Evolutionary analyses aimed at detecting the molecular signature of selection during crop domestication and/or improvement can be used to identify genes or genomic regions of likely agronomic importance. Here, we describe the DNA sequence-based characterization of a pool of candidate genes for crop-related traits in sunflower. These genes, which were identified based on homology to genes of known effect in other study systems, were initially sequenced from a panel of improved lines. All genes that exhibited a paucity of sequence diversity, consistent with the possible effects of selection during the evolution of cultivated sunflower, were then sequenced from a panel of wild sunflower accessions an outgroup. These data enabled formal tests for the effects of selection in shaping sequence diversity at these loci. When selection was detected, we further sequenced these genes from a panel of primitive landraces, thereby allowing us to investigate the likely timing of selection . We ultimately identified seven genes that exhibited the signature of positive selection during either domestication or improvement. Genetic mapping of a subset of these genes revealed co-localization between candidates for genes involved in the determination of flowering time, seed germination, plant growth/development, and branching and QTL that were previously identified for these traits in cultivated × wild sunflower mapping populations. To help avoid designing primers across splice sites, we used a tblastx-based intron finding Perl script (http://www.citrusgenome.ucr.edu/usa/ucr/Files.php) with Arabidopsis genome sequence information (v. 10) available from TAIR (http://www.arabidopsis.org/) to identify putative intron positions in sunflower unigenes. The resulting primer sequences can be found in To identify candidates for genes underlying traits related to sunflower domestication and improvement, we searched the literature for genes influencing relevant aspects of floral development and flowering time, seed/fruit development, seed germination, plant growth/development, and branching. We then performed BLAST searches of these genes against sunflower expressed sequence tags (ESTs) from the Compositae Genome Project EST Database , and 14 improved lines  in the improved panel were retained for sequencing in the wild panel and the outgroups, thereby enabling tests for selection; and (3) when a gene showed evidence for selection in the improved panel, it was also sequenced in the panel of primitive accessions. This last piece of data allowed us to further infer the likely timing of selection .The focus of this study was a collection of 28 ed lines . Achenes2, 125 µM of each dNTP, 0.2 µM reverse primer, 0.2 µM forward primer and 2 units of Taq polymerase. The PCR conditions involved a ‘touchdown’ protocol, as follows: 3 min at 95C; 10 cycles of 30 s at 94C, 30 s at 65C and 45 s at 72C, annealing temperature decreasing to 55C by 1C per cycle, followed by 30 cycles of 30 s at 94C, 30 s at 55C, 45 s at 72C, followed by 20 min at 72C. PCR products were checked for single-banded amplification via electrophoresis on 1% agarose gels. Amplification conditions were modified for loci that exhibited weak or non-specific amplification  by either decreasing  or increasing  the annealing temperature.PCR was performed in a total volume of 20 µL containing 5 ng of template DNA, 30 mM Tricine pH 8.4-KOH, 50 mM KCl, 2 mM MgClEscherichia coli , and screened for the presence of an insert. At least five positive colonies per individual were then sequenced as above except that vector primers [T7 and SP6] were used. Sequences have been deposited in the National Center for Biotechnology Information (NCBI) as BioProject PRJNA248055.PCR products were treated with 4 units Exonuclease I and 0.8 units Shrimp Alkaline Phosphatase (USB) at 37C for 45 min followed by enzyme denaturation at 80C for 15 min to prepare for sequencing using BigDye v3.1 (Applied Biosystems). The sequencing reactions were cleaned using Sephadex (Amersham) before being run on an ABI 3730xl (Applied Biosystems). In cases where direct sequencing results were unclear due to unresolvable heterozygous bases, indels, or short repeats, PCR products were TA-cloned into pGEM-T vectors (Promega), transformed into competent π), Watterson's θ), number of segregating indels, and the synonymous genetic distance from the outgroup for each gene. The distance (D), from the outgroup was determined by calculating the number of synonymous segregating sites in all pairwise comparisons among sequenced individuals within each panel and the outgroup and then averaging to obtain D . We also compared levels of genetic diversity amongst the three panels: wild, primitive (for those genes with preliminary evidence of selection), and improved. For these comparisons, S and the angular transformation of π and θ values were analyzed. For all genes that were sequenced in all three panels, two-factor ANOVAs (panel and locus) were performed using JMP version 9  and posterior Tukey-Kramer tests were used to test for significant differences amongst means.For all genes, sequences were aligned using Sequencher version 4.10 (GeneCodes), and FASTA files were generated for each. These FASTA files were then imported into DnaSP version 4.50.2 For the selection analyses, we used the maximum likelihood (ML) version We first confirmed that the collection of neutral genes utilized herein were not themselves under selection. To do this, we performed a “round-robin” test of all 11 putative neutral genes against each other to confirm selective neutrality . For each gene, this entailed using the ML-HKA test to compare two models, one model in which all 11 genes were assumed to be neutral, and one in which 10 genes are neutral and one is selected. This process was repeated with five different seed numbers for each of the 11 putatively neutral genes in each of the panels and none exhibited evidence of selection.Following confirmation that our control loci were indeed behaving in a neutral fashion, we performed the ML-HKA test for each of the candidate genes of interest. This initially involved comparing the levels of polymorphism and divergence in the wild panel and the improved panel. As noted above, when a gene showed significant evidence of positive selection in the improved lines, we also sequenced it in the primitive panel and tested for selection at that stage. All ML-HKA tests were performed with five different seed numbers and a chain length of 100,000 as recommended by the authors. The resulting maximum likelihoods were averaged across the five replicates and used to perform a likelihood-ratio test.http://sunflower.uga.edu/cmap/) to identify markers that flanked the QTL of interest  and were also present on the sunflower consensus map Candidate genes that demonstrated evidence of positive selection during domestication and/or improvement were screened for polymorphism in eight arbitrarily chosen individuals of one of two recombinant inbred line (RIL) mapping populations via Sanger sequencing: an improved x wild sunflower cross  were shown to be under selection in the wild. After sequencing these genes in the primitive landraces, we found that LATERAL SUPPRESSOR (LAS) showed evidence for selection in both the primitive and improved lines. Five other genes, including LOW PHOSPHATE ROOT (LPR), MORE AXILLARY GROWTH 2 (MAX2), PHENYLALANINE AMMONIA-LYASE 1 (PAL1), PHYTOCHROME B (PHYB), and RGA-LIKE 2 (RGL2), showed evidence of selection in the improved lines only. We thus conclude that LAS likely experienced selection during the initial phase of sunflower domestication, whereas selection on the remainder was likely restricted to the subsequent period of improvement. Note that a reliable PCR product was not obtained for ISOPENTENYLTRANSFERASE 5 in the primitive lines. We were thus unable to investigate the timing of selection for this gene.As expected, we observed an overall, progressive loss of genetic diversity in the primitive and improved sunflower lines as compared to their wild progenitor . Of the PHYB, is a photoperiod response gene that is thought to play a role in the transition from vegetative to reproductive growth . Though PHYB was found to have identical predicted protein sequences in the parents of an improved x wild sunflower mapping population that exhibits extensive variation in flowering time, this gene was consistently expressed at higher levels in the cultivar parent. We also found that PHYB co-localized with a previously identified QTL in an improved x wild sunflower mapping study , one during improvement (MAX2), and one with unknown timing (IPT5)  co-localized with a previously identified QTL for branching on LG 17.Like flowering time, plant architecture changed dramatically during the evolution of cultivated sunflower. Initially, selection for increased apical dominance is thought to have resulted in a complete loss of branching g (IPT5) . LAS is LPR and PAL1) showed evidence of positive selection during improvement. LPR is a multicopper oxidase affecting root growth/development in ArabidopsisPAL1 is a component of the phenylpropanoid pathway having broad effects on plant growth/development RGL2, which is a DELLA protein that represses germination in ArabidopsisRGL2 co-localized with a QTL for seed dormancy in an improved x wild sunflower cross RGL2 gene in particular, a promising target for functional studies involving seed dormancy and germination.Of the genes selected for analysis due to their potential role in other aspects of plant growth and development, two . Though early studies of linkage disequilibrium (LD) in sunflower found that it decayed relatively rapidly e.g., , more reWe would like to thank Eleni Bachlava and Jeff Roeder for assistance in the laboratory and Mark Chapman for help with the selection analyses. We would also like to thank two anonymous reviewers for comments on a previous version of this manuscript.Table S1List of candidate genes, reference taxa where the gene was identified, and functional traits of interest.(DOCX)Click here for additional data file.Table S2Primer sequences and information for candidate genes and control loci (second tab).(XLSX)Click here for additional data file.Table S3Statistics for candidate and neutral genes for wild, primitive, and improved individuals.(XLSX)Click here for additional data file.


