophin-3 (NT-3) in spinal cord tissue. Compared to the sham group, the expression levels of NT-3 were significantly decreased and EA was shown to upregulate the expression of NT-3. The present study suggests that the role of EA in neuroprotection and dorsal neuronal function recovery after SCI in rats, especially EA stimulation at GV 14 and GV 4, can greatly promote neuronal function recovery, which may result from upregulating the expression of NT-3. Spinal cord injury (SCI) is an accidental tragedy, causing unexpected suffering physically and emotionally, and is costly to patients . TraumatAt present, mainstream treatments for SCI include pharmacotherapies, neurotrophic factors, cell-transplantation, gene therapy, and biological materials transplantation , 4. TradAcupuncture is a therapeutic technique used in TCM. Since its development several thousand years ago, acupuncture has made many contributions to health care and medical treatment. Electroacupuncture (EA) is a type of therapy in which a needle inserted into an acupoint is attached to a trace pulse current with the purpose of producing a synthetic electric needling stimulation. The application of EA for the treatment of SCI has shown promising results in the alleviation of the patients' suffering . PreviouTCM believes that Governor Vessel injury is the main reason of SCI. Malnutrition of tendons and muscles due to obstruction of qi and blood induces disuse and atrophy. Therefore, Governor Vessel acupoints are the first choices for treating SCI. “Dazhui” (GV 14) is the confluence of Governor Vessel and the hand and foot three Yang meridians. Acupuncture stimulation in GV 14 can activate and inspire Yang-qi in the whole body to dredge the meridians. “Mingmen” (GV 4) is also a Governor Vessel acupoint and is the intersection of the Governor Vessel and Belt Vessel. It gathers genuine-Yin and genuine-Yang, which are the root of Yuan-qi and the gateway of life. Stimulating GV 4 can regulate channels and activate collaterals, tonify Yang, and strengthen the kidneys. In previous experiments using SCI rat models, acupuncture stimulation in GV 14 and GV 4 has been adopted .EA on the Governor Vessel has been shown to alleviate secondary damage after SCI in both patients and animal models , 11. TheNeurotrophic factors (NTFs) are protein molecules which are essential to neuron survival and growth . NGF, BDThis study aims to explore whether EA can promote a suitable microenvironment for recovery of neurological function after SCI by increasing endogenous NT-3 secretion and expression at the injured area and adjacent tissues after performing EA on the Governor Vessel during the early phase of SCI.All experiments were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. 165 rats  were randomly assigned into three groups at a ratio of 1 : 1 : 1. The sham-operation group (S) received only a laminectomy. The remaining two groups were modeled as spinal cord injury at the T10 spinal segment. The control group (C) received no treatment. The EA group received EA treatment at GV 14 and GV 4 acupoints. Rats in the control group and EA group were randomly placed into the following subgroups: 1 d, 3 d, and 7 d. There were 20 rats per group in 3 d and 7 d, respectively, and 15 rats in group 1 d. Each of the animals was housed in separated cages with free access to food and water. Room temperature was set at 25 ± 3°C.μ, twice daily. No analgesics were used. Rats that underwent SCI received specialized care consisting of manual bladder expression three times daily and cleansing for the duration of the experiment.The surgical procedure for inducing SCI was conducted according to established methods . The rat∗25 mm, Zhongyan Taihe brand, Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) were inserted into GV 14 (oblique downwardly) and GV 4 (oblique upwardly), respectively, as deep as 5–7 mm for both points. Following the insertions, electrodes were connected to the handle of the needles . Electric simulation parameters were at a frequency of 2 Hz and an intensity of 1 mA for 20 minutes. The rats received EA treatment 2 hours after the SCI model was established and anesthesia recovery. One hour before euthanasia, at the 24th hour of postsurgery, the rats in 1 d EA group were given the 2nd treatment. The rats in 3 d and 7 d EA group were given the same treatment once per day until they were euthanized at the appointed time. The sham and control animals did not receive treatment. Finally, 11 rats per group were analyzed. 6 rats per group were perfused to fixation and fresh tissue was taken from the other 5 rats per group. All the above procedures were conducted after anesthesia.GV 14 and GV 4 acupoints were utilized during EA treatment. The GV 14 acupoint is located in the posterior midline and in the depression below the spinous process of the 7th cervical vertebra in prone position. GV 4 is also located in the posterior midline and in the depression below the spinous process of the 2nd lumbar vertebra in prone position. Rats were placed within a cloth bag without anesthesia during EA administration. Sham and control animals were captured for bondage when the EA group received treatment, for a duration of 20 minutes each time, to ensure the same processing conditions. Sterilized disposable stainless steel needles  hind limb locomotor rating scale test. The BBB rating scale is a 21-point system based on operationally defined behavioral features, which follow the recovery progression from complete paralysis to normal locomotion. The rating scale ranges from 0 to 21, with a score of 0 indicating complete hind limb paralysis and a score of 21 denoting completely normal locomotor function.μm were obtained. To assess the histopathologic change, each one of six sections obtained was subjected to Nissl staining. Surviving neurons were characterized by blue staining Nissl bodies. Quantification was performed by counting the quantity of survival neurons in five randomly chosen fields within each slide from each section in the same part of the structure at 400x with an Olympus  optic microscope. Two observers, blind to the experiment, counted the surviving neurons.Six of eleven rats in each group were euthanized at the specified time of postsurgery and perfused transcardially with 0.9% sodium chloride and then with 4% paraformaldehyde in 1x phosphate buffered saline (4% PFA) for 30 min. Spinal cords were surgically removed and kept in 4% PFA for postfix overnight. After dehydration, the spinal cords were embedded with paraffin, and serial coronal sections with thickness of 4 To detect apoptosis, each one of six coronal sections obtained in the above experimental procedure was further subjected to terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining. To assess the apoptotic cells, we measured 6 samples in one section chosen at random from each spinal cord. Apoptotic cells were characterized by dark brown staining of nucleus and nuclear membrane. Quantification was performed by counting the quantity of positive cells in five randomly chosen fields within each slide from each section in the same part of the structure at 400x with an Olympus  optic microscope. The index of apoptosis was calculated as the ratio of overall apoptotic cells. Two observers, blind to the experiment, counted the apoptotic cells.