Obesity in pregnancy is associated with systemic inflammation, immunological changes and adverse maternal-fetal outcomes. Information on the association between maternal obesity and breast milk composition is scarce. This study describes changes and relationships between biochemical and immunological parameters of colostrum and serum of overweight and obese women.Colostrum and blood samples were collected from 25 normal weight, 24 overweight and 19 obese women for determination of glucose, total protein, triglycerides, cholesterol, immunoglobulins, complement proteins (C3 and C4), fat and calorie content and C-reactive protein (CRP).p = .002). In normal weight and obese women, total protein content was higher in colostrum than in serum (p = .001). Serum triglycerides (p = .008) and cholesterol (p = .010) concentrations were significantly higher in overweight and obese women than in their normal weight counterparts, but in colostrum their concentrations were similar across the three groups. Secretory IgA (sIgA) in colostrum and IgA in serum concentrations were significantly higher (p = .001) in overweight and obese mothers, whereas IgG and IgM concentrations did not vary among the groups (p = .825). Serum C3 (p = .001) and C4 (p = .040) concentrations were higher in obese women. No differences in colostrum complement proteins were detected among the groups. Calorie content (p = .003) and fat (p = .005) concentrations in colostrum and serum CRP (p = .002) were higher in obese women.Glucose was higher in colostrum of obese women (The results corroborate the hypothesis that colostrum of overweight and obese women undergoes biochemical and immunological changes that affect its composition, namely increasing glucose concentrations, calorie content, fat and sIgA concentrations. Breastfeeding promotion is one of the main strategies for reducing child mortality worldwide . Breast In recent decades, due to changes in lifestyle the incidence of overweight and obesity are increasing among breastfeeding mothers. Obesity and overweight are common metabolic disorders and their growing frequencies worldwide are a major public health concern . ObesityThe risks associated with obesity become even more relevant in women of reproductive age , 21 becaOther studies show that chronic inflammation caused by obesity may be related to an exaggerated inflammatory response in the placenta of pregnant women, with an accumulation of macrophages and pro-inflammatory mediators . HoweverChanges in maternal metabolism and immunological response may affect the developing immune system of newborns because of the intense interplay between mother and child during pregnancy and breastfeeding. To clarify this issue, the present study investigated the association between maternal weight and immunological, biochemical and nutritional parameters of colostrum.n = 25); overweight (n = 24) and obese (n = 19). Group definition was based on the World Health Organization [This cross-sectional study evaluated 68 mothers (18–36 years of age), divided into three groups according to their prepregnancy body mass index (BMI): normal weight  women with breasts without nipple fissures or mastitis; (b) who were exclusively breastfeeding their babies; and (c) signed a Consent Form. Women with multiple pregnancies, fetal malformations and deliveries before the 37th week of gestation were excluded.About 48–72 h post-partum, 10 mL colostrum was collected from the volunteers. Supernatant was obtained by colostrum centrifugation at 160 × g for 10 min at 4 °C. The upper fat layer was discarded, and the aqueous supernatant was stored at −80 °C for later biochemical and immunological analyses.Samples of 10 mL of blood were collected prior to the beginning of labor from each mother in tubes without anticoagulant and the blood samples were centrifuged at 160 × g for 15 min, until serum separation. Serum samples were stored individually at −80 °C for further glucose, enzyme and protein determination.Glucose concentrations of colostrum supernatant and maternal serum were determined by the enzymatic system . SamplesTotal protein of colostrum supernatant and maternal serum was determined by the Biuret colorimetric method . SamplesCholesterol concentrations of colostrum supernatant and maternal serum were determined by enzymatic colorimetric method . SamplesTriglycerides concentrations in colostrum supernatant and maternal serum were determined by the enzymatic colorimetric method . SamplesThe immunoglobulin (Ig), complement protein (C) 3 and 4 concentrations in colostrum and serum were determined by turbidimetric method .For sIgA in colostrum and IgA in serum determinations, the samples were diluted at 1:5 (v/v) with saline solution (9 g/L), for IgM at 1:11 (v/v) and for IgG at 1:15 (v/v), and the antibody concentrations were determined using IgA , IgM  and IgG antiserum  diluted at 1:12 (v/v). A calibration curve obtained by the Multical  calibrator was used to determine the standard curve for each immunoglobulin. Samples of colostrum, serum, standards, positive and negative control sera were placed in 500 μL of buffer solution . The suspensions were mixed and incubated at 37 °C for 10 min. The reactions were read on a spectrophotometer at 340 nm.For C3 and C4 concentration determination the samples (colostrum and serum) were diluted at 1:12 (v/v) with saline solution (9 g/L), and the C3 and C4 concentrations in sample supernatants were determined using C3 and C4 antiserum  diluted at 1:12 (v/v). A calibration curve obtained by the Multical , Ref K064) calibrator was used to determine the standard curve. Ten microliter samples of