The application of biochar to soil is considered to have the potential for long-term soil carbon sequestration, as well as for improving plant growth and suppressing soil pathogens. In our study we evaluated the effect of biochar on the plant growth of soybeans, as well as on the community composition of root-associated bacteria with plant growth promoting traits. Two types of biochar, namely, maize biochar (MBC), wood biochar (WBC), and hydrochar (HTC) were used for pot experiments to monitor plant growth. Soybean plants grown in soil amended with HTC char (2%) showed the best performance and were collected for isolation and further characterization of root-associated bacteria for multiple plant growth promoting traits. Only HTC char amendment resulted in a statistically significant increase in the root and shoot dry weight of soybeans. Interestingly, rhizosphere isolates from HTC char amended soil showed higher diversity than the rhizosphere isolates from the control soil. In addition, a higher proportion of isolates from HTC char amended soil compared with control soil was found to express plant growth promoting properties and showed antagonistic activity against one or more phytopathogenic fungi. Our study provided evidence that improved plant growth by biochar incorporation into soil results from the combination of a direct effect that is dependent on the type of char and a microbiome shift in root-associated beneficial bacteria. Glycine max L.; Tagoe et al., Phaseolus vulgaris; Rondon et al., Mimosa debilis) and open areas  in the Amazon region. However, despite numerous reports on microbial changes induced by biochar application in various cropping systems, there have been no studies on how biochar affects the diversity and physiological activity of plant growth stimulating rhizobacteria, especially in legumes.Biochar is a fine-grained charcoal that is rich in organic carbon, produced by pyrolysis or by heating biomass in a low oxygen environment and has been used worldwide as a soil amendment to increase soil fertility  as an important grain legume because it is a source of protein, oil, animal feed, and biodiesel in many countries worldwide, with an annual production of 276.4 Mio tIn our study, we focused on soybean −1; K content, 1.25 g (100 g soil)−1; and Mg content, 0.18 g (100 g soil−1).The soil used for pot experiments was from an experimental arable field under irrigation (V4) operated by the Experimental Field Station of Leibniz Centre for Agricultural Landscape Research (ZALF), Müncheberg, Germany. The selected chemical and physical properties of soil are as follows: clay and fine silt, 7%; coarse and medium silt, 19%; sand, 74%; Corg – 570 mg 100 gThe three types of char were supplied from the Leibniz-Institut for Agrartechnik Potsdam-Bornim e.V. (ATB) and used for pot experiments  were surface-sterilized using 10% v/v NaOCl for 5 min and 70% ethanol for 5 min, and then rinsed five times with sterile distilled water. Surface-sterilized seeds were transferred on paper tissue soaked in 0.5 mM CaSO4 and germinated for 5 days in a dark room at 25°C. The germinated seeds were transferred to pots containing 800 g of soil with four replicates. The treatments were control plants without biochar, soil amended with biochar  and HTC char at rates of 1, 2, and 3% (w/v). The plants were grown under greenhouse conditions  and were watered when necessary. After 6 weeks, the plants were harvested, the roots were separated from shoots and the dry weight was determined.The soil was mixed with crushed chars (particle size < 3 mm) at increasing rates of 1, 2, and 3% (w/v) just before planting pre-germinated soybean seeds. All pots were arranged in a randomized block design. The soybean seeds  were prepared, and 100 μl from appropriate dilutions was dispensed on Tryptic Soy Agar  for bacterial culture and Peptone dextrose agar  for fungal culture. The plates were incubated at 28°C for 2 days, and the total numbers of bacteria and fungi were counted. The colonies of bacteria that displayed differentiable colony morphologies were picked from plates and were re-streaked on fresh agar plates for purification. One hundred bacterial cultures were selected from each treatment and maintained at 4°C for further study.Among the biochar types under study, HTC char showed stimulatory effects on soybean plants in previous experiments and thus was used for further study. Three plants from each treatment, soil without biochar and soil amended with HTC char (2%) were collected for bacterial isolation. Excess soil was removed from the root by shaking, and only tightly adherent soil remained for study. The root samples (10 g for each treatment) were added to 100 ml of PBS buffer  supplemented with cycloheximide  at a final concentration of 100 μg ml 7 cells/ml. Germinated seeds were placed in the bacterial suspension with sterile forceps and shaken gently. After approximately 10 min, the inoculated seeds were aseptically planted into the potting soil. Three seeds were sown per plastic pot  to a depth of approximately 1.5 cm. After germination, plants were thinned to one per pot, and pots were set-up in a randomized design with six replications. The plants were grown under greenhouse conditions  for 1 month. At harvest, the plants were removed from the pots, and the dry weights of roots and shoots were determined. A total 32 strains from control soil and 43 strains from HTC char amended soil were selected based on their plant growth promoting abilities and were further identified and characterized.To test whether bacterial isolates were capable of stimulating plant growth, a pot experiment was conducted in the greenhouse using loamy sand. The seeds were surface-sterilized and inoculated with bacterial strains as described above. The sterility of the seeds was previously tested on TSA agar by incubating the plates for 3 days at 28°C. No contaminants were found, indicating that the surface-sterilization was effective. Two hundred bacterial strains isolated from the rhizosphere of soybeans were grown overnight in Tryptic Soy Broth (TSB), and one milliliter of each culture was pelleted by centrifugation ; the supernatant was discarded. Non-inoculated plants were used as negative controls. Cell pellets were washed with 1 ml of PBS and re-suspended in PBS. Cell suspensions corresponded to a cell density of 10The identification of isolated strains was performed using whole-cell matrix assisted laser desorption/ionization (MALDI)–time of flight (TOF) mass spectrometry. Sample preparation was carried out according to the ethanol/formic acid extraction protocol recommended by Bruker Daltonics . The isolates were cultured on tryptic soy agar  for 24 h, and approximately 10 mg of cell mass was suspended in 300 μL of water and vortexed to generate a homogenous suspension. The suspension was mixed with 900 μL of ethanol and centrifuged. The pellet was resuspended in 50 μL of 70% formic acid and subsequently carefully mixed with 50 μL of acetonitrile. After centrifugation, aliquots of 1 μL of supernatant were placed immediately on spots of a MALDI target. Each spot was allowed to dry and subsequently overlaid with 1 μL of matrix (α-ciano-4-hydroxycinnamic acid in 50% aqueous acetonitrile containing 2.5% trifluoroacetic acid). Mass spectra were acquired using a MALDI-TOF MS spectrometer in a linear positive mode  in a mass range of 2–20 kDa. A bacterial test standard  was used for instrument calibration. The raw spectra were imported into MALDI Biotyper™ software  and then processed and analyzed using standard pattern matching against the reference spectra in the MALDI Biotyper™ reference database .7cells/ml) was added to 10 ml of TSB amended with tryptophan (100 μg/ml). After 3 days of cultivation, 2 ml aliquots of bacterial cultures were centrifuged at 13.000 × g for 10 min. One milliliter of supernatant was transferred to a fresh tube to which 100 μg/ml of 10 mM orthophosphoric acid and 2 ml of reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) were added. After 25 min, the absorbance of the developed pink color was read at 530 nm. The IAA concentration in culture was calculated using a calibration curve of pure IAA as a standard.Production of IAA (indole 3-acetic acid) was determined as described by Bano and Musarrat . BrieflyThe phosphate-solubilizing activity of the bacterial strains was determined on Pikovskaya agar . A sterilized filter paper saturated with a 1% solution of picric acid and 2% sodium carbonate was placed in the upper lid of the Petri plate. The Petri plate was sealed with Parafilm® M and incubated at 28°C for 3 days. The change in the paper color from yellow to dark blue was recorded as an index of HCN production Castric, .in vitro for their antagonistic activities against the pathogenic fungi Fusarium solani, F. culmorum, F. graminearum, Alternaria infectoria, and A. teniussima. The bacterial isolates were grown in TSB broth for 3 days and 50 μL of bacterial culture was dropped into the hole of a PDA plate (4 mm in diameter). Fungal strains were grown in peptone dextrose agar (PDA) plates at 28°C for 5 days, and disks of fresh fungal culture (5 mm diameter) were cut out and placed 2 cm from the hole filled with bacterial filtrate. The plates were sealed with Parafilm® M and incubated at 28°C in darkness until the fungi had grown over the control plates without bacteria. Antifungal activity was recorded as the width of the zone of growth inhibition between the fungus and the test bacterium.The bacterial isolates were tested t-test. Mean comparisons were conducted using a least significant difference (LSD) test (P = 0.05).Data were tested for statistical significance using the analysis of variance package included in Microsoft Excel 2007. Comparisons were performed using Student's The response of the soybeans to the type of biochar and to different concentrations was investigated under greenhouse conditions. Our study showed that shoot and root biomass of soybeans were not significantly affected by either MBC or WBC amendments in all concentrations  and 5.3 × 107 CFU (per gram fresh weight) in soil with 2% HTC char. Furthermore, a remarkably greater number of fungi (1.8 × 104 CFU per gram fresh weight) were observed in the rhizosphere of the plants grown in soil without biochar compared with the plants grown in soil amended with 2% HTC char (0.9 × 104 CFU per gram fresh weight).The results of the pot experiments showed that HTC char at a concentration of 2% stimulated the growth of soybeans and thus was used for the characterization of root-associated plant growth promoting bacteria. The bacteria were enumerated after 48 h in the plate count agar and fungi after 5 days in PDA medium. The total numbers of cultivable bacteria isolated from the rhizosphere of plants grown in soil without biochar were 1.5 × 10In total, 200 bacterial strains were isolated from the rhizosphere of soybeans. Among these, 90 isolates were selected from plants grown in control soil, and 110 isolates, from plants grown in HTC char amended soil. All strains were tested for their abilities to stimulate root and shoot growth of soybeans under greenhouse conditions in loamy sand soil. Root and shoot growth stimulating abilities (>20%) were observed in 27–32% of isolates from plants grown in soil without biochar and in 45–57% of isolates from soil amended with 2% HTC char, respectively. A total of 32 isolates from the control plants and 43 isolates from the HTC amended soil induced stimulatory effects on plant growth compared