μL lysis buffer. Samples were centrifuged at 10000 r/min for 10 min at 4°C. Protein concentrations were determined using the Bradford method . Ho. Ho24]. ∗∗p < 0.01, versus sham group; ##p < 0.01, versus control group; n = 6 animals per group). This suggested that EA can reduce apoptosis, promote the survival of neurons, and has a certain protective effect.In the sham group, there were almost no TUNEL positive cells . In the p > 0.05, p < 0.05, p < 0.01, p > 0.05, We used Western blotting analyses to determine the expression of NT-3 semiquantitatively for assessing the progression of SCI and the protective effects of EA. In the sham group, NT-3 demonstrated expression. In the control group, the expression of NT-3 decreased at 1 d and 3 d without statistical significance (In this research, the BBB scores of control group and EA group which received SCI operation had statistical difference compared with that of sham group in 1 d, 3 d, and 7 d, which proved that the models of SCI were successfully established as the motor function of rats' hind limbs appeared paralyzed. At 7 d after SCI, the BBB scores began to show significant statistical difference between EA group and the control group, which indicated that the hind limbs' motor function of rats was gradually recovering, and the recovery of rats that received EA treatment was better than that of control rats without EA treatment. This signifies that EA treatment could promote motor function recovery after SCI, which was also shown by Nissl staining, TUNEL, and Western blotting.In addition to the damage at the site of the spinal cord injury, secondary pathological changes occur in the following order: edema, ischemia, calcium overload, lipid peroxidation, microcirculation obstruction, and apoptosis , 26. UnlOur results showed that less postinjury neuronal death was occurring in the EA group compared with the control group, which was interpreted as neuroprotection. Furthermore our results still indicated that EA could maintain the cell morphology of neurons, which contributed to the functional recovery of the injured spinal cords of the rats.Neurotrophic factors enter axons by way of its receptor mediated endocytosis at the end of axons, then reach neurons through plasma flow reverse transportation, which can cause different signaling pathway activation or inhibition in the cell, and regulate relative protein expression, to play its effect of supporting neuron development, survival, growth, and function integrity . NT-3 isNT-3 is widely distributed in peripheral and central nervous systems, mainly in the hippocampus, dorsal root ganglia, brainstem, and spinal cord et cetera . Chen anThe present study found that the level of NT-3 in the injured spinal cords of the control group and EA group decreased in comparison with that in sham group rats' spinal cord cell where it was still synthesized and secreted. This might be injured spinal cord induced cell death or the decline of damaged cells' function in the damaged region and adjacent tissues which synthesize and secrete NT-3; therefore the NT-3 levels would be decreased . We alsoIn addition, NT-3 plays important roles in oligodendrocyte development , 36. HuaAccording to TCM, GV 14 and GV 4 are points pertaining to the Governor Vessel. The spinal cord and Governor Vessel have direct contact on channels and collaterals. Based on the principle of “where meridians pass, indications for acupoints of this meridian could be considered,” selecting the Governor Vessel to treat paraplegia related to spinal cord injury conforms to the TCM saying “searching for the primary cause of disease in treatment” , 39.Governor Vessel EA on GV 14 and GV 4 could improve functional recovery by reducing apoptotic cell death after SCI. Meanwhile, the neuroprotective effects of EA treatment might be mediated by the level of NT-3 in the microenvironment around the spinal cord after injury. In addition, the present study suggested that stimulating GV 14 and GV 4, especially with electroacupuncture, might be an effective therapeutic strategy in spinal cord injury.


A woman in her mid-60s presented with a bulky mass on the anterior abdominal wall. She had a previous incidental diagnosis of endometrial adenocarcinoma FIGO stage IB following a vaginal hysterectomy. Physical exam and imaging revealed a well circumscribed bulging tumour at the umbilical region, measuring 10 × 9 × 9 cm, with overlying intact skin and subcutaneous tissue. Surgical resection was undertaken, and histological examination showed features of endometrial carcinoma. She began chemotherapy and is alive with no signs of recurrent disease one year after surgery. This case brings up to light an atypical location of a solitary metastasis of endometrial carcinoma. Uterine cancer is the most frequently diagnosed gynaecological malignancy in developed countries. In 2008, more than 288,000 new cases were registered in the world, with a mortality rate of 2.2 per 100,000 women [The majority of women with endometrial carcinoma are diagnosed with localized early stage disease, yielding a high survival rate. The first step of treatment is complete surgical staging that can be followed by adjuvant radiotherapy and chemotherapy or both. In early stage disease, adjuvant radiotherapy has shown to significantly reduce locoregional recurrence, although it does not appear to increase overall survival or distant recurrence rates. Adjuvant chemotherapy can be considered in high-risk patients, but compared to radiotherapy alone, no differences were found in overall survival and progression-free survival, although further studies are needed. Combined modality treatment in high-risk patients was associated with a reduction in risk of recurrence or death in two randomized clinical trials and is the subject of the ongoing PORTEC 3 study . For medFollowing primary treatment, the overall recurrence risk ranges from 8 to 19% . TreatmeThe authors describe a case of an atypical location of endometrial carcinoma metastasis on the anterior abdominal wall without other signs of advanced disease.2) presented 3 years before with stage IV uterine prolapse and occasional vaginal spotting related to ulceration of the protruding cervix. Preoperative evaluation included a pelvic ultrasound that showed an enlarged uterus (L 89 × AP 58 × W 61 mm) with various small fibroids (around 20 mm) that distorted endometrial cavity hindering an accurate measurement of endometrial thickness. Both ovaries were normal in shape and size, and no malignancy was suspected. The optimization of the underlying diseases delayed surgery for several months. In July 2012, a vaginal hysterectomy was performed. Intraoperatively, inspection of both ovaries was unsuspicious. The histopathology examination revealed an enlarged uterus distorted by the presence of multiple fibroids and an intracavitary tumour invading more than half of the myometrium. The tumour was grade 3, poorly differentiated