colostrum, serum, standards, positive and negative control sera were placed in 500 μL of buffer solution . The suspensions were mixed and incubated at 37 °C for 15 min. The reactions were read on a spectrophotometer at 340 nm.Colostrum samples were water-bath-heated at 40 °C for 15 min and subjected to vortex mixing. Capillary tubes 2 μL) were filled to approximately three quarters with the samples, sealed with sealing wax and centrifuged for 15 min. Centrifugation separated the samples into cream and serum [ μL were C-Reactive Protein (CRP) concentrations in human colostrum and serum were measured using the PCR Turbilatex Kit  by turbidimetric method . Samplesp-value was less than .05.Two-way Analysis of variance (ANOVA) with calculation of F statistic and Tukey’s multiple comparison test were used to evaluate glucose, total protein, cholesterol, triglycerides, antibody concentration, complement protein, CRP, calories and fat considering the BMI status as one factor and the biological materials (colostrum or serum) as the other. Statistical significance was considered when the Clinical characteristics from all groups are shown in Table We evaluated biochemical Table  and immup = .002) than in overweight and normal weight groups. Serum glucose concentrations were higher (p = .001) than colostrum glucose concentrations in overweight mothers (Table p = .758) across the groups. In normal weight and obese groups, total protein concentrations were higher in colostrum (p = .001) than in maternal serum (Table Colostrum glucose concentrations were higher in obese p = .00 than in p = .008) in the obese group. Irrespective of the BMI, triglyceride concentrations were higher (p = .001) in colostrum than in serum (Table p > 0.05) in cholesterol concentrations were detected in colostrum. However, obese women exhibited higher cholesterol concentrations in serum (p = .010) than normal weight individuals. In the obese group, cholesterol concentrations were higher in serum (p = .001) than in colostrum  and calorie (p = .003) content of colostrum was higher in the obese group. Colostrum CRP concentrations were similar among the groups, but in serum, they were significantly higher in the obese group (p = .002) than in the other groups. The highest concentrations of CRP (p = .001) were found in serum  colostrum sIgA and serum IgA concentrations than normal weight women. IgM concentrations did not vary (p = .825) among the groups and between serum and colostrum samples (p = .177). IgG concentrations were significantly higher in serum (p = .001) than in colostrum (Table The obese group showed significantly higher (um Table .p = .001) and C4 (p = .040) were significantly higher (p = .010) in the obese group than in normal weight and overweight groups, but in colostrum did not differ among the groups. Irrespective of weight status, C3 concentrations were significantly higher in serum (p = .001) than in colostrum, and in normal weight and overweight groups, C4 concentrations were significantly higher in serum (p = .001) than in colostrum samples (Table In serum, the concentrations of complement proteins C3 (The immune protection that breast milk provides and its nutritional importance has been widely described, and the composition of this secretion is known to undergo inter-and intra-individual variations . These vOther studies report that maternal anemia, hypertension and diabetes can change nutritional and immunological features of breast milk , 30–32. The association between maternal obesity and dyslipidemia has been extensively described –37, and via placenta during uterine life and then via colostrum and breast milk after birth is important in reducing this deficiency [The immaturity of the immune system of newborns makes it susceptible to infections by viruses and bacteria. Accordingly, the transfer of antibodies ficiency . In the ficiency . The preficiency . IL-6 isficiency , 42.Unlike IgG, which is transferred transplacentally, the action of immunoprotective components of colostrum and milk is usually local, in the newborn’s intestinal mucosa . sIgA isThe increased serum C3 concentrations in overweight and obese women and serum C4 in the obese group was not accompanied by an increase in these concentrations in colostrum. It was previously reported that obese individuals exhibit higher concentrations of circulating C3 , 46 and Obese mothers exhibited higher concentrations of serum CRP, but not in colostrum. CRP secretion by the liver is stimulated by several inflammatory cytokines, which are released in response to trauma, infection and inflammation, and this protein rapidly reduces the resolution of these conditions . AnotherIt should be considered that these data were evaluated in one period of collection and only one milk maturation stage that may be considered a limitation of this study. It is necessary to continue investigations focusing on other factors that may be involved during breastfeeding of the mothers with BMI alterations.The data obtained in the present study support the hypothesis that metabolic changes promoted by obesity can change the biochemical and immunological parameters of breast milk. Nevertheless, we did not observe any changes that could cast doubt on the protection that breastfeeding provides to newborns or that could reflect the inflammatory state observed in maternal serum, because the only immunological component that increased in serum the obese women was IgA, which is known to be a non-inflammatory antibody. However, the increased calorie and fat content and glucose concentrations detected in colostrum from obese women deserve further attention.