with the non-treated control plants.Bacillus was the predominant genus, which was followed by the genera Arthrobacter and Rhizobium. Furthermore, isolates affiliated with the genera Cellulosimicrobium, Enterobacter and Pseudomonas were also found. The most abundant species were identified as Rhizobium radiobacter , followed by the species Arthrobacter globiformis , Bacillus megaterium , Cellulosimicrobium cellulans , Enterobacter asburiae , and Pseudomonas chlororaphis . Only one isolate was identified as Burkholderia terricola , Ochrobactrum intermedium , Pseudoxanthomonas kaohsiungensis , and Stenotrophomonas sp. . Members of the genera Achromobacter, Brevibacillus, Chryseobacterium, Microbacterium, Ochrobactrum, Paenibacillus, Pseudoxanthomonas, Sphingobacterium and Stenotrophomonas were not found among isolates from the control soil.A total of 13 bacterial genera were isolated from the rhizosphere of soybeans grown in HTC char amended soil, whereas 12 isolates were identified at the species level Table . The isoAll bacterial strains isolated from the rhizosphere of soybeans grown in HTC char amended soil and without biochar were screened for multiple plant growth promoting traits. Most of the bacterial isolates exhibited one or more plant growth-promoting activities Tables .Arthrobacter  and Bacillus . Three isolates belonging to the genus Stenotrophomonas  from HTC char amended soil showed IAA activity, followed by the genera Cellulosimicrobium , Pseudomonas  and Rhizobium .The production of the phytohormone IAA by bacterial isolates is shown in Table Fusarium solani, F. culmorum, F. graminearum, Alternaria infectoria, and A. teniussima. The proportions of isolates with antagonistic activity to one or more pathogens was higher for the HTC char amended soil (51%) than for the control soil (28%). As shown in Table Pseudomonas chlororaphis strains, C27 and C28, from control soil and six isolates, Stenotrophomonas maltophilia H66, Stenotrophomonas sp. H92, Cellulosimicrobium cellulans H90, Bacillus megaterium H82, Paenibacillus polymyxa C44, and Pseudomonas putida 73, from HTC char amended soil exerted the highest inhibition of mycelial growth of the genus Fusarium.Positive P-solubilization was observed in 7 strains from 4 genera (20%) originating from plants grown in control soil and 16 strains from 11 genera (37%) originating from HTC char amended soil. All bacterial isolates were screened for their ability to suppress plant pathogenic fungi, such as in vitro, eight isolates  originating from HTC char amended soil and six isolates  originating from control soil were selected for plant growth stimulation under greenhouse conditions.The ability of isolates to produce cell wall degrading enzymes, as well as proteases and lipases, was also determined. The isolates from the HTC char amended soil exhibited a higher proportion of enzyme producers than the control soil, where lipase, protease, pectinase and cellulase activity were detected in 14, 33, 40, and 26% of the isolates, respectively. The percentage of enzyme producing bacteria isolated from control soil was lower, where only 6% of isolates exhibited lipase, 11% protease, 20% pectinase, and 29% cellulase activity. Out of isolates that exhibited plant growth-promoting activities Pseudomonas chlororaphis (C28) and the shoot dry weight increased up to 44% with Enterobacter asburiae , Pseudomonas putida (H73), Stenotrophomonas maltophilia (H66) and Stenotrophomonas sp. (H75) showed significantly higher plant growth stimulation, from 40 to 63%  after amendment with biochar from citrus wood. In our study, the analysis of cultivable root associated bacteria demonstrated that HTC char amendments increased bacterial populations in the rhizosphere of soybeans compared with control plants, whereas fungal growth was decreased over the control, in agreement with Chen et al.  is able to form IAA, solubilize phosphate and stimulated growth of chili plants , cucumber (Cucumis sativus), and tomato  was observed after HTC char addition. The increased proportion of bacteria capable of inhibiting fungal pathogens following amendment of HTC char suggests that the observed suppression of the fungal population was due to antagonistic interactions of microbes. The phytohormone IAA is a naturally occurring auxin which has a major role in the regulation of plant growth. The stimulation of the growth of various plants by inoculation with PGPR and IAA producing ability is well-documented (Egamberdieva, in vitro such as phytohormone production and suppression of fungal pathogens. Thus, we provided evidence that improved plant growth by hydrochar incorporation into soil is mostly an indirect rather than a direct effect that depends on the type of char and the activity of plant-associated beneficial soil bacteria. The stimulation of certain plant-beneficial bacteria by biochar also suggests the possibility of developing combined approaches of biochar treatment and biological control solutions (Berg et al., From our study, we conclude that increased plant growth in response to soil amendment with biochar is based on the type of char, i.e., HTC application increased growth of soybean but not in case of either MBC or WBC. Moreover, HTC application was shown to alter the community composition of root associated microbes exhibiting plant growth-promoting activities DE, SW, and GB did experimental design work. DE and UB conducted experiments. EA analyzed the data. DE, SW, and GB wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pig, which shares greater similarities with human than with mouse, is important for agriculture and for studying human diseases. However, similarities in the genetic architecture and molecular regulations underlying phenotypic variations in humans and swine have not been systematically assessed.PLAG1 as a causal gene for limb bone length. This finding is consistent with GWAS findings for human height and supports the common genetic architecture between swine and humans. By leveraging a human protein-protein interaction network, we identified two putative candidate causal genes TGFB3 and DAB2IP and the known regulators MESP1 and MESP2 as responsible for the variation in rib number and identified the potential underlying molecular mechanisms. In mice, knockout of Tgfb3 and Tgfb2 together decreases rib number.We systematically surveyed ~500 F2 pigs genetically and phenotypically. By comparing candidates for anemia traits identified in swine genome-wide SNP association and human genome-wide association studies (GWAS), we showed that both sets of candidates are related to the biological process “cellular lipid metabolism” in liver. Human height is a complex heritable trait; by integrating genome-wide SNP data and human adipose Bayesian causal network, which closely represents bone transcriptional regulations, we identified Our findings show that integrative network analyses reveal causal regulators underlying the genetic association of complex traits in swine and that these causal regulators have similar effects in humans. Thus, swine are a potentially good animal model for studying some complex human traits that are not under intense selection.The online version of this article (doi:10.1186/s12864-015-1240-y) contains supplementary material, which is available to authorized users. BRCA1-associated mammary carcinogenesis. Genetic studies of pig models of human diseases led to the identification of novel quantitative trait loci for cutaneous melanoma and a novel mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia [RACK1 as a potential marker of malignancy for human melanocytic proliferation [The pig is one of the most important agriculture animals, which has provided the largest amount of consumable red meat protein. The pig is also a valuable model for studying human diseases because pigs are more similar in genomic structure to human than mice are . Pigs arerolemia . Gene exferation . The pigHowever, the genetic similarity between pig and human has not been assessed in large, systematic studies. Thus, pigs may be underused as models for human diseases. Here we surveyed about 500 F2 animals in a swine cross using a high density 60 K SNP array and examined phenotypic traits of interests to both agriculture and human diseases, including anemia traits, limb bone length, and number of ribs.Integrated network or systems biology approaches, which combine genetic, genomic, and phenotypic data into network views, have been applied to understand obesity ,5, cancePLAG1 for limb bone length (corresponding to human height). We then integrated genetic association result and human protein-protein interaction network to identify two novel candidate causal genes TGFB3 and DAB2IP as well as the known regulators MESP1 and MESP2 as responsible for the variation in rib number and illustrated the potential underlying molecular mechanisms. Tgfb3 knockout together with Tgfb2 in mice decreases number of ribs, which supports TGFB3 as a regulator for rib number in pig.By applying integrated analysis on phenotypic traits and genotype data, we show that both swine and human genome wide association candidates for anemia traits are related to lipid metabolism in liver. By integrating phenotype and genotype data with human adipose Bayesian causal network, we identified A large-scale F2 intercross comprising 1,912 pigs was constructed by crossing the western breed White Duroc and the Chinese breed Erhualian . These bP = 4.85 × 10−6), there were 12, 5, and 2 quantitative trait loci (QTLs) for anemia, bone length, and rib number traits, respectively. Then, we applied systems biology approach to identify potential causal genes underlying QTLs of phenotype traits.High density SNP genotypes for 497 F2 animals were successfully generated. Of 52,183 SNPs , we used MCH as the representative of anemia traits in the downstream analysis. We previously identified a significant QTL for MCH at day 240 in a 3-cM region on SSC8 using 194 microsatellite markers [KIT, a finding which suggests KIT is the causal gene for MCH at locus SSC8:43.43 Mb. KIT regulatory mutations, including a gene duplication and a splice mutation that leads to the skipping of exon 17, are responsible for the dominant white phenotype in pigs [ACO1, which encodes aconitase 1. Also known as iron regulatory element binding protein 1 (IREB1), ACO1 regulates cellular iron homeostasis and is linked to anemia in human [Hematopoietic disorders are associated with a variety of human diseases such as coronary heart disease, diabetes, and liver diseases. One founder breed of our F2 resource population, the White Duroc pig, has the dominant coat color and lower hemoglobin concentration, an indicator of macrocytic anemia . Two blo markers . In the  in pigs . These r in pigs . The secKIT, was present in the candidate sets from human GWAS and from our swine cross. Bone marrow, kidney, and liver are important tissues for red blood cell production and homeostasis. Instead of directly comparing the two candidate sets from swine and human, we examined how these genes are regulated in a human liver transcriptional network  while subnetworks around human GWAS candidates were enriched in the GO biological processes immune response and lipid metabolism  . The enrichment of multiple GO biological processes in the human GWAS candidates subnetwork explains why human GWAS candidates were not significantly co-regulated in the human liver network as a whole . The swine candidate subnetwork and human candidate subnetwork overlapped significantly , and genes in the GO biological process cellular lipid metabolism were even more enriched when considering the two subnetworks together   and 5 genes , respectively. Leg length is generally proportional to height. Our results are similar to those of GWAS of human height. The syntenic regions on human genome of the two loci we identified in pigs matched perfectly with two human loci (8q12 and 6p21) associated with human height in GWAS [HMGA1 (at SSC7:35.18 Mb for pig and 6p21 for human) has been suggested as the causal gene for height, possibly through a mechanism involving modification of chromatin structure [Two loci were strongly associated with limb bone length . Osteoblasts and adipocytes are very close in cell lineage [LYN and one on PLAG1. Only PLAG1 subnetwork overlapped with the height-related OMIM gene subnetwork: 4 of 10 nodes were in the OMIM subnetwork  and is centered on marker ALGA0044022 at 105.38 Mb , and C14orf118. The chromosome 1 locus (association P= 3.56 × 10−11) is centered on the marker DRGA0002465 at 293.93 Mb. The flanking region (200 kb on each side of the marker) contains one gene, DAB2IP.The chromosome 7 locus had the strongest association with rib number . In the early development of vertebrate embryos, the thoracic spine forms during somitogenesis, a process controlled by segmentation clock, whose key regulators include Notch, Wnt, and FGF [TGFB3 and DAB2IP interact with genes in the Wnt signaling pathway in the PPI subnetwork  are not identified in a specific physiological state and thus may capture interactions or regulations during embryonic development. We therefore collected PPIs from multiple sources . The PPI and FGF  , there was one locus whose genotype and interaction with the TGFB3 locus was associated with the residual variance (P= 9.8 × 10−6)  Figure . The flaogenesis , during  pathway . Thus, MHMGA1 and PLAG1 are candidate genes for bone length, which corresponds to human height.This study shows that causal genes for traits of interests to both agriculture and human diseases can be identified by combining high-density SNP genotyping and Systems Biology approaches. By comparing pig and human candidate genes from GWAS for phenotypic traits related to anemia, bone length , and rib number, we identified putative causal genes and uncovered the mechanisms for these phenotypes. Specifically, we showed that both human and pig causal genes for anemia are related to lipid metabolism in liver. We confirmed that In genomic structure  and physEven though there are 112 naturally occurring disease-causing mutations common to humans and pigs , a largeTGFB3 and DAB2IP as well as the known genes MESP1 and MESP2. Using network approaches, we not only identified candidate genes, but also further dissected the mechanisms that mediate their effects on the phenotype. Abnormal rib number is associated with at least 19 different human diseases and syndromes , such as Herrmann-Opitz craniosynostosis, spondylocostal dysostosis, and Campomelia Cumming type. In humans, one extra rib in human can increase cancer risks 120-fold [For the rib number trait, we identified candidate genes in the Wnt and Notch pathways, which are known to influence rib development including 120-fold . Our netNR6A1 has been implicated as the causal gene for vertebrae number at the chromosome 1 locus [NR6A1 SNP and those around NR6A1 on the 60 K chip were similar  were 90% identical. In addition, the NR6A1 SNP and the neighboring SNPs on the 60 K SNP array had similar association results, and the NR6A1 SNP explained less variation in rib number than DRGA0002465  until 120 days of age. Piglets were then transferred to a performance test station, where growth and feeding traits were recorded with an ACEMA 64 electronic auto-feed intake recording station, which phenotypes feeding behavior and food intake circadianly . At a mean age of 240 ± 3 days, the pigs were sent to a commercial slaughter facility and processed according to Chinese industry standards. All animal work was conducted in accordance with the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China.A large-scale F2 resource population was constructed by crossing two divergent pig breeds, White Duroc and Chinese Erhualian, as described previously . BrieflyPhenotypic values of growth, fertility, carcass, meat quality, immune capacity, behavior and morphological traits were recorded in this F2 pig populations. At the time of slaughter, blood samples were collected from the major artery vessels near the heart and morphological traits (including the number of ribs and the limb bone length) and blood biochemical traits were measured. Rib number was counted from the carcass. Both forelimb and hind limb were removed from the right side carcass of F2 animals and dissected from the limb. A large caliper was used to measure the length of five limb bones: humerus , scapula , femur , ulna (length from the olecranon process to the styloid process) and tibia .−6) and X-linked SNPs likely to be autosomal (odds > 1000) were excluded from further analysis. The associations between phenotypes and genome-wide SNP genotype data were analyzed with the single-marker association model in the GenABEL package [mmscore function of GenABEL in R package. Sex and batch were considered as fixed effects, and genetic co-variances among samples were considered by fitting a kinship matrix derived from genotypes of whole-genome SNP markers as y ∼ sex + batch + g + G , where y is a trait, G is kinship estimated based on genome-wide SNP data, and g is the genotype at a query SNP marker.502 F2 animals were successfully genotyped with a 60 K pig SNP chip (Illumina)  and an i package  based onThe human liver transcriptional network is the union of molecular Bayesian causal networks constructed from two human genetic gene expression studies ,64. In op(cis − > trans) = 1, but genes with trans eQTLs were not allowed to be parents of genes with cis eQTLs, p(trans − > cis) = 0. Bayesian Information Criterion (BIC) was used in the reconstruction process. One thousand Bayesian networks were reconstructed using different random seeds to start the reconstruction process. From the resulting set of 1000 networks generated by this process, edges that appeared in more than 30% of the networks were used to define a consensus network. In this consensus network, an edge was removed if 1) it was involved in a loop, and 2) it was the most weakly supported of all edges making up the loop.The selected genes were input into a Bayesian network reconstruction software package, RIMBANet-Reconstructing Integrative Molecular Bayesian Network , based oThe final human liver transcription network used in the anemia trait analysis is the union of the two Bayesian networks constructed based on the two data sets, which consists of 12,875 genes and 64,253 connections.No large coherent set of bone expression data was available for building a human bone transcription network, so we used networks of tissues that are molecularly similar to bone. Adipocytes, osteoblasts, and chondrocytes share common progenitors and are close in lineage , and adiThe human PPI network was constructed by integrating human PPIs from several molecular interaction databases, both public  and commercial . Identifiers for the interacting genes identified in these databases were mapped to Entrez Gene IDs to obtain a unified naming system. Both directed regulations  and undirected interactions  in these databases were mapped to undirected edges in our PPI network, which consisted of 19,800 nodes and 65,056 interactions.To construct a subnetwork for a set of genes, we used genes in the input set as seeds and selected d-step neighbors of seeds . When fewer than 50 seeds were used, d was set as 2; when more than 50 seeds were used, d was set as 1. Seeds and their d-step neighbors of seeds were nodes in the subnetwork, and links among nodes in the subnetworks were the same as in the whole Bayesian network.http://www.genome.gov/gwastudies/), which covers 3 studies [−8. Twenty-two unique loci and genes mapped to these loci were retrieved and listed in Additional file Human GWAS candidates for traits mean corpuscular hemoglobin (MCH) / mean corpuscular volume (MCV) were retrieved from the NHGRI human GWAS catalog   and comp


Objective. To elucidate the optimum preoperative biliary drainage method for patients with pancreatic cancer treated with neoadjuvant chemotherapy (NAC). Material and Methods. From January 2010 through December 2014, 20 patients with borderline resectable pancreatic cancer underwent preoperative biliary drainage and NAC with a plastic or metallic stent and received NAC at Hiroshima University Hospital. We retrospectively analyzed delayed NAC and complication rates due to biliary drainage, effect of stent type on perioperative factors, and hospitalization costs from diagnosis to surgery. Results. There were 11 cases of preoperative biliary drainage with plastic stents and nine metallic stents. The median age was 64.5 years; delayed NAC occurred in 9 cases with plastic stent and 1 case with metallic stent (p = 0.01). The complication rates due to biliary drainage were 0% (0/9) with metallic stents and 72.7% (8/11) with plastic stents (p = 0.01). Cumulative rates of complications determined with the Kaplan-Meier method on day 90 were 60% with plastic stents and 0% with metallic stents . There were no significant differences between group in perioperative factors or hospitalization costs from diagnosis to surgery. Conclusions. Metallic stent implantation may be effective for preoperative biliary drainage for pancreatic cancer treated with NAC.  Radical surgery is the treatment of choice for pancreatic cancer. However, since pancreatic cancer is already at an advanced stage at the time of diagnosis in many cases, surgery may not be possible, and it tends to have a poor prognosis . CombineThe metallic stent (MS) is the standard stent used in biliary drainage for bile duct obstruction due to pancreatic head cancer in cases of unresectable pancreatic cancer since it has a longer patency period and is associated with fewer complications than the plastic stent (PS) . HoweverThus, the purpose of the present study was to elucidate the optimum drainage technique for PBD performed in cases of pancreatic cancer treated with NAC.The subjects were 20 patients with BR cancer who underwent NAC with PBD at Hiroshima University Hospital between January 2010 and December 2014. Based on the drainage procedures performed, patients were divided into a plastic stent (PS) group and a metallic stent (MS) group. We then investigated delayed NAC and the rate of complications due to the use of bile duct drainage ; the effect of stent type on perioperative factors  and postoperative complications; and the cost of hospitalization from diagnosis to surgery (excluding outpatient chemotherapy). This study received approval from the Hiroshima University Hospital Ethics Committee.Prior to bile duct drainage, all subjects underwent imaging with multidetector row computed tomography (MDCT) and cancer staging. To obtain histological confirmation of pancreatic carcinoma, brushing cytology of the pancreatic duct stricture and pancreatic juice cytology were performed at the time of biliary drainage. When histological confirmation was not possible via endoscopic retrograde cholangiopancreatography, we performed endoscopic ultrasound fine-needle aspiration (EUS-FNA). Histological confirmation was obtained for all subjects prior to NAC.As a rule, endoscopic nasobiliary