endometrial adenocarcinoma, without vascular space invasion, FIGO stage IB. Given her general condition and absence of signs or symptoms of the disease, despite incomplete surgical staging and high intermediate-risk disease, it was decided by a multidisciplinary team of gynecologic surgeons, pathologists, medical, and radiation oncologists to proceed with adjuvant radiotherapy. Chemotherapy was not considered due to her medical and social background. The patient received 50 Gy (25 fractions of 2 Gy) of external pelvic irradiation and 24 Gy (4 fractions of 6 Gy) of brachytherapy to the vaginal cuff.A Caucasian woman in her mid-60s, gravida 1 para 1, with toxic multinodular goiter with an intrathoracic extension, hypertension, chronic anaemia, and obesity , lactate dehydrogenase mildly raised (333 UI/L), and elevated CA 125 (478 UI/mL). Magnetic resonance imaging (MRI) Figures  revealedSurgical resection of the tumour was performed followed by peritoneal washing and bilateral salpingo-oophorectomy. Reconstruction of the abdominal wall was accomplished with the use of a synthetic mesh. The cross section of the tumour is shown in 2) and carboplatin (300 mg/m2) and completed six cycles. At the end of treatment, radiologic imaging with thoracic and abdominal CT and pelvic MRI did not show any signs of recurrence. More than one year after surgery, the patient is alive and asymptomatic.She began chemotherapy one month after surgery with paclitaxel , spleen (1%), central nervous system (<1%), extra-abdominal lymph nodes , and, more rarely, adrenals, pancreas, and appendix –13.In this case and taking into account its location near the umbilicus, the hypothesis of an umbilical metastasis, also termed Sister Mary Joseph's nodule, was raised. Sister Mary Joseph's nodule most typically presents as an irregular lump on the umbilicus, ranging in size from 0.5 to 2 cm, although there are reports with nodules reaching up to 10 cm. It may be ulcerated and necrotic and have a bloody, mucinous, serous, or purulent discharge. It can be detected before or during diagnosis of the primary tumour or after treatment. This umbilical metastasis is found in 1–3% of patients with gastrointestinal or genitourinary malignancy, including endometrial cancer , 14. TheAbdominal wall metastases have also been linked to surgical incision, regardless of the surgical approach (laparotomy or laparoscopy). Although rare, it has been described in cases of endometrial carcinoma –23. The Park and Hwang reported a case of a postmenopausal woman with an abdominal wall metastasis in stage IA serous endometrial adenocarcinoma, eight months after surgical staging. Treatment consisted of surgical excision and chemotherapy, and three years after surgery she is alive with no signs of disease . CompariThis case highlights an unusual location of a solitary metastasis of endometrial cancer on the soft tissue of the abdominal wall. Almost all cases reported in the literature are diagnosed after primary treatment and are related to the surgical incision. In this case, it is difficult to know exactly how neoplastic cells implanted in the soft tissue and developed into a mass. Surgical resection was feasible and the patient completed chemotherapy with no signs of recurrence after one year.


In the recent years, colour sorting machines are used in order to separate red lentils according to their colours. The goal is to select the items which are discoloured, not as ripe as required, or still with hull even after dehulling of lentil seed. During the sorting, a new byproduct called “sorting byproduct” leftover is obtained. The byproduct is cleaner and is of a higher quality than other lentil byproducts. This experiment was conducted to study the effects of the inclusion of different levels of lentil byproduct on laying quail performance. The experimental treatment included 10% or 20% lentil byproduct in the diet, and this was fed to quails aged between 11 and 22 weeks. The inclusion of 10% and 20% levels of lentil byproduct in the diet significantly increased egg production, but feed intake and feed conversion ratio were not significantly affected. Egg weight decreased significantly following the inclusion of 20% lentil byproduct. The inclusion of lentil byproduct in the diet increased the deposition of yellow yolk pigments and decreased malonaldehyde formation in the yolk.One hundred and sixty-eight 11-week-old laying quails  Lens culinaris L.) is primarily used for food, it also provides an excellent protein-rich (22%–29%) feedstuff for domestic livestock. In addition, crop residues and byproducts from the food processing industry can be effectively utilized by livestock, particularly poultry. Lentil byproducts are an excellent source of macronutrients and contain phytochemicals [Leguminous seeds used in human nutrition are processed before being made available for consumption, and during this process various byproducts are generated, such as pea byproduct, lentil byproduct, and sunflower meal. These byproducts are used in animal rations as cheap feed sources. Red lentil's peel is removed and cleaned while it is processed in the factories as edible product. After this process, in the recent years, color sorting machines are used in order to separate harvested food stuffs such as red lentils according to their colors. The goal is the separation of items that are discoloured, not as ripe as required, or still with hull after dehulling lentil seed . During ein-rich 2%–29% fehemicals . Lentil hemicals . Until rhemicals . Phenolihemicals . High cohemicals . Lentil hemicals . BecauseCoturnix coturnix japonica) were fed one of the following three diets over an 11-week period: control: basal diet with no lentil byproduct; inclusion of 10% lentil byproduct; inclusion of 20% lentil byproduct. Each treatment comprised 4 replications with 4 cages (14 quails per cage), amounting to 56 quails per treatment group. A photoperiod of 17 h/day was maintained, and egg production was recorded daily. During the laying period, a random sample of 10 eggs/replicate was collected on 2 consecutive days per week for determination of egg weight. Feed intake was recorded on a weekly basis and feed conversion ratio (FCR) was calculated as the ratio of grams of feed consumed per gram egg weight. Levels of production variables such as feed intake and egg production were adjusted for mortality rates, mortality being recorded daily. All diets were isocaloric and isonitrogenous (in mash form), with water being provided ad libitum. At the end of the experiment (22 weeks of age), 24 eggs were collected from each treatment and analyzed for yolk color with a Minolta Chroma Meter CR-300 using the CIE  scale. L*, a*, and b* values reflect lightness , redness , and yellowness  of the samples, respectively. The albumen was removed from the yolk before yolk color evaluation.All animal protocols received prior approval of the Institutional Animal Care and Use Committee, and all experiments were performed in accordance with relevant guidelines and regulations. One hundred and sixty-eight 11-week-old laying quails (n = 8) were weighed into 50 mL test tubes