C. latifolia were inoculated on MS medium supplemented with various concentrations of BAP and IBA, alone or in combinations, to achieve in vitro plant regeneration. Subsequently, antioxidant and antibacterial activities were determined from in vitro and in vivo plant developed. No response was observed from seed culture on MS media with various concentrations of PGRs. The highest percentage of callus was observed on tuber explants (94%) and leaf explants (89%) when cultured on MS media supplemented with IBA in combination with BAP. A maximum of 88% shoots per tuber explant, with a mean number of shoots (8.8 ± 1.0), were obtained on MS medium supplemented with combinations of BAP and IBA (2.5 mg L−1). The best root induction (92%) and mean number (7.6 ± 0.5) from tuber explants were recorded on 2.5 mg L−1 IBA alone supplemented to MS medium. The higher antioxidant content (80%) was observed from in vivo tuber. However, tuber part from the intact plant showed higher inhibition zone in antibacterial activity compared to other in vitro and in vivo tested parts.Leaf, seed, and tuber explants of Curculigo and Hypoxis are the two main genera of this family. The genus Curculigo is distributed in the tropical region of Africa and Asia rainforest's, particularly Malaysia and Singapore [ C. latifolia, C. capitulations, C. racemes, and C. orchioides [There are more than 20 species in the family Hypoxidaceae whereingapore . The fouCurculigo latifolia Dryand, Lemba in Malaysia, is one of the important traditional Chinese medicinal plants. These species are propagated by rhizomes [β-amylase) have been known and extracted from this species. However, the phenols and phenolic glycosides from this plant are categorised as benzyl benzoate glycosides, followed by phenol glycosides and simple phenol. The 20 unique saponins are cycloartane triterpenoids and could be found in this species [rhizomes  and may rhizomes . Howeverrhizomes . In addirhizomes . Yamashirhizomes  revealedrhizomes , 9 whichrhizomes . To date species . in vitro acetone extract of Salvia officinalis exhibited antioxidant properties. However, many authors stated that in vitro and in vivo plant extracts showed antioxidant activities [Grzegorczyk et al.  reportedtivities –15. in vitro technique is being widely applied to produce identical quality and disease-free plants [ C. latifolia from seeds, but we reported that it is being propagated through rhizome [ in vitro or tissue culture technique is mandatory.The increase in demands of this plant for commercial use requires an alternative rate of proliferation. During these years,e plants . There i rhizome . However in vitro method for propagation of the species C. orchioides was established as a rare and endangered species in India [ in vitro culture of this species has been reported [Anin India –20. Howereported . in vitro plant regeneration, and the role of regeneration plantlets in antioxidant and antibacterial effects to compare with in vivo C. latifolia and the subsequent transplantation of the plantlets to natural environmental conditions.The present study is advancement over the earlier protocol, because it describes the PGRs regulation, C. latifolia, grown at Genting Highlands, Malaysia, were collected in the middle of September 2014. The tiny black seeds were obtained from dry fruits. Some botanists identified the plant materials and some pots were deposited at The University of Malaya . The seed explants of C. latifolia were surface sterilized according to Taha [The fresh mature fruits of to Taha  with somThe seeds were treated with 70%, 50%, 20%, and 10% (v/v) commercial bleach (Clorox) for 1 min at each concentration. The treated seeds were submerged in 70% (v/v) ethanol and finally by 3 times rinsing with sterile distilled water. Two drops of Tween-20 were also added during the treatment with 100% (v/v) Clorox to facilitate the sterilization process and reduce surface tensions.Leaves and tubers were collected from (5-6 months old) seedlings, which were grown in Genting Highlands, Malaysia. Both types of explants were washed thoroughly under running tap water for 20 min. The leaves were soaked with commercial bleach or Clorox (70%) for 3 min under laminar flow and were rinsed 2 times with sterile distilled water. Treated leaves were submerged in 70% (v/v) ethanol and finally were rinsed 5 times with sterile distilled water.The tubers were dipped in 30% citric acid to remove phenolic content and were immersed in 75% Clorox for 10 min. The tubers were sterilized by ethanol 100% (v/v) containing 0.1% (V/V) Tween 20 for 5 min and finally were rinsed five times with sterile distilled water.