drainage was the first drainage procedure performed before histological confirmation was obtained. After histological confirmation was obtained, either PS (the end of May 2013) or MS (as of June 2013) was inserted. In the PS group, 7 Fr, 7 cm straight type biliary drainage stent  was placed. In the MS group, 10 mm, 6 cm fully covered metallic stent  was placed . Two subAccording to the NCCN guideline, BR tumors are defined by tumor abutment (≤180° or ≤50% of the vessel circumference) of the superior mesenteric artery (SMA) or celiac axis, short segment abutment or encasement (>180° or >50% of the vessel circumference) of the common hepatic artery  that is amenable to segmental resection and primary repair, or segmental venous occlusion with an adequate superior mesenteric vein (SMV) below and portal vein (PV) above the area of tumor-induced occlusion to allow for interpositional grafting , 20 Fig.2 was administered on days 1 and 8, while S-1 80 mg/body (<1.25 m2/body surface area) or 100 mg/body (≥1.25 m2/body surface area) was administered on days 1–14. After the three cycles were completed, MDCT was performed. Surgery was performed on cases that were classified as showing stable disease (SD) or a better response according to the Response Evaluation Criteria in Solid Tumors (RECIST).We administered three cycles of gemcitabine and S-1 combination therapy (GS therapy) for BR pancreatic cancer. Gemcitabine 1000 mg/mIn all cases, there was a 3-week waiting period between the completion of NAC and surgery.Based on previous reports on PBD with NAC, we assumed that the onset of complications caused by bile drainage would be 65% for PS and 10% for MS. Therefore, we set the alpha error at 0.05 and detectability at 80%, which resulted in nine required subjects per group .U test and the chi square test. Statistical significance was set at p < 0.05.Statistical analysis was performed with the Mann-Whitney p = 0.66). There were no significant differences between groups in any other patient background factor . Of these, one subject in the PS group and one subject in the MS group experienced delayed NAC due to bone marrow suppression. Significantly fewer patients in the MS group versus the PS group experienced delayed NAC due to PBD .Our investigation of delayed NAC due to all types of complications indicated that nine subjects in the PS group and one subject in the MS group experienced delayed NAC  and 72.7% in the PS group (8/11). Specifically, occlusion occurred five times, migration occurred two times, and retrograde cholangitis occurred one time , respectively; the operative time was 364 minutes and 469 minutes (p = 0.24), respectively; and the length of postoperative hospitalization was 18 days and 21 days (p = 0.13), respectively. There were two cases of postoperative complications in the PS group (18%): specifically, one case of hepatic necrosis and one case of cholangitis. There were three cases of postoperatibe complications in the MS group (29%): specifically, one case of intra-abdominal abscess, one case of cholangitis, and one case of intestinal necrosis (p = 0.61), indicating that there was no significant difference in perioperative factors (Blood loss in the MS and PS groups was 960 mL and 1450 mL ( factors .USD) was 1,3650 in the PS group and USD 1,0580 in the MS group, indicating no significant difference between the groups  on surgery and since the per-stent cost is high, PS are generally used in PBD procedures. Recent studies have reported on cases in which neoadjuvant therapy was administered to patients with BR to improve the long-term outcome of pancreatic cancer. If the period until surgery is delayed as a result of neoadjuvant therapy, there is an increased frequency of procedural accidents related to bile duct drainage, such as stent occlusion and cholangitis, which leads to the clinical problem of an increase in the number of cases in which surgery and neoadjuvant therapy must be delayed . Mullen The effect of MS use in the perioperative phase of PBD procedures has not been elucidated and few studies have reported on its effect in surgery , 25, 26.There have been almost no studies of the one drawback associated with the use of MS in PBD procedures: its cost. In their investigation of the cost from the start of PBD until surgery, Kubota et al. reported that the use of PS in PBD procedures cost USD 11,545 and that the use of MS cost USD 11,773, indicating no significant difference between the two . A simulFinally, we obtained extremely interesting results from our investigation of the R0 resection rate. When NAC was performed at our facility, the R0 resection rate was 44.4% when PS was used and 85.7% when MS was used. Several studies reported that the R0 resection rate improves after neoadjuvant therapy , 28, 29.Because this study was limited by its retrospective and nonrandomized design in a single facility, future multi-institution and prospective randomized controlled trials will be necessary.In conclusion, this study elucidated the fact that since the use of MS in PBD procedures does not lead to increased perioperative procedural accidents and significantly decreases the onset of complications that occur in association with bile duct drainage during chemotherapy, it can contribute to safer treatment. Although its per-unit cost is high, its reintervention rate is low. This suggests that, compared to PS, MS use may not be disadvantageous in terms of medical cost. This suggests the superiority of MS as the first choice in PBD performed in conjunction with NAC.Metallic stent implantation may be effective for preoperative biliary drainage for pancreatic cancer treated with NAC.


Camellia japonica is a traditional garden plant, and its flower and seed have been used as a base oil of traditional cosmetics in East Asia. The present study was carried out to evaluate the effect of C. japonica fruit extracts (CJF) in a high fat diet- (HFD-) induced hypercholesterolemic rat model. CJF was administered orally at three different doses: 100, 400, and 800 mg·kg−1·day−1 . Our results showed that CJF possessed strong cholesterol-lowering potency as indicated by the decrease in serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL), accompanied by an increase in serum high-density lipoprotein (HDL). Furthermore, CJF reduced serum lipid peroxidation by suppressing the formation of thiobarbituric acid reactive substance. In addition, oil red O (ORO) staining of rat arteries showed decreased lipid-positive staining in the CJF-treated groups compared to the control HFD group. Taken together, these results suggest that CJF could be a potent herbal therapeutic option and source of a functional food for the prevention and treatment of atherosclerosis and other diseases associated with hypercholesterolemia.Hypercholesterolemia is a well-known etiological factor for cardiovascular disease and a common symptom of most types of metabolic disorders. A high blood level of cholesterol, known as hypercholesterolemia, accelerates the oxidation of serum lipids and is known to contribute to the disruption of the circulatory systems homeostasis by a variety of chemical and physical processes. Hypercholesterolemia is generated by multiple factors such as unhealthy dietary practices, obesity, genetic disposition, and insufficient daily exercise. Previous studies have already highlighted the prevalence of hypercholesterolemia that could ultimately affect the majority of the adult population of developed countries . ParticuFurthermore, various clinical studies have shown strong correlations between elevated circulating triglyceride (TG), total cholesterol (TC), and reduced HDL levels as major predictors of obesity, diabetes, and hyperlipidemia , 7. The Camellia japonica is a popular ornamental plant that usually appears as a colony in wild environmental conditions. Geographically, it is mainly distributed in East Asia and China [ C. japonica have various biological activities such as antihuman immunodeficiency virus (HIV) activity [ in vitro and ex vivo data suggested that the ethanol extract of C. japonica fruit (CJF) showed beneficial effects on the cardiovascular system [nd China . A previactivity , antioxiactivity , antiphoactivity , and hypactivity . Howeverr system . These er system .However, the biological effects of CJF on the cardiovascular physiology are still unclear. Hyperlipidemia and obesity are known as the main causes of atherogenesis , 20. The C. japonica plant parts used in this study were collected from the southern parts of the Korean Peninsula, and a voucher specimen was deposited at the Herbarium of the Jeonnam Forest Resources Research Institute, Korea. The C. japonica fruit extract (CJF) was prepared as previously described [ C. japonica was extracted twice with 70% ethanol for 4 h, and then this crude extract was dried by using a rotatory evaporator under vacuum to obtain the final extract at a yield of 93.4 g . For daily administration of CJF during the experimental period, various doses were freshly prepared with sterile water.The differentescribed . Brieflyn = 8). The normal and control groups were fed a normal and 60% HFD diet , respectively, while the CJF 100, CJF 400, and CJF 800 groups were given 100, 400, and 800 mg·kg−1·day−1, respectively, in a HFD for 1 month. The formula with the diet ingredients and their energy values are shown in Tables All the experimental procedures were conducted in accordance with the animal care guidelines and were approved by the relevant Committee of the Mokpo National University. Male Wistar rats (6-week-old) were purchased from Orient Bio  and were acclimatized for 1 week with free access to water and standard rodent chow under a 12 h light-dark cycle. Then, the rats were divided into five groups . Briefly, 300 μL of each of the serum and separating solution was mixed and centrifuged at 3000 rpm for 10 min, and then 100 μL of each of the supernatant and the HDL standard solution was mixed with 3 mL of enzyme solution. Then, the absorbance values of all the resultant colored products from the TC, TG, and HDL assays were measured within 1 h using the Epoch microplate spectrophotometer at a wavelength of 500 nm. The serum LDL level was calculated according to the Friedewald formula [The rat serum TC and TG levels were analyzed by using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer's protocol. Briefly, 20  formula .