and 18 mL of 3.86% perchloric acid was added. The samples were homogenized with a vortex, with butylated hydroxytoluene added to each sample during homogenization to control lipid oxidation. The homogenate was then filtered through Whatman 1 filter paper and the filtrate was (2 mL) mixed with 2 mL of 20 mM TBA in distilled water. The filtrate was incubated in a darkened cabinet at room temperature overnight (15–17 h), with absorbance determined at 531 nm. TBA values were expressed as milligrams of malonaldehyde (MDA) per kilogram of yolk. Ten eggs from each replicate were collected at the end of treatment (40 eggs from each dietary treatment) for pH analysis. The eggs (yolk + white) were homogenized and pH was measured with a pH meter. Condensed tannins in the lentil byproduct were assayed in accordance with the butanol-HCl method of Makkar [P < 0.05.To investigate the effects of diet on shell eggs lipid oxidation during refrigeration, 10 eggs from each replicate collected at the end of treatment (40 eggs from each dietary treatment) were placed in a refrigerated cabinet at 4°C for analysis of yolk thiobarbituric acid (TBA) content at 0, 7, 14, 21, and 28 days of storage. TBA values were determined as previously described by Cherian et al. [f Makkar . Red lenThe effects of dietary lentil byproduct on egg production, feed intake, FCR, and egg weight are presented in P < 0.01) hen-day egg production above that of the control diet, with egg production being similar for both diets including lentil byproduct. However, the inclusion of lentil byproduct in excess of 10% had no additional beneficial effect on egg production. In contrast to our results, Kılıçalp and Benli [P > 0.05) in feed intake and FCR between treatments over the 11-week period. Similar results were reported by Kılıçalp and Benli [L*) was increased with the inclusion of dietary lentil byproduct compared with the control diet, but the inclusion of lentil byproduct in excess of 10% had no additional beneficial effect.There were significant effects on egg production and egg weight. The inclusion of 10% and 20% lentil byproducts in the diet increased  parameter of yolk was not significantly affected by the dietary inclusion of lentil byproduct; the b* (yellowness) was increased by the inclusion of 20% lentil byproduct, but not by 10%. This result showed that inclusion of lentil byproduct in the diet increased deposition of yellow pigments into the yolk. The effects of these dietary inclusions on the oxidative stability of shell egg measured at different time periods, and on pH, are shown in The P < 0.01) among dietary treatments, but did not change with storage time. MDA formation in eggs decreased with increasing dietary content of lentil byproduct. Total antioxidant capacity and the mechanism of action of tannin in lentil were analyzed in vitro studies. The lentil was shown to have a marked total antioxidant capacity, likely related to its high content of condensed tannins [P < 0.01).The level of lipid oxidation, as measured by MDA formation, differed ( tannins , 5, 6. HThe inclusion of lentil byproduct in the diet of laying quail was beneficial in terms of increased egg production and deposition of yellow yolk pigments. Moreover, lipid oxidation was reduced in shell egg. Additional research is needed to investigate the antioxidant constituents of tannin in lentil byproduct when deposited in egg yolk.


This network meta-analysis aimed to investigate the effectiveness and safety of 100 mg BID and 200 mg QD oral celecoxib in the treatment of OA of the knee or hip. PubMed, Embase and Cochrane Library were searched through from inception to August 2014. Bayesian network meta-analysis was used to combine direct and indirect evidences on treatment effectiveness and safety. A total of 24 RCTs covering 11696 patients were included. For the comparison in between the two dosage regimens, 100 mg BID oral celecoxib exhibited a greater probability to be the preferred one either in terms of pain intensity or function at the last follow-up time point. For total gastrointestinal (GI) adverse effects (AEs), both of the two dosage regimens demonstrated a higher incidence compared to the placebo group. Further analyses of GI AEs revealed that only 200 mg QD was associated with a significantly higher risk of abdominal pain when compared with placebo. Furthermore, 100 mg BID showed a significantly lower incidence of skin AEs when compared with 200 mg QD and placebo. Maybe 100 mg BID should be considered as the preferred dosage regimen in the treatment of knee or hip OA. Osteoarthritis (OA) is the arthritis from with the highest prevalence in the world, affecting about 40% of the total population aged over 7035et al. conducted two randomized controlled trials (RCTs) and demonstrated that celecoxib 200 mg daily and 100 mg twice a day were equally effective and well tolerated in patients with knee OA8et al.According to the official instructions of celecoxib, 200 mg QD and 100 mg BID are the two recommended dosage regimens for the treatment of OA. A few years ago, Williams Classical meta-analysis was limited due to the lack of multiple comparisons. Bayesian network meta-analysis is a method combining all available direct (studies compared 200 mg QD with 100 mg BID directly) and indirect evidences (studies compared 200 mg QD with 100 mg BID via the placebo group) on the relative treatment effects, enabling a unified; and coherent analysis of all RCTs121314http://www.controlledtrials.com/), ClinicalTrials.gov  and the World Health Organization International Clinical Trials Registry . In addition, references of the retrieved papers and reviews were also manually identified.Searches of electronic databases of PubMed, Embase and Cochrane Library were done using a series of logic combination of keywords and text words related to OA to identify interested interventions and RCTs (see web extra appendix 1) dated up to August 2014. The database search was then supplemented by subsequent periodic scrutiny of unpublished and ongoing studies from the following websites: Current Controlled Trials  large RCTs (at least 100 patients per arm)The modified oxford score19The primary outcome of this study was the effectiveness of pain relief and function improvement from the baseline to the end of the treatment by applying 200 mg QD or 100 mg BID oral celecoxib therapy for knee or hip OA. If a study reported multiple pain scales, the highest one on the hierarchy of pain scale related to outcomes was adopted, as described by Jüni and colleaguesGI AEs: dyspepsia, diarrhea, abdominal pain, nausea, constipation, flatulence, vomit.CV AEs: chest pain, hypertension, thrombotic CV AE, stoke, myocardial infarction, congestive heart failure, CV events excluding chest pain.CNS AEs: headache, dizziness, insomnia, depression, nervousness, anorexia.Musculoskeletal AEs: arthralgia, back pain, myalgia, sciatica.Infections: upper respiratory infection, nasopharyngitis, influenza, urinary tract infection, sinusitis, bronchitis, rhinitis, pharyngitis.Skin AEs: rash, pruritus, exanthema, erythema, itching, dry skin, skin irritation, urticarial, allergic