−1 Gelrite  and supplemented with various concentrations of IBA either alone or in combination with BAP (0.5–4 mg L−1).For all treatments, MS basal medium  containiAll the media were adjusted to pH of 5.7 ± 0.1 with 0.1 N KOH prior to autoclaving at 121°C for 20 min. The media were dispensed into 60 mL specimen presterilized containers under laminar flow in aseptic condition.2 pieces, removing leaf ribs, and any other major leaf veins before being placed on the culture medium. The explants (3 per plate) were arranged horizontally and were pressed lightly into the surface of the culture medium.The leaves were further cut into approximately 0.5 cm2 and placed on the surface of the culture medium. In order to avoid the death of explants due to phenolic exudation, the tuber explants were subcultured three times every 3 days to the same media and PGRs.Tubers (3 per plate) were cut into 2 cmμmol m−2 s−1 provided by cool daylight fluorescent lamps.The leaves and tuber explants were inoculated on MS basal media and supplemented either alone or in combination with IBA and BAP. The cultures were maintained at 25°C under a 16–8-hour photoperiod at a photon flux rate of 60 Callus cultures were optimized and measured for its biomass and secondary metabolic content. By applying various concentrations of PGRs, fresh and dry weights of the callus were determined at 8 weeks. At the end of the period, for all the treatments, each callus was harvested by careful separation from media using metal spatulas, and fresh and dry weight were promptly recorded.μmol m−2 s−1 provided by cool daylight fluorescent for 8 weeks. The plantlets were transferred to greenhouse conditions after 8 weeks.The regenerated plantlets were transplanted to 6 cm plastic pots filled with perlite and pit (3 : 1) and were kept in a controlled condition chamber with 80–90% relative humidity under a 16/8 h (light/dark) of photoperiod at a photon flux rate of 60 In vivo samples from young and healthy leaves and tuber (5-6 months old) were collected. The in vitro developed friable callus (without roots and shoots) from leaf and tuber explants was collected and both in vivo and in vitro samples were dried separately at room temperature for 4 days. To produce a fine homogenous powder, the samples were ground by electric blender. The 5 g of dried in vivo sample and in vitro regenerated callus of C. latifolia was extracted five times with ethanol [−1) was dissolved in phosphate buffered saline (PBS) and was kept at 4°C. ethanol . The ethIn order to study antioxidant properties, radical scavenging and superoxide dismutase assay were applied and the obtained results were compared.∗  free radical scavenging capacity assay was achieved using the protocol described by Rafat et al. [μL) at a concentration of 90 μM was mixed with 50 μL plant extract (10 g L−1), and the volume was adjusted to 4 mL using ethanol (95%). The solution was incubated for 120 min at room temperature in the dark condition. Scavenging of DPPH reduced the color of the solution and was measured using a spectrophotometer at 515 nm.DPPHt et al. . DPPH (9Comparison of the reduction of color in the examined samples with the blank (solution without plant extract) was used to measure the potential of scavenging capacity of the plant extracts using the following formula :(1)Radic−1 were added to 200 μL of the kit working solution. The mixture, after gentle shaking, was incubated at 37°C for 20 min after adding 20 μL of the kit enzyme working solution. The absorbance of the mixtures was measured at 450 nm using a microplate reader  and the SOD activity was calculated based on the following equation [−1) and BHT or tert-butylated hydroxytoluene (1 g L−1) were employed as the positive controls in this study:1 = blank  of  mixture  working  solution + enzyme  working  solution + double  distilled  water, blank2 = blank  of  mixture  plant  extract + working  solution + dilution  buffer + double  distilled  water, blankAsample = blank  of  mixture  plant  extract + working  solution + dilution  buffer.Superoxide dismutase (SOD) determination kit (19160), ascorbic acid (A4544) from Sigma-Aldrich , and Tert-butylated hydroxytoluene (34750) from Fluka (Spain), were used for this part of the study. Plant extracts with a concentration of 10 g Lequation . Ascorbi C. latifolia was investigated based on the paper disc diffusion method adopted from Farzinebrahimi et al. [Staphylococcus aureus and Bacillus cereus) and two gram-negative pathogenic bacteria (Pseudomonas aeruginosa and Klebsiella sp.) were obtained from Microbiology Division of Institute of Biological Sciences, University of Malaya, and maintained in a nutrient broth medium to produce a final concentration of 107 colony forming units (CFU) per mL. The test bacteria (0.1 mL) were streaked by sterile cotton swab on Mueller Hinton medium (MH) plates. Sterilized filter paper discs were soaked in extracts (10 g L−1) and then placed at the centre of test bacteria plates. The diameters of the inhibition zones were recorded after 24 hours of incubation at a temperature of 37°C overnight. Ampicillin (30 μg) was applied as positive and negative controls, respectively. Sterile paper disks were put in samples; sterile distilled water and kanamycin antibiotic were as control for two hours.The antibacterial potential ofi et al.  with minThe experiments were conducted in a factorial based on randomized completely design with 4 blocks and 16 treatments. For all treatments, the mean and standard error were calculated. The data were analyzed by