μL of 10 μM copper sulfate (CuSO4) and 0.6 mL phosphate-buffered saline (PBS) for 4 h at 37°C. Then, 0.3 mL of this mixture was mixed with 1 and 3 mL of freshly prepared 0.67% TBA and 0.05 N hydrochloric acid (HCl), respectively. The resulting mixture was heated in boiling water for 30 min, and then the test tubes were collected and placed on ice for 5 min to stop the reaction. After cooling, the resulting chromogen was extracted with 4 mL of a mixture of 85% butanol and 15% methanol. The organic phase was separated by centrifugation at 3000 rpm for 10 min, and the absorbance was recorded at a wavelength of 540 nm using the Epoch microplate spectrophotometer. A freshly prepared malondialdehyde (MDA) solution by the hydrolysis of 1,1,3,3,-tetraethoxypropane was used as the standard. The concentration of serum TBARS was calculated by comparing with the standard curve obtained by serial dilutions of MDA [Acontrol and Asample are the absorbance values of the control (standards) and serum samples, respectively.The serum lipid peroxidation was measured by using the thiobarbituric acid (TBA) reactive substance (TBARS) assay. First, 0.3 mL of each of the rat serum samples from each group was incubated with 50 s of MDA (1)TBARSμThe ORO staining was carried out according to the protocol described by Maganto-Garcia et al. . BrieflyP values ≤ 0.05 were considered statistically significant.The statistical package for the social sciences (SPSS) software  was used to perform the data analysis. All data were presented mean ± standard error (SE). The groups were compared by using a one-way analysis of variance (ANOVA) followed by Duncan's post hoc test of multiple comparisons while The lipid peroxidation induced by oxidizing agents is indicated by the production of MDA that ultimately binds with TBA to produce TBARS. Furthermore, MDA production is known to be a marker of the serum oxidative lipid levels, and the color of the end product of the reaction depends on the amount of lipid present in the serum. The result shows that treatment with CJF dose-dependently decreased subsequent formation of TBARS and the serum lipid accumulation Figures , resp.. C. japonica leaves and seeds, there are very few studies reporting the beneficial effects of its fruit, especially the cardiovascular actions. Therefore, our finding supports the notion that CJF could be a novel herbal source of potential drug therapies to target diseases associated with abnormal lipid metabolism.The present study demonstrated that the CJF ameliorated the discrepancies in the lipid profile of the HFD animal model by lowering the serum TC, TG, and LDL while increasing serum HDL. Moreover, the administration of CJF inhibited the lipid accumulation in the inner layer of the arteries and lipid peroxidation. Although previous studies have explored the pharmacological activity of in vivo model for studying cholesterol metabolism [ via endothelium-dependent relaxation of porcine coronary arteries and inhibition of vascular smooth muscle cell migration [−1·day−1 decreased the rat body weight compared to that of the control group rats fed with the HFD. This dose of CJF is within the safe dose obtained in a preliminary in vivo toxicity study and, therefore, indicates that the CJF could be used for its potential antiobesity effects.HFD-induced hypercholesterolemic rats are used as antabolism , 25. Thetabolism . Our preigration . In thisigration , 28. In igration , 30. In igration . However via endothelial NO synthase (eNOS) acts as a potent vasodilator and inhibits vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion [−), which react with superoxide anions to induce lipid peroxidation and endothelial dysfunction. In our previous study, CJF significantly upregulated eNOS phosphorylation in primary endothelial cells extracted from porcine coronary arteries [The accumulated serum LDL can easily undergo oxidation by ROS. The oxidative products are then engulfed by macrophages in the tunica intima of the blood vessels and are subsequently converted into fatty streak foam cells that facilitate atherogenesis . The resadhesion . In contarteries . In contDuring hypercholesterolemia, lipids accumulate on the inner layer of the blood vessel walls as the primary step in atherogenesis. There is a direct correlation between increased serum lipids, especially cholesterol, LDL, and other lipoprotein particles and the accumulation of lipids within the arterial wall, leading to atherosclerotic plaque development . HoweverC. japonica is a popular garden plant in East Asia, and its different parts including the flower, seed, and leaves are currently used as traditional medicines and commercialized as a source of cosmetics. Previous studies have indicated that the extracts of different parts of the C. japonica plant have various biological activities. It has been reported that extracts of C. japonica flowers possess antioxidant effects via scavenging of ROS and induction of antioxidant enzymes [ C. japonica leaves have shown antihuman HIV activity by inhibiting the HIV-protease enzyme, which is required for cleaving newly synthesized polyproteins essential for virion maturity [ via free radical scavenging potency mainly due to the presence of tannins [ C. japonica have been focused on the seeds, flowers, and leaves, but not the fruits. The constituents of C. japonica such as saponins in the seeds [ C. japonica has strong cardiovascular protective effects and could be a good potential candidate for development as a natural-based medicine for the prevention and treatment of cardiovascular diseases. Despite these beneficial effects of the fruit, there is very little information available on its constituents compared with the seeds and leaves. Recently, Uddin et al. isolated oleanane-type triterpenes from the fruit peels of C. japonica, which inhibited protein tyrosine phosphatase 1B [ enzymes . Extractmaturity . In addi tannins , antipho tannins . Further tannins . The stuhe seeds , flavonohe seeds , 43, andhe seeds  have beeatase 1B . TherefoIn this study, we demonstrated the antiatherogenic activity of CJF mediated by the lowering of the serum TC and TG and increasing the serum HDL. Similarly, CJF inhibited the lipid accumulation on the inner blood vessel walls and significantly decreased lipid peroxidation, thereby providing evidence of its efficacy in reducing the progression of atherosclerosis. Taken together, these results suggest that CJF could be a potential herbal therapeutic option for lowering hypercholesterolemia and preventing atherosclerosis progression triggered by abnormal lipid metabolism. However, further studies are necessary to identify the active phytochemicals in CJF and elucidate their underlying vascular protective mechanisms.


Solanum tuberosum L.) caused by the oomycete Phytophthora infestans (Mont.) de Bary, is one of the most important bottlenecks of potato production worldwide. Cultivars with high levels of durable, race unspecific, quantitative resistance are part of a solution to this problem. However, breeding for quantitative resistance is hampered by the correlation between resistance and late plant maturity, which is an undesirable agricultural attribute. The objectives of our research are (i) the identification of genes that condition quantitative resistance to P. infestans not compromised by late plant maturity and (ii) the discovery of diagnostic single nucleotide polymorphism (SNP) markers to be used as molecular tools to increase efficiency and precision of resistance breeding. Twenty two novel candidate genes were selected based on comparative transcript profiling by SuperSAGE  in groups of plants with contrasting levels of maturity corrected resistance (MCR). Reproducibility of differential expression was tested by quantitative real time PCR and allele specific pyrosequencing in four new sets of genotype pools with contrasting late blight resistance levels, at three infection time points and in three independent infection experiments. Reproducibility of expression patterns ranged from 28 to 97%. Association mapping in a panel of 184 tetraploid cultivars identified SNPs in five candidate genes that were associated with MCR. These SNPs can be used in marker-assisted resistance breeding. Linkage mapping in two half-sib families (n = 111) identified SNPs in three candidate genes that were linked with MCR. The differentially expressed genes that showed association and/or linkage with MCR putatively function in phytosterol synthesis, fatty acid synthesis, asparagine synthesis, chlorophyll synthesis, cell wall modification, and in the response to pathogen elicitors.Late blight of potato ( Phytophthora infestans (Mont.) de Bary, is one of the most important bottlenecks of potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum) cultivation worldwide  were identified in wild potato species and introgressed into advanced cultivars. However, this type of resistance proved not durable, as new P. infestans races having virulence alleles compatible with host resistance genes evolved after few years of widespread cultivation of the resistant varieties. The alternative to single R genes is polygenic or quantitative resistance to late blight, which is in essence the natural, quantitative variation of a compatible host-pathogen interaction. It allows the pathogen to multiply in certain degree but slows down the rate of disease progression, thereby reducing the selection pressure on the pathogen. This type of resistance is more durable as the pathogen has to undergo multiple mutations to overcome polygenic resistance. Quantitative resistance is also largely race unspecific , defense signaling and defense responses   analysis. SuperSAGE generates from each transcript present in a biological sample 26 base pair sequence tags, the frequency of which is proportional to the frequency of the corresponding transcript. Individual tag frequencies are determined by next generation sequencing of complex tag libraries. Transcripts are identified by mapping the tags to annotated EST (expressed sequence tag) collections and/or annotated genome sequences. SuperSAGE permits the simultaneous detection and quantification of the transcriptome both from the host and the pathogen in infected samples  in the corresponding loci.P. infestans and for plant maturity in 3 years in the field at Ebstorf (the BNA subpopulation) and Windeby (the SKP subpopulation) as described , were used. They originated from the crosses Phy20 × Phy14 (SL1 family) and Phy20 × Phy16 (SL2 family) . These 24 genotypes were infected under controlled conditions with a complex isolate of P. infestans and used for expression analysis of candidate genes.The 12 most resistant and susceptible genotypes were selected from the 111 SL genotypes based on the MCR values in the field 2010  set at 80% humidity, 100% light, 16 h light at 22°C and 8 h dark at 18°C and acclimated for few days before infection with P. infestans. Four plants per genotype were grown for four different treatments. One plant was used to collect leaf tissue immediately prior to inoculation (T0), the second plant was sprayed with water as negative control to check that water alone did not cause any blight symptoms. The remaining two plants were inoculated with a complex field isolate of P. infestans. The isolate was virulent on plants carrying the major late blight resistance genes R1, R2, R3, R4, R6, R7, R10, and R11, and avirulent on R8 and R9 carrying plants.P. infestans mycelium was weekly propagated on fresh leaflets of the susceptible cultivars Grata and Granola. Sporangia were collected in a sterile beaker by rinsing the infected leaves with sterile tap water. The concentration was adjusted to 20 sporangia/μl and zoospores were released after 1–2 h incubation in the dark at 4°C.o10007245, neoLab, Heidelberg, Germany). Infected and control plants were covered with transparent plastic bags to maintain high humidity. On the second (T2) and third (T3) day after inoculation, the fourth and fifth leaves were collected using a sterile blade, rapped in aluminum foil, immediately frozen in liquid nitrogen and stored at −80°C until use. Leaf samples were collected in the morning between 9 and 10 a.m. After collecting the leaf samples, the plants were kept in the growth chamber for 1 week to visually monitor P. infestans growth and symptom development on the third compound leaf of each genotype. Disease progression was scored as percent infection  of the P. infestans ribosomal gene RL23a (PITG_02694) as described  of each plant were marked with rings and sprayed with the sporangia/zoospore suspension or water two to three times using a 20 ml spraying bottle  