dermatitis.Peripheral edema.Safety and tolerability were also examined by comparing the number of patients who withdrew due to AEs and who suffered from serious AEs (SAEs) and the incidence of common reported AEs. No standard was set in this study for identifying SAEs; they were defined by their original research. AEs were classified into eight kinds of events in order to summarize their variety in all included studies: GI AEs, CV AEs, central nervous system (CNS) AEs, musculoskeletal AEs, infections, skin AEs and peripheral edema. Below is the detailed description of the AEs classification:Meanwhile, six most common reported GI AEs  were analyzed separately due to their high incidence during the treatment. Odds ratios (OR) were calculated to determine the difference between compared groups.http://www.mtm.uoi.gr/ and http://www.bristol.ac.uk/social-community-medicine/projects/mpes/ . The overall effect sizes (ORs or SMDs) were generated from the median of the posterior distribution. The 2.5th and 97.5th percentiles of the posterior distribution were considered as the lower and upper limit of the traditional corresponding 95% credible interval (95%CI) respectively. A significant difference could be identified by 95%CI which did not include 1 for OR, or 0 for SMD. Inconsistency is defined by the differences between direct and indirect effect estimates for the same comparison, and was evaluated in this study by using the ratio of two odds rations (RoR) from direct and indirect evidences in one loop. A RoR value closer to 1 suggests that the two estimations are consistent to each other. Loops with the lower 95%CI limit of RoR value do not reach the 1 line represent statistically significant inconsistency262 statistics, which measures the percentage of the total variation across various studies (I2 ≥ 50% was considered heterogeneous). Publication bias was evaluated by Begg’s test, and a P value equal to or less than 0.05 represents the existence of publication biasA Bayesian random effect model of network meta-analysis was used to compare the overall effect size among different celecoxib dosage regimens and placebo for knee and hip OA. The advantage of network meta-analysis is that it combines the evidences of both direct and indirect comparisons in all primary trials221524All statistical analyses were implemented by using WinBUGS software , R version 3.0.2  and STATA software .8930313233343536373839404142434445p = 0.00, I2 = 80%). There was no publication bias among various studies.The results of network meta-analysis and pairwise meta-analysis were reported in p = 0.00, I2 = 75%). There was no publication bias among various studies.The results of tolerability and AEs  were reported in Six kinds of GI AEs  were analyzed separately . It is nFor Skin AEs, 100 mg BID showed a significantly lower incidence compared to the placebo group , while 200 mg QD showed a significantly higher incidence compared to the 100 mg BID group  . No evidFor all the rest kinds of AEs, no evidence of significant difference was observed in this network meta-analysis. The probability distribution of each treatment for non-significant AEs was shown in Appendix 3, and all the forest plots of pairwise meta-analysis were presented in Appendix 4.Several meta-analyses and systematic reviews on celecoxib have been published24474849505152532450515153As described in the introduction of this study, 3 RCTs suggested that there was no difference between celecoxib 200 mg QD and 100 mg BID in efficacy or safety for the management of OA, providing flexibility to patients and physicians in choosing a dosing regimen89However, Solomon and colleagues conducted a pooled analysis of 6 RCTs and came to a noteworthy speculation that 400 mg QD celecoxib might be safer than 200 mg BID because the former regimen was associated with a shorter duration of exposure to susceptible atherosclerotic tissueThe findings of this network meta-analysis are extremely important to clinic practice. On one hand, this study provided evidence to support the two evidence-based guidelines (OARSI and AAOS)3A single study is impossible to solve all the problems pertinent to the evaluation of oral celecoxib for knee or hip OA. Considering the small number of direct evidence comparing 200 mg QD with 100 mg BID, further high quality RCTs of direct comparison, especially industry independent trials, are needed. Additional questions to be addressed include the determination of the best treatment duration (shortest in the case of effective). Furthermore, the follow-up time of the subsequent studies also needs to be extended in order to see whether the effects may diminish and whether there were delayed AEs, especially for CV AEs.As far as we know, this is the first network meta-analysis on two different official dosage regimens of oral celecoxib (200 mg QD and 100 mg BID) for the treatment of knee or hip OA. It combined evidences from both direct and indirect comparisons while fully preserving randomisation for evaluating the relative effectiveness in pain relief, function improvement and safety. In addition, this is also the first meta-analysis that restricted subjects to OA patients for examining the effect and safety of oral celecoxib. Furthermore, a comprehensive literature search was performed in several major databases and sources to cover as many eligible trials as possible, so the chance of missing any relevant trial was fairly low. With the pre-stated inclusion criteria on the sample size threshold, only large-scale RCTs were included in this network meta-analysis, which enhanced the robustness of the resultsNevertheless, the limitations of this study should not be ignored. Firstly, variations in duration and final follow-up time point might contribute to the evidence of significant heterogeneity, particularly for the possible duration-response patterns which could affect the performance of celecoxib. Fortunately, no obvious evidence of inconsistency was observed in this network meta-analysis. Secondly, all of the included trials measured the pain or function parameters at a time point too shortly after the completion of the treatment courses. It is uncertain whether these effects might diminish over a period of time. Thirdly, none of the included trials was industry independent trial, so there is a possibility that the effect size was overestimated31This network meta-analysis indicated that 200 mg QD and 100 mg BID oral celecoxib are both effective in pain relief and function improvement for the management of knee or hip OA. Overall, 100 mg BID exhibited a greater probability of being the preferred regimen in pain relief and function improvement, and it also showed a significantly lower risk of abdominal pain and skin AEs when compared with 200 mg QD, so maybe 100 mg BID oral celecoxib should be considered as the recommend dosage regimen in the treatment of knee or hip OA. Further RCTs are needed to confirm this result.How to cite this article: Zeng, C. et al. Comparison between 200 mg QD and 100 mg BID oral celecoxib in the treatment of knee or hip osteoarthritis. Sci. Rep.5, 10593; doi: 10.1038/srep10593 (2015).