ANOVA followed by mean comparison using Duncan multiple range test (DMRT) . Data weThe seeds did not respond to MS media and different PGRs either alone or in combination after eight weeks of seed inoculation. It may be due to the hard seed coat, immature embryo, rudimentary embryo, and inhibitor substances. However, the callus initiation did not form without PGRs  in tuber and leaf explants.The best callus induction from tuber explants was recorded in MS media with combinations of IBA and BAP (94%), whereas the same media supplemented with IBA and BAP alone produced 71% and 79% callus, respectively. This pattern was observed in callus formation from leaf explants, when combination of IBA and BAP produced higher percentage of callus (89%) as compared with IBA (82%) and BAP (83%).−1) and BAP (4.0 mg L−1) induced nature callus with a maximum of fresh and dry weight. However, the highest callus was formed in leaf explants when IBA and BAP (4.0 mg L−1) were applied alone or in combination  showed the highest number of shoots (8.8 ± 1.0). However, the best root numbers (7.6 ± 0.5) were formed in the same media added with IBA (2.5 mg L−1) alone after eight weeks of culture.As illustrated in −1 IBA alone. However, combinations of BAP and IBA (2 mg L−1) produced 5.7 ± 0.8 shoots/explants (The highest number of roots/explants was obtained from leaves (7.3 ± 0.1), when leaf explants were cultured on MS media supplemented with 2 mg Lexplants .−1) showed the optimum result of root and shoot length (6.61 ± 0.68 cm and 5.35 ± 1.31 cm), respectively (−1 IBA and BAP (The leaf explants cultured in MS media supplemented with combination of IBA and BAP (2 mg Lectively . The len and BAP .−1 IBA and BAP showed the higher weight based on fresh (45.589 ± 1.45 g) and dry weight (12.805 ± 0.57 g). The leaf explants inoculated on the same media and PGRs showed higher fresh and dry weight , respectively  in a chamber with 80% humidity. The plants were transferred to the open place and gradually were acclimated to outdoor condition. The survival rate was measured as 89%. C. latifolia was compared with leaves from in vivo plants, butylated hydroxyl toluene (BHT) and ascorbic acid or vitamin C (1 mg L−1) as a positive control.The antioxidant activity of plant extracts  of in vivo tuber and leaf extract showed 80% and 60%, respectively. The same pattern was observed when SOD activity was applied  and callus (in vitro) showed higher inhibition zone compared to other extracts on gram-negative bacteria, especially against Klebsiella spp. and P. aeruginosa.The various extract of10 g L−1 . However in vitro establishment and regeneration of plants are influenced by various factors, such as explants type, the physiological status of in vivo plants, genotype, species, and media composition, type and combination of plant growth regulators, and culturing conditions.It is well known that in vitro regeneration was only obtained from leaf and tuber explants. The explants cultured in MS medium in the absence of PGRs have showed no response to shoot and root induction. The response of tuber explants to in vitro regeneration in Hypoxidaceae species varied considerably.In this study, among the different types of explants such as leaf, tuber, and seed, H. obtusa (Hypoxidaceae) on MS medium supplemented with (1 mg L−1) BAP. Based on Page and Van Staden [ H. rooperi (Hypoxidaceae) were formed on MS media added with (1 mg L−1) BAP between six and ten weeks, respectively.Vinesi et al.  reportedn Staden , more th H. colchicifolia reported on MS medium containing 2 mg L−1 BAP compared to other PGRs [However, the best in multiplication response ofher PGRs .Appleton and van Staden  and Nsibin vivo). According to Farzinebrahimi et al. [ H. hemerocallidea from this family showed antioxidant properties via hydroxyl scavenging ability [ In vitro studies indicated good ability to scavenge free radicals (hydroxyl ions) [ C. latifolia. Antibacterial effect against gram-negative and gram-positive bacteria could be as natural source for producing pharmacological products. The results of the current study supported the traditional treatment by medicinal plants and proposed antibacterial agents from plant extracts with antibacterial properties.Based on our research, the antioxidant activities of this plant from leaves and tubers have not been reported. Even then, slight differences in the antioxidant activities do occur that solely depend on varieties, location, and growth conditions. Overall, in the estimation of the antioxidant capacities and the free radical scavenging assays showed positive results. The percent of inhibition in the DPPH assay and SOD activity were found more from tuber and leaf extraction  . The antyl ions)  C. latif C. latifolia tuber extracts in vivo and in vitro as compared with leaf extract, respectively.The maximum activity was observed against gram-negative and gram-positive bacteria for C. latifolia (in vivo and in vitro) showed strong activity against most of the tested bacterial strains. The results were compared with standard antibiotic drug.Antimicrobial properties of