according to the supplier's protocol. Four sets of RNA pools (Pools_1 to Pools_4)  before performing cDNA synthesis. First strand cDNA was synthesized using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) following the supplier's protocol.Pools_1 were constructed by pooling RNA from the 12 most resistant and susceptible SL genotypes that were selected based on MCR in the field in 2010 (see above). In Pools_2, RNA was pooled from the six most resistant and susceptible SL genotypes, based on the quantification by qRT-PCR of P. infestans. The details of the genotype pools, infection experiments, SuperSAGE libraries, and data analysis are described by Draffehn et al.  in at least five of the six comparisons between groups A2, A1, and B2 were selected. Transcript sequences matching the tags were retrieved from the DFCI (Dana-Farber Cancer Institute) potato EST (expressed sequence tag) database and checked for alignment with the tag sequence. Tags and transcript sequences were matched by BLAST searches to the potato genome reference sequence (version 4.03) at http://solanaceae.plantbiology.msu.edu/. From the matches in the potato genome sequence, the annotated genes aligning with the corresponding tag sequence (minimum 25 of 26 bp) were selected. Genomic DNA sequence, locus name, intron-exon structure and genomic position of the candidate gene were collected from the genome browser. When no match of the tag and transcript sequence was found in the potato genome reference sequence, the transcript sequence from DFCI was used in further analysis. A heatmap was constructed for the differential expression of the selected unique 26 bp tags using a custom made R script.The genes analyzed in this study were selected based on differential expression detected by SuperSAGE  analysis, which generates 26 bp tags from the transcripts present in a biological sample  or on a CFX384 Touch Real-Time PCR (Bio-Rad) using 2.5 μl of 1:5 diluted  first strand cDNA and the Power SYBR® Green PCR Master Mix (Applied Biosytems) according to the supplier's protocol. PCR cycling conditions were as described by Draffehn et al. , 0.2 mM dNTP Mix, 0.4 μM each forward and reverse primers, 0.5 U Taq DNA Polymerase (Ampliqon), high purity water  and 50 ng template DNA. The PCR reaction was run with 2 min initial denaturation at 94°C, followed by 50 cycles with 45 s denaturation at 94°C, 45 s annealing at the temperature specified for each primer pair , 0.2 mM dNTP Mix, 2.5 mM MgCl2, 0.2 μM each forward and reverse primers, 0.2 U Taq DNA Polymerase (Ampliqon), high purity water , and 50 ng template DNA. The PCR reaction was run with 2 min initial denaturation at 94°C, followed by 35 to 40 cycles with 40 s denaturation at 94°C, 45 s annealing at the temperature specified in Supplemental File Gene specific primers were designed . The P-values were corrected for multiple testing using the R package “q-value,” using the method described by Storey (Pair wise linkage disequilibrium (LD) was analyzed by testing for independence of two loci with a chi-square test using the statistical software R  using the linear model function In a previous study , one (T1), and two (T2) days after infection, Draffehn et al., P. infestans , one (PME) was consistently up and three  were down regulated. The remaining four genes  showed minor or inconsistent responses to infection. For each of the 14 genes, a unique 26 bp tag  with ag Table . The expMPP, HPR) and one gene (AspS) were down and up regulated, respectively, upon infection with P. infestans. The response to infection of the allelic tags of the remaining five genes was inconsistent. The tag SNPs in MPP and RLPK were non-coding, as they were located in the 3' untranslated region of the gene. The tag SNPs caused synonymous changes in DSD, HPR, BCCP and AspS, and non-synonymous changes in UPA18 and MADS, resulting in a non-conservative (methionine/isoleucine) and a conservative (aspartate/glutamate), respectively, amino acid change  the loci corresponding to the differential tags were unambiguously identified by in silico mapping of tag and transcript sequences to the potato genome sequence version 4.03  mapped equally well to two additional loci, PGSC0003DMG400021473, a short gene fragment annotated as “late blight resistance protein” and PGSC0003DMG400043121, a “Hypothetical gene of unknown function” with unknown genomic position. The locus PGSC0003DMG400008099 (Kiwellin) was chosen for further analysis. It is a member of a clustered family of six “Kiwellin” genes (KiTH-2) on chromosome XI, one of which (PGSC0003DMG400008101) was strongly induced upon infection with P. infestans and consistently expressed at higher level in resistant genotype pool A2 vs. both susceptible pools A1 and B2  for EIF and SABP. Contradictory to their expression in SuperSAGE, these genes were expressed at higher level in S than in R pools.Expression of 14 genes was analyzed by qRT-PCR in three independent infection experiments . Six genes were tested in all four pools and eight were tested in Pools_1 and Pools_2. Measuring expression in all four sets of pools yielded 72 expression values: four each resistant (R) and susceptible (S) pools at T0, T2, T3, and three biological replicates. When testing two pools, 36 samples were analyzed. The reproducibility of the differential expression observed in SuperSAGE based on 36 or 18 qualitative comparisons between R and S genotype pools is shown in Table P. infestans similar as in SuperSAGE, taking into account that absolute expression levels and infection kinetics can vary considerably between independent infection experiments. Contradictory results were obtained for ACRE20 and Cgu transcripts. ACRE20 was transiently up regulated upon infection in SuperSAGE but consistently down regulated in the samples analyzed in this study. Vice versa, Cgu was up regulated at T1 and down regulated at T2 in SuperSAGE, while it was continuously up regulated in this study  : the “resistant” allele was expressed at higher level in R pools compared with S pools . In the case of AspS and BCCP, the “susceptible” allele was either absent in some genotype pools  or its frequency was very low . Nevertheless, the expression pattern of AspS was completely reproducible in Pools_1 and _2 in accordance with the allele frequency, but not in Pools_3 and _4. Expression of BCCP was completely consistent with SuperSAGE only in Pools_4, although only the “resistant” allele was present in both R and S pools. In four cases  the frequency of the “resistant” allele was higher in the S pool than in the R pool  had shown >60% reproducibility of differential expression in R vs. S pools. The expression patterns of the three remaining genes  as determined by qRT-PCR or pyrosequencing were less consistent with the SuperSAGE results. Gene specific primers were developed for amplicon sequencing and SNP detection  at the loci DSD, SMO, CCAP, and AspS that were linked with QTL for MCR or AUDPC or both  in the PIN184 population and were therefore excluded in the association analysis. One linked SNP (AspS_SNP6303) in the AspS locus caused the conservative amino acid substitution arginine/lysine  or pyrosequencing (9 SNPs). Linkage analysis with AUDPC, MCR and PM in the combined SL families identified 13 SNPs  as well as genotype at the StAOS2 locus. The principle of bulked segregant analysis , signaling and regulation , chloroplast processes , cell wall modification , protein biosynthesis, degradation, transport, and conformation protection , lipid metabolism (BCCP), sterol biosynthesis , photorespiration (HPR), and nitrogen/amino acid metabolism (AspS). The selected genes were evaluated first for reproducibility of differential expression in genotype pools assembled in a way slightly different than the ones used in SuperSAGE and in independent infection experiments with P. infestans, and subsequently for association of SNPs at corresponding genomic loci with resistance to late blight and plant maturity.The analysis of gene expression, either in response to pathogen infection or between pathogen resistant and susceptible plants, is a long standing molecular genetic approach to discover and functionally characterize genes that play a role in host pathogen interactions  AUDPC, the latter two evaluated in a growth chamber, showed remarkable consistency in sorting 24 SL genotypes into two groups with higher and lower late blight resistance levels  confirms the observation that P. infestans growth measured by qRT-PCR during the first 3 days after controlled infection, before disease symptoms become visible, is an indicator for field resistance  containing the target SNP is sequenced from a single strand template using a third sequencing primer. These further steps could increase the specificity of pyrosequencing compared to qRT-PCR. As stated by Draffehn et al. , sensitiACRE20, Pubq, SLP, PME, SMO, DSD, MPP, HPR, UPA18) was reproducible in more than 70 percent of all comparisons between R and S genotype pools and that of four genes  was reproducible in 60 to 70 percent (Tables When tested in two or four pairs of genotype pools with contrasting resistance to late blight (R and S pools), at three infection time points and in three independent infection experiments, the SuperSAGE expression pattern of nine genes  is a key enzyme in the biosynthesis of phytosterols, important membrane components and precursors of brassinosteroid plant hormones  of tobacco encodes a putative EF-hand calcium binding protein  is post-translationally attached to other proteins (ubiquitination), which among other processes, targets them for degradation by the 26S proteasome  was first described in maize where it is associated with the plasma membrane  is a molecular chaperone that is induced in response not only to abiotic stress  complex, which catalyses the first committed step of fatty acid biosynthesis in chloroplasts  functions downstream of SMO in sterol biosynthesis. Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids  is involved in chlorophyll biosynthesis  is part of the photorespiratory cycle, one of the major pathways of plant primary metabolism which also plays a role in defense responses  is a pepper gene of unknown function, which was induced within 8 h by the AvrBs3 effector of Xanthomonas campestris pv. vesicatoria (Xcv) upon infection  differential expression in R and S genotype pools  and three with lower reproducibility (< 60%)  yielded nine SNPs in seven genes  which were associated with rAUDPC and/or MCR but not with plant maturity. Only one SNP in the CCAP gene showed association with PM  was only 50%, one SNP in this gene was associated with rAUDPC. The tomato and Arabidopsis homologs of Chp are annotated as ATP-dependent, thylakoid-bound Zn protease (FtsH) that is important in the biogenesis and maintenance of photosystem II , as the size of each family alone was too small for QTL mapping. Linkages with resistance alleles inherited from either Phy14 or Phy16, the seed parents of family SL1 and SL2, respectively, were probably not detectable. Nevertheless, 13 SNPs in four genes, eight of those in the AspS gene, showed linkage with MCR and/or AUDPC and none was linked with PM , which were inherited from both resistant parents of the SL families but were very rare in the PIN184 association panel. This indicates that one or more rare resistance alleles for MCR segregated in the SL families. In the case of rare alleles, linkage mapping in experimental populations is superior over association mapping, where rare alleles are discarded or do not reach statistical significance  is an interesting candidate gene based on functional analysis in other plant species. AspS is a key enzyme in the biosynthesis of asparagine and is required for nitrogen assimilation and defense responses against microbial pathogens. Hwang et al. (Capsicum annuum asparagine synthetase1 (CaAS1) was rapidly induced in leaves upon infection with Xanthomonas campestris pv. vesicatoria (Xcv). Silencing CaAS1in pepper plants resulted in enhanced susceptibility to Xcv. Arabidopsis plants overexpressing CaAS1 exhibited enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and to the oomycete pathogen Hyaloperonospora arabidopsidis. AspS might be involved in induction of reactive oxygen species (ROS) and nitric oxide (NO)  as novel candidates for playing a functional role in resistance to late blight not compromised by late maturity. Gene silencing by targeted genome editing using new technology like CRISPR/Cas9  into cultivars. Whether the AspS locus itself is responsible for the resistance phenotype or another, physically closely linked gene requires further studies.Linkage mapping of SNPs in 11 candidate genes identified five SNPs in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Obesity impacts utilization of healthcare resources. The goal of this study was to measure the relationship between increasing body mass index (BMI) in patients undergoing total hip arthroplasty (THA) with different components of operating room (OR) time.Methods. The Stanford Translational Research Integrated Database Environment (STRIDE) was utilized to identify all ASA PS 2 or 3 patients who underwent primary THA at Stanford Medical Center from February 1, 2008 through January 1, 2013. Patients were divided into five groups based on the BMI weight classification. Regression analysis was used to quantify relationships between BMI and the different components of total OR time.Results. 1,332 patients were included in the study. There were no statistically significant differences in age, gender, height, and ASA PS classification between the BMI groups. Normal-weight patients had a total OR time of 138.9 min compared 167.9 min (P < 0.001) for morbidly obese patients. At a BMI > 35 kg/m2 each incremental BMI unit increase was associated with greater incremental total OR time increases.Conclusion. Morbidly obese patients required significantly more total OR time than normal-weight patients undergoing a THA procedure. This increase in time is relevant when scheduling obese patients for surgery and has an important impact on health resource utilization. Using the World Health Organization’s (WHO) weight-class definitions, two-thirds of the adult population in the United States above the age of 60 years are “pre-obese” or “obese” . FurtherThe goal of this study was to measure the relationship between increasing obesity in patients undergoing total hip arthroplasty (THA) with the various components of OR time in order to determine how obesity contributes to overall case duration. The expected result is an increase in resource utilization for obese surgical patients as measure by perioperative times.Following IRB approval, the Stanford Translational Research Integrated Database Environment (STRIDE) was utilized to identify all patients who underwent primary THA at Stanford Medical Center from February 1, 2008 through January 1, 2013. STRIDE is a standards-based informatics platform supporting clinical and translational research and includes a clinical data warehouse based on the HL7 Reference Information Model . This ob2), Group 2—pre-obese (BMI = 25–29.9 kg/m2), Group 3—obese (BMI—30–34.9 kg/m2), and Group 4—severely obese (BMI = 35–39.9 kg/m2) and Group 5—morbidly obese (BMI > = 40.0 kg/m2).Only American Society of Anesthesiologists Physical Status (ASA PS) class 2 or 3 patients undergoing primary elective unilateral THA procedures under general anesthesia, with or without spinal for post-operative pain control were included in the study. ASA 1, 4 and 5 patients were removed from the study since they represent a small number of the total hip arthroplasties. Patients were divided into five groups based on WHO BMI classification . Group 1The following parameters were retrieved: age, gender, height, weight, surgeon (14 surgeons), anesthesiologist (116 anesthesiologists) and ASA PS. Patients who had placement of central lines or arterial lines were excluded from the study since these interventions could be a confounding variable for increased length of perioperative time.Time stamps were retrieved from the STRIDE database. The data points collected were time of (a) Admission to pre-op Unit, (b) Entry into operating room, (c) Anesthesia handoff, (d) Surgery start, (e) Surgery end, (f) Out of operating room, (g) Post-Anesthesia Care Unit (PACU) admission, (h) PACU discharge, and (i) Hospital discharge day and time.The primary outcome variable of the study was total OR Time, which is the time interval beginning when a patient entered the OR (b) until the moment the patient physically left the OR (f).Secondary outcome variables included:1.Induction Time defined as the time from Entry into OR (b) to Anesthesia Handoff (c). 2.Operation time defined as the interval from Surgery Start (d) to Surgery End (e).3.Emergence Time defined as the interval from Surgery End (e) to Out of Operating Room (f).4.Recovery Room Time defined as the interval from PACU Admission (g) to PACU Discharge (h).5.Total Hospital Length of Stay defined as the interval from the Admission to Pre-op Unit (a) to Hospital Discharge (i).All time data points were collected and recorded into the electronic medical record by pre-operative, operating, and recovery room nurses who were unaware of the study, as data was analyzed retrospectively.2 or less than 18.5 kg/m2 were removed from the study due to a paucity of data points (96 patients removed). These two procedures would cause a wide variability in induction times and could be representative of inability to get a non-invasive blood pressure or peripheral intravenous access or could be due to severity of illness. By removing these few occurrences from our dataset, we reduce the possibility of variability due to covariates and had a total of 1,332 patients remaining. All data points for the time intervals were available for these patients, except 1 was missing for induction time , 4 were missing for operation time , and 568 were missing for recovery time (764 observations). The patients were grouped by body mass index as defined by the WHO classifications (stated above in this section). For the primary and each secondary outcome variable the mean and standard deviations were calculated. We estimated regression parameters separately for each time component of the case. In the data analysis BMI is an indicator variable. The database accessible covariates that can affect case duration were included as regressors   BMI patients to each successively higher BMI grouping. This analysis was repeated for the primary and each secondary outcome variables.Observations taken from the STRIDE database, which already excluded patients based on ASA, central line and/or arterial line placement, and regional anesthesia, resulted in 1,613 patients. The top 1% and bottom 1% times for each data interval were excluded as a means of removing incorrectly entered data points and ensuring outliers did not drive the results (185 patients removed). Patients with a BMI of greater than 50 kg/mication) . These w2) and total operating room time, induction time, total operation time, emergence time, recovery room time, and length of hospital stay. Using Stata  a kernel weighted local polynomial regression to examine potential non-linearity in the relationship between BMI, the continuous variable, and procedure duration was performed. In the graphical analysis the covariates are not controlled for. This method traces out average total OR time and induction time for each BMI value without making any parametric assumptions.A graphical regression analysis was performed to quantify the relationship between BMI  patients undergoing THA had a mean Total OR Time of 138.9 min, Group 3 (obese) patients 146.3 min, and Group 5 (morbidly obese) patients 167.9 min , Fig. 1.t-test between higher BMI groups when compared to Normal BMI group. Incremental BMI group increase was associated with a greater incremental time increase at higher BMIs.There was also a direct association between increasing BMI and the length of many of the individual components of total OR Time based on Patients in Group 1 had a mean Induction Time of 21.4 min, Group 3 patients 23.7 min, while Group 5 patients required 28.2 min . Group 5As the number of obese patients continues to rise so does the demand for joint arthroplasty procedures. Therefore, elucidating the impact of obesity on perioperative costs is becoming ever more relevant . Our stuThe reasons increasing weight lengthens both anesthesia and surgical times are multifactorial but could be related to difficulties transferring patients to the OR table, with airway management, with moving the patient to the lateral decubitus position once anesthetized, and because excessive fat can interfere with intraoperative surgical exposure . ObesityIt is estimated that by 2030, the number of primary THA operations will rise by 174%, and a significant number of these patients will be obese . Health Throughout the US there is wide variability reported for actual operating room costs depending on whether fixed costs or variable costs are included and on the specific resources consumed by each patient . BesidesIn our analysis, the mean operation and induction times plateau and begin to decrease as the extremes of BMI are reached. The confidence interval widens as the BMI increases, which is a result of a limited number of morbidly obese patients undergoing hip arthroplasty as compared to normal BMI, overweight, and obese patients undergoing this operation.t-test analysis is an inherent limitation in that we can only view each t-test alone, since there is inherent correlation between time intervals. We decided to report the most straightforward and easily understood test in the presentation of the data. Despite this, it is apparent from the data presented that the higher the BMI the longer the OR time will be. This will hold true however the data is reported. An inherent limitation in retrospective non-randomized studies is the fact that causal inferences can be mistakenly made, but we are confident in this study, which is supported by current available literature. As can be seen in This study has inherent limitations in the fact that it is a single-center retrospective electronic chart-review. The time points collected by preoperative, OR, and recovery room nurses may have some errors, but should affect the BMI groups similarly. In the case of recovery times, a large number of the entered values were not recorded by the STRIDE database properly limiting the power of our recovery time results. The multiple We found that for patients undergoing THA, increasing BMI was associated with increased total OR time. Also increasing BMI was associated with longer hospital stays after THA. Operating room scheduling and plans for resource utilization should recognize that the same THA procedure will require more time in a morbidly obese patient than in a normal-weight or pre-obese patient. These considerations can potentially reduce the increased healthcare costs associated with performing surgery on obese patients.