Shiraia bambusicola is a parasitic fungus which is widely used in China for curing severaldiseases with little side-effects. However, the mechanisms are not well understood yet.The aim of this study was to further understand the pharmacological mechanisms of Shiraia bambusicola and investigate whether it can be used for curing gastric cancer. Gastric cancer is a major health issue worldwide. Using a therapeutic approach, with minor side-effects, is very essential for the treatment of the gastric cancer.Shiraia bambusicola on BGC823, A549 and HepG2 cells. We usedMTT assay to test cell viability. We also analyzed morphologic changes caused by apoptosisusing Hoechst 33342 fluorescence staining, as well as cell cycle status and apoptosis ratio using flow-cytometer. In addition, protein expression level was tested by Western-blotting assay. In this experimental study, we mainly tested the effect of activecomponents extracted from Shiraia bambusicola. Meanwhile, these active components could induce BGC-823 cellsapoptosis and retard the cell cycle in S/G2 phase. We also determined that two criticalprotein markers cleaved Poly(ADP-ribose) polymerase-1 (PARP-1) and FLICE-inhibitoryprotein (FLIP), involved in apoptosis process, were regulated by these active components. BGC-823 cell proliferation was specifically inhibited by active components ofShiraia bambusicola can be a good therapeutic candidate for treatment of this malignancy.These data shed light on the treatment of human gastric cancer and concludethat  Gastric cancer can be initiated in any part of the stomach and may spread throughout this organ and many others, particularly esophagus and the small intestine . This tyPrognosis of the patients with gastric cancer is related to tumor extent, including both nodal involvement and direct tumor extension beyond the gastric wall. Currently different types of therapeutic approach are utilized for treatment of this disease including surgery, chemotherapy and neo-adjuvant, adjuvant, and palliative therapies. Using the choice of surgical approach in gastric cancer depends on the size, location, and locally invasive characteristics of this malignant tumor . CurrentShiraia bambusicola P. Henn. is a parasitic fungus on twigs of several kinds of bamboos, including Brachystachyum densiflorum and relevant species in China as well as Bambusa species in Japan  polymerase-1 (PARP-1) is a nuclear chromatin-associated enzyme involved in several important cellular processes, particularly in the DNA repair system . Within In the present research, using human gastric cancer cell line (BGC823), lung carcinoma cell line (A549) and hepatocellular carcinoma cell line (HepG2), we attempted to investigate the effects of recruited active components from Shiraia bambusicola on different cell models as well as the potential mechanisms involved in these processes. Shiraia bambusicola was milled and mixed with freshly prepared 80% ethanol  in erlenmeyer flask, followed by smoothly stirring in a digital orbital shaker for 6 hours at 70˚C. After filtering the mixture and discarding debris, the solvent named group 1 was filtrated in a rotary evaporator. When incubated with cells, the resulting solid was suspended in 80% EtOH and preserved in a concentration of 5 mg/ml. Group 1 component was further extracted by Polyamide column chromatography and the solvent was mixed with 95% EtOH, alternatively named group 3. When incubated with cells, the resulting solid (group 3) was suspended in 80% EtOH and preserved in a concentration of 2 mg/ml. This experimental study was approved by the Ethical Committee of Second Xiangya Hospital of Central South University. The 2 . BGC823 cell line was purchased from the Shanghai Cell Collection (China). In addition, A549 and HepG2 cell lines were prepared from American Type Culture Collection . Cells were cultured according to previously described method . The cel4 cells/well. They were subsequently treated with different concentrations of components obtained from Shiraia bambusicola. At the end of treatment, the medium was carefully removed and 200 μl of medium containing 20 μl MTT  was added to each well. The cells were then incubated for 4 hours at 37˚C, followed by removing the medium and adding dimethyl sulfoxide (100 µl) to each well. The absorbance of each well was read at 490 nm. MTT assay was used to assess cell viability. The cells were grown in a 96-well plate for 48 hours, with a density of 2×10Shiraia bambusicola, the cell culture medium was removed and the cells were fixed using 0.5 ml stationary liquid , for 10 minutes. By removing the stationary liquid, the cells were rinsed twice with PBS after 3 minutes incubation. The slides containing cells were then stained by adding 0.5 ml Hoechst 33342  and incubation for 5 minutes, followed by three times rinsing with PBS before cell observations by microscope. In this experiment, morphological changes leading to condensed or fragmented nuclei were considered as apoptotic cells  placed in advance. After treatment with different concentrations of the extracted components from ic cells . For western blotting, the cells were lysed in RIPA buffer  with protease inhibitor cocktail . Protein concentrations were determined using the BCA protein assay kit . Samples of cell lysate supernatant (50 μg protein) were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred into nitrocellulose membranes. The membranes were then probed at 4˚C overnight with the following dilutions of the indin cated antibodies: cleaved-PARP  and FLIP . Anti-rabbit secondary antibodies  were used at a 1:4000 dilution. Immunocomplexes were detected by incubating with enhanced chemiluminescence  and subsequent exposure to x-ray film. Anti-β-actin polyclonal antibodies  were incubated with the same membrane, following the membrane stripping. 6cells/ml concentration. 