medicinal plants are being increasingly stated from various parts of the world. Based on The World Health Organization report, the plant active constituents are used as folk medicine in traditional therapies of 80% of the world's population. In this study, the tuber extracts obtained from Curculigo latifolia Dryand has the most effective antibiotics against all the studied bacteria compared with other explants.The effect of antibacterial in medicinal plants varies intensely depending on the phytochemical features of plant families and subfamilies and even the grown area , 34. Our in vivo gave higher results for antioxidant activity and inhibition zone against gram-negative bacteria compared to the same concentration of callus extract from tuber and leaf.Combinations of BAP and IBA in MS media exhibited the highest average numbers of root and shoot formation from leaf and tuber explants. Our data revealed that frequent subculture was effective in reducing phenolic exudation; also, pretreatment of tubers with citric acid could eliminate browning during culture period. This study also showed that tuber extracts from in vivo or in vitro, are recommended to determine the characteristics of this species.Further and more specific studies,


The infusion of a combination of IBU and FUR increased the fractional removal of IS from 6.4 ± 0.1 to 18.3 ± 0.4%. IAA removal rose from 16.8 ± 0.3 to 34.5 ± 0.7%. TRP infusion increased the removal of IS and IAA to 10.5 ± 0.1% and 27.1 ± 0.3%, respectively. Moderate effects were observed on HIPA removal. Pre-dialyzer infusion of PBUT binding competitors into the blood stream can increase the HD removal of PBUTs. This approach can potentially be applied in current HD settings.Protein-bound uraemic toxins (PBUTs) cause various deleterious effects in end-stage kidney disease patients, because their removal by conventional haemodialysis (HD) is severely limited by their low free fraction in plasma. Here we provide an experimental validation of the concept that the HD dialytic removal of PBUTs can be significantly increased by extracorporeal infusion of PBUT binding competitors. The binding properties of indoxyl sulfate (IS), indole-3-acetic acid (IAA) and hippuric acid (HIPA) and their binding competitors, ibuprofen (IBU), furosemide (FUR) and tryptophan (TRP) were studied in uraemic plasma. The effect of binding competitor infusion on fractional removal of PBUT was then quantified in an  With better detection technologies, a broad spectrum of retained solutes in dialysis patients’ blood has been identified and characterized in recent studies12356787The removal of PBUTs is a major challenge for current HD technology. The removal of such solutes in conventional HD primarily relies on diffusion of the free molecules into the dialysate, which is severely limited for PBUTs due to their low free fraction and hence small diffusion gradient. For some strongly bound uraemic toxins, clearance is undetectable during a regular HD session101213Several new approaches have been reported in recent publications to improve the dialytic removal of PBUTs. Longer dialysis sessions1312in vitro using human serum albumin solutionex vivo dialysis model, and to determine whether the presence of red blood cells or endogenous albumin ligands in human plasma has any appreciable impact on the effect of PBUT displacement in comparison to previous in vitro studies with human serum albumin.Here we propose another innovative method for improving dialytic removal of PBUTs. Our method is based on the observation that albumin-binding ligands can influence the binding properties of albumin to other ligands through direct competition for binding sites, or by allosteric mechanisms1819181921222324Ibuprofen, which possesses the highest binding affinity among the displacers tested in the study, increased the free fraction of both IS and PCS approximately 3-fold in uraemic plasma, higher than the free fraction generated by tryptophan (about a 2-fold increase) and furosemide (about a 1.3-fold increase) . Additio27L-Tryptophan shares the same primary site as IS and PCS, while furosemide shares the same primary binding site as hippuric acid (HIPA). Thus, tryptophan displaces IS and PCS more efficiently than HIPA . In contWhile furosemide alone at a concentration of 0.18 mmol/l did not induce a significant increase in the free fraction of IS , the samin vitro HD model (P < 0.0001). Tryptophan infusion was able to increase the removal of IS and IAA by a factor of 1.4 (P < 0.0001) and 1.3 (P < 0.0001), respectively. HIPA removal, on the other hand, was only slightly increased by infusion of the ibuprofen/furosemide combination, and virtually unaffected by infusion of tryptophan  when used in dialysis patients, as well as effective at displacing PBUTs. The ideal displacer would induce a significant increase in PBUT removal during HD, and yet be rapidly metabolized into biologically inert compounds that are readily removed by HD or eliminated via biliary excretion.We successfully demonstrated the feasibility of a novel potential therapeutic method of enhancing the removal of highly protein-bound uraemic toxins in HD with the use of displacers. Non-protein-bound substances are not affected by the described displacement method. Of note, we are not advocating the routine clinical use of these particular displacers in a clinical setting. They serve merely as model compounds to demonstrate the concept of displacement therapy for lowering PBUT levels during HD. The primary purpose of this study was to demonstrate the feasibility and efficacy of the PBUT displacement approach in a setting comparable to the one encountered in clinical routine HD. Future clinical pilot trials will be required to document the Rapid Equilibrium Dialysis (RED) cartridges  were purchased from Pierce Thermo Fisher . Acetonitrile (HPLC grade) was obtained from Fisher Chemical . Furosemide, IAA, HIPA, IS, indole, tryptophan, p-cresol and PBS were obtained from Sigma . PCS was synthesized following the protocol of Feigenbaum and NeubergNormal human plasma with heparin (from a six-donor pool) was purchased from Bioreclamation . Twenty-four heparinized uraemic plasma samples from 18 HD patients (obtained from a de-identified bio-repository) were pooled to create a uraemic plasma pool. The pooled uraemic plasma contained endogenous  23.6 ± 5.6 μmol/l tryptophan, 181.9 ± 6.2 μmol/l IS, 326.5 ± 74.1 μmol/l HIPA and also PCS at a level not quantitated. De-identified human whole blood and informed consent were obtained in cooperation with a local blood bank from healthy volunteer donors  on the morning of the experiments. The whole blood was heparinized with 15 IU/ml unfractionated heparin .All experiments were performed in accordance with relevant guidelines and regulations.2 surface area with the same pore size distribution as Optiflux® F160NR fibers and 15.5 cm fiber length. Peristaltic pumps (Watson-Marlow 120U) were obtained from Watson-Marlow Fluid Technology Group North America . A syringe pump for the displacer infusion was purchased from Kd Scientific Inc. . Plasma and dialysate samples were collected using the Sarstedt S-Monovette® Blood Collection System (EDTA) .Dialysis experiments were performed using miniature polysulfone dialyzers produced by the Biotechnology Research Group, Dialyzer R&D, of FMCNA in Ogden, Utah, USA. The specifications of the dialyzers are 0.039 mWe evaluated the albumin binding of uraemic toxins and their interaction with displacers in human uraemic plasma utilizing the RED device. Candidate displacers were mixed with uraemic plasma before incubation. The test mixture (300 μl) was added into the sample chamber, while the buffer chamber was filled with 500 μl PBS. After 4 hours of incubation , samples were collected from both chambers for HPLC analysis. The concentration of all displacers and negative control (antipyrine) in the incubations was 1 mmol/l. For furosemide, incubations at final concentration of 0.18 mmol/l were also included since this is the clinically relevant concentration for intravenous administration.Normal plasma was spiked with IS (150 μmol/l) and mixed with ibuprofen over a concentration range from 0 to 931 μmol/l to generate a dose-dependent displacement curve using the RED device. Further experiments were performed with the addition of furosemide , to test the hypothesis that addition of furosemide to ibuprofen would produce greater displacement of IS.Ibuprofen and furosemide doses were calculated to achieve a range of plasma concentrations that one might encounter when administering these drugs into the extracorporeal circuit following clinical patient-dosing protocols. Infusion of 800 mg ibuprofen into the arterial line over 30 min, with a Qb of 200 ml/min and a hematocrit of approximately 31%, would result in a plasma concentration of approximately 931 μmol/l . Infusion of 250 mg furosemide over 30 min in this setting would results in a plasma concentration of 182 μmol/l.Dialysis experiments were conducted with a volume of 300 ml of heparinized human whole blood spiked with uraemic toxins, referred to as uraemic blood in the study. IS, IAA and HIPA were added to achieve final plasma concentrations of 150 μmol/l, 15 μmol/l and 400 μmol/l, respectively, except in the experiments with tryptophan as a displacer, where a HIPA concentration of 1 mmol/l was used.Experiments were conducted at 37 °C, with a Qb of 12.5 ml/min and counter-current dialysate flow at a Qd of 25 ml/min. Blood and dialysate were perfused through each compartment in single-pass. An infusion pump was connected between the blood pump and dialyzer blood inlet. The tube segment between the infusion site and the dialyzer blood inlet was 45 cm long, with a 3 mm inner diameter, allowing a 15-second transit time between displacer infusion site and dialyzer inlet.The system was primed prior to each experiment, initially with saline, and then with “blank” whole blood without uraemic toxins in the blood circuit, and with dialysate in the dialysate circuit. With the blank blood in recirculation, we manually adjusted the trans-membrane pressure between two circuits so there was zero net ultrafiltration at the beginning