1 ml of the cells were transferred into a 15 ml polypropylene tube on ice and 3 ml cold (20˚C) absoo lute ethanol was added. The cells were incubated for at least 1 hour at 4˚C, for fixation. They were subsea quently washed twice with PBS. Later, 1 ml of propidium iodide  staining solution was added to the cell pellets and mixed well. Ultimately, cell cycle and apoptosis rate of the BGC823 cells was analyzed by flow-cytometer  using manufacturer’s protocol. The cells were harvested and prepared as single cell suspension in washing buffer. They were then washed twice and re-suspended in a 2×10Statistical analysis of the results was carried out using one-way ANOVA as well as Student’s t test. P<0.05 was considered statistically significant. Data from all experiments are presented as mean ± SD of at least three independent experiments, performed in duplicate for each construct. in vitro to investigate potential treatment strategy for human gastric cancer, lung carcinoma and hepatocellular carcinoma, respectively. Using BGC823, A549 and HepG2 cells treated with extracts of Shiraia bambusicola, we found that only BGC823 proliferation can be inhibited by the active components group 1 and group 3 .Shiraia bambusicola has been used for the treatment of different diseases for long time, among Chinese population. Thus far, very rare side-effects have been determined from this parasite, turning that into a reliable treatment approach for long time administration. Previous reports implicated on several active components isolated from sporophores of Shiraia bambusicola (Fungus busicola , howeverin vitro models, to investigate the function of active components isolated from Shiraia bambusicola. Our results showed that both group 1 and group 3 can specifically inhibit proliferation and stimulate apoptosis of the BGC823 cells. These results suggest that Shiraia bambusicola could be a candidate for human gastric cancer treatment. Since group 3 is a component extracted from group 1, the percentage of useful materials from group 3 may be higher compared to group 1. Hence, we focused on the effects of group 3 on BGC823 cell cycle blockage and apoptosis. Our findings confirmed that group 3 can dose-dependently promote BGC823 cell apoptosis and retard cell cycle in the S/G2 phase. In this experiment, we used different types of human cancer cell line, as 2, Caspases, PARP-1 and FLIP can participate in apoptosis process (Cell apoptosis is a process of the programmed cell death that may occur in multicellular organisms and be involved in many biochemical events, leading to characteristic cell morphology changes and death. Many critical genes, such as P53, Bcl process ,23. TherShiraia bambusicola can induce BGC823 apoptosis and we propose that Shiraia bambusicola might be a good candidate for treatment of human gastric cancer. However, it is noteworthy to indicate that these active components could only have effect on some types of gastric cancer. Further investigations are required in vitro and in vivo to determine the precise effect of Shiraia bambusicola on human gastric cancer. It is very promising that the active components of Shiraia bambusicola, which can inhibit BCG823 proliferation, block cell cycle on S/G2 phase and induce cell apoptosis, might be useful for treatment of this malignancy. Meanwhile, we confirmed that PARP-1 and FLIP proteins play important roles during apoptotic process of the BGC823 cells, induced by active components of Shiraia bambusicola. Together, our data shed light on the treatment of human gastric cancer. The active components of


Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs  It consists of three layers; epithelium, stroma, and endothelium. The corneal epithelium covers the front of cornea and acts as a barrier. The stroma is the thickest layer of the cornea and provides the cornea structural strength. Finally, the corneal endothelium is a monolayer of hexagonal cells on the inner surface of the cornea, attached to its basement membrane termed Descemet’s membrane. Cells consisting of endothelial layers  play an essential role in maintaining the corneal transparency by their barrier and pump function through Nar chamber,2.in vivo have a limited capacity in proliferation since they are arrested at the G1-phase of the cell cycle[in vivo can be compensated by migration and enlargement of remaining CECs. However, loss of a significant number of CECs caused by injury[CECs ell cycle,4. Loss by injury,6 or inhby injury cannot bby injury, which rex vivo expanded CECs[The most effective way to treat these serious disorders in clinical settings at this moment is corneal transplantation, where endothelial failure is one of the most common indications. Howevernded CECs, or multnded CECs or cornended CECs and embrnded CECs.+-K+-ATPase[in vivo and in vitro and the small number of cells existing in vivo.Because cultured CECs have a limited proliferative and passaging ability, inductiK+-ATPase and N-caK+-ATPase, are useK+-ATPase–21  and used in the following analyses.CAGE  is a method to obtain transcriptome profiles by sequencing 5’-ends of capped RNAs, which identifies transcription starting sites (TSSs) and quantifies their activities. The FANTOM5 project surveyed TSSs in a large collection of primary cells, tissues, and cell lines, which consists of 975 human samples including more than 180 cell types. On the As shown in human samples were handled according to the tenets of the Declaration of Helsinki. Research-grade corneoscleral rims and whole eyeballs from cadaver human donors considered unsuitable for transplantation were procured from Sight Life .All A donor cornea was preserved and transported in Optisol-GS  at 4°C, and was used within four days from preservation. The age of donor was 58 years old.The corneoscleral rim was washed with phosphate-buffered saline (PBS) three times, then it was placed under the dissecting microscope  with the endothelium side up in a Petri dish, and the endothelium and Descemet's membrane were carefully dissected from the cornea along Schwalbe's line. The Descemet's membrane with its attached corneal endothelium was rapidly transferred into RNA later RNA Stabilization Reagent . Total RNA was extracted by Qiagen miRNeasy Mini Kit (QIAGEN Inc.) according to the manufacturer's protocol.To prepare RNA extracts from human ocular tissues, four whole eyeballs from two donors were preserved and transported in moist chamber at 4°C, and were used within five days from preservation. The ages of donors were 75 and 79 years old.The whole globes were first divided into the anterior segments and the posterior cups. Each tissue was carefully isolated using sterile forceps. After the endothelium and Descemet's membrane were peeled, the corneoscleral rims were punched out by using an 8.0-mm diameter trephine. Central corneal parts and limbal parts were treated with Dispase I  overnight at 4°C, and corneal epithelium and limbal epithelium were separated from stroma. All the isolated tissues were rapidly transferred into Isogen RNA extraction reagent . Total RNA was extracted using the Isogen RNA extraction kit according to the manufacturer's protocol.The RNA samples from non-ocular primary tissues were purchased . In addition to this panel, Human Kidney Total RNA  and Human Pancreas Total RNA  were also purchased.2. Total RNA was extracted using the Isogen RNA extraction kit. All the cells used for RNA extraction were harvested at full confluence during the first passage.To prepare samples for CEC culture, four donor corneas were preserved and transported in Optisol-GS at 4°C, and were used within 6 days from preservation. The ages of donors ranged from 14–25 years. Descemet's membrane with corneal endothelium was isolated as mentioned above. The isolated tissue was incubated for 1 hour at 37°C in a medium containing Dulbecco’s modified Eagle’s medium , 1.2 U/mL dispase II (Godo Shusei) and 1% Antibiotic-Antimycotic . Hereby, CECs were separated from Descemet’s membrane. After gentle centrifugation, the cells were suspended in culture medium containing DMEM, 50 U/mL penicillin, 50 μg/mL streptomycin, 10% fetal bovine serum , and 2 ng/mL basic fibroblast growth factor . The cells were incubated on dishes coated with cell attachment reagent  in an incubator at 37°C with humidified atmosphere of 10% CO(ACTB) as an internal control. The thermocycling program was performed as follows: an initial cycle at 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec, 60°C for 30 sec and 72°C for 30 sec. Data were obtained from duplicate experiments, and Ct values above 40 were regarded as not expressed.Total RNAs were inputted into reverse transcription-polymerase chain reaction with the SuperScript III First-Strand Synthesis System , and cDNA was used as a template for quantitative PCR. Quantitative PCR was performed using the ABI Prism 7500 Fast Sequence Detection System . SYBR Pre-mix Dimer Eraser  was used, and expression values were normalized to the housekeeping gene β-actin The list of primers used in this study is indicated in A donor cornea was preserved and transported in Optisol-GS at 4°C, and was used within 11 days from preservation. The age of donor was 27 years old.Fresh corneal tissues were embedded in optimal cutting temperature (OCT) compound and frozen sections were cut using a microtome-cryostat  in 10 μm. After drying for 30 minutes at room temperature, tissue sections were washed with Tris-buffered saline  3 times, and incubated with TBS containing 5% donkey serum and 0.3% Triton X-100 for 1 hour to block non-specific reactions. Each section was then incubated with primary antibodies listed in in vivo, we obtained RNA-seq data produced by Chen et al.[KRT3 and KRT12[We started our analyses from a collection of publicly available transcriptome data. As a resource of expressed genes in CECs en et al., which iand KRT12,33 to coand KRT12 that conand KRT12,35. The PPIP5K1, CLRN1, MRGPRX3, GLP1R) in adult CECs, three genes  in fetal CECs, and six genes  in both of the adult and fetal CECs . Our approach based on careful curation of CEC transcriptome data and the use of wide coverage of expression atlas generated by FANTOM5 as a background set enabled us to identify novel CEC marker candidates which rarely express other than CECs. Although the set of FANTOM5 transcriptome profiles represent the largest number of cell types assayed in a single project, there remain missing cell types and we cannot reject possibilities that the identified candidates may be active in those cells. Our follow-up experiments based on qRT-PCR for CECs and several tissues enabled us to select five markers by excluding eight genes active in the tissues, which demonstrate both of the power and the limitation in our approach using computational screening. Since the qRT-PCR experiments in in vivo. CLRN1 is known as the causative gene product of Usher syndrome type IIIa which causes deafness and visual impairment phenotypically similar to retinitis pigmentosa[To our knowledge, this is the first report of the presence of these proteins in CECs igmentosa. Its expigmentosa. MRGPRX3igmentosa,39, HTR1igmentosa, GRIP1 iigmentosa, and ZP4igmentosa in whichigmentosa. Most ofMRGPRX3 is highly and exclusively expressed in CECs, and this gene should be the first choice as a molecular marker to discriminate CECs from other cell types. It is noteworthy that MRGPRX3 is completely absent in corneal stroma which is adjacent to CECs and important to be discriminated because corneal stroma also originates from cranial neural crest as is the case for CECs. Interestingly, it is expressed at quite low level in heart, salivary gland and thymus that are all derived from neural crests.Based on the qRT-PCR results shown in ZP4 is evidently expressed only in CECs and not expressed in any other non-ocular tissues. Notably, the expression level of ZP4 in cultured CECs is higher than CECs in vivo. Similarly, expression level of HTR1D was also higher in cultured CECs than CECs in vivo. Cultured CECs are essential for the investigation of physiology and pathology of CECs, and these two markers should be more useful as markers of cultured CECs than other candidates. Intriguingly, furthermore, the RNA-seq data showed that these two are expressed more in fetal CECs than in adult CECs, which suggests that they are more highly expressed in undifferentiated state of CECs. These data indicate that ZP4 and HTR1D should be ideal markers for the discrimination of actual CECs during differentiation induced from multipotent stem cells.The FANTOM5 expression atlas and our qRT-PCR analysis indicate that CLRN1 was rarely expressed in other non-ocular tissues. Although CLRN1 was expressed in some other ocular tissues at RNA level, immunofluorescence staining analysis demonstrated its distinct expression pattern to CECs which enabled us to distinguish CECs from corneal stroma by itself. On the other hand, the remaining four genes were completely absent in corneal stroma according to the qRT-PCR analysis.Our analysis also indicated that SLC4A11, COL8A2 and CYYR1 could be useful to identify CECs. Cheong et al.[GPC4 and CD200. They analyzed the gene expression of CECs and corneal stroma by using RNA-seq, and identified genes that were highly expressed in CECs but lowly or not expressed in corneal stroma. Indeed, these markers could be useful to distinguish CECs from corneal stroma, but they were expressed in many other tissues according to the FANTOM5 expression atlas as shown in Previously, several marker candidates of CECs have been reported. Recently, Chng et al. reportedng et al. aimed toCLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers to discriminate CECs at the RNA and protein levels. Our results demonstrated their expression specificities, which would be applicable as CEC marker in particular for cell cultures consisting of unknown or mixtures of cell types. This is also the first report to examine the expression of these genes in vivo ocular tissues in depth, which may provide a clue to novel aspects of CEC functions and its transcriptional states. Given that the approach taken here is not very specific to CECs necessarily, our study sheds possibilities to explore novel molecular markers specific in other cell types, which is becoming highly important in clinical research.In conclusion, we identified five genes, S1 FigCECs: corneal endothelial cells, A-brain: adult brain, F-brain: fetal brain, SC: spinal cord, SG: salivary gland, BM: bone marrow, SM: skeletal muscle, A-liver: adult liver, F-liver: fetal liver, SI: small intestine. Y axis indicates % ACTB, and error bars represent standard deviation of technical duplicates.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.