of the experiment. Blank whole blood in the circuit was replaced with uraemic blood at the start of the experiments. Details of the experimental model setup are illustrated as in Human whole blood containing uraemic toxins was circulated for 10 minutes without displacer to measure the baseline uraemic toxin single-pass removal. At 10 min, tryptophan, ibuprofen/furosemide, or PBS were infused into the blood line. The target level of tryptophan was 1 mmol/l in plasma, and 650 μmol/l ibuprofen and 129 μmol/l furosemide in whole blood. The infusion concentrations of ibuprofen and furosemide were determined according to the manufacturer’s prescription recommendation as described above. The infusion rate was 70 μl/min for ibuprofen (116 mmol/l) /furosemide (23 mmol/l), 138–150 μl/min for tryptophan (50 mmol/l), and 146 μl/min for PBS.One milliliter samples for analysis were collected from the dialyzer blood inlet, blood outlet and dialysate outlet at 1-minute intervals throughout the experiment.Blood samples were centrifuged at 13200 rpm for 3 mins. The plasma layer was collected and stored at −80 °C. Plasma samples (50 μl) were precipitated with 100 μl of ice-cold acetonitrile to precipitate the proteins. The samples were then vortexed thoroughly and centrifuged at 3000 rpm for 10 minutes. The supernatant, containing the uraemic toxins and displacers, was collected and further diluted 1:1 with deionized water before HPLC injection. The dialysate samples (100 μl) were diluted with 100 μl water before HPLC injection.Measurements were calibrated with standard curves for all analytes, and were linear over the concentration range of the experiments. Measurements were performed on an Agilent 1100 system  equipped with a C18 column . Mobile phase A: ammonium formate, 20 mmol/l, pH 4. Mobile phase B: acetonitrile. The gradient profile for mobile phase B was: 0–2 min, 15%; 11–14 min, 100%; 14.2–16 min, 15%; and the flow rate gradient was 0–11.2 min, 0.4 ml/min; 11.2–16 min 0.5 ml/min. The fluorescent detector was programmed with the following timetable: 0–9 min, excitation at 280 nm and emission at 360 nm, for detection of tryptophan and IS; 9–10.5 min, excitation at 214 nm and emission at 309 nm for PCS; and then for the remainder of the HPLC run, excitation at 280 nm and emission at 360 nm, for detection of IAA and ibuprofen. HIPA and furosemide were detected at 230 nm with the UV-Vis detector. The injection volume was 5 μl.Urea was measured on Envoy clinical biochemical analyzer using Thermo Fisher Infinity urea liquid stable reagent.PBUT removal from whole blood was expressed as fractional removal, calculated as the amount per unit of time leaving the dialysate outlet, as a percentage of the amount per unit of time entering at the blood inlet, in accordance withwhere Cpi and Cdo are the concentrations of each uraemic toxin in the plasma inlet and dialysate outlet streams, respectively. Qd is the dialysate flow rate, and Qp is the plasma flow rate calculated from blood flow rate and hematocrit according to Qp = Qb·(1-Hct). Urea removal was specified as the single pass extraction ratio (ER) in percent, calculated aswhere Cpi and Cpo are the plasma concentrations in the blood inlet and blood outlet streams, respectively.For PBUT displacement in RED assays, Dunnett’s post-analysis comparisons to PBS were performed by one way ANOVA using GraphPad Prism .For the PBUT displacement experiments with the HD model, data were converted into the percent change during the intervention phase, compared to the baseline phase for that experiment. After pairing similar observations in the two phases for an individual experiment  and calculating the paired differences, the value for each observation was divided by the corresponding paired difference SD. This transformation created independent observations with theoretically equal variances (within patients and between patients) for the paired differences under the null hypothesis. Each paired t test was two-tailed; a P value less than 0.05 was taken as statistically significant. The data from the first 2 minutes of each phase (baseline and intervention) were excluded to restrict the analysis to data with stable values during that phase. Calculations were done with SPSS 16.0 on an IBM-PC.How to cite this article: Tao, X. et al. Improved dialytic removal of protein-bound uraemic toxins with use of albumin binding competitors: an in vitro human whole blood study. Sci. Rep.6, 23389; doi: 10.1038/srep23389 (2016).


Plasmodium falciparum  from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression.Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate  P. falciparum malaria was typically based on the detection of a P. falciparum-specific antigen, namely, the histidine rich protein II 7189P. falciparum does not naturally express the P. falciparum HRPII gene8For diseases of unknown causes , proteomics12325Antibodies in serum are ideal biomarkers for diagnosis to avoid the false-negative problem described above. Epitopes, as the antibody recognition region of the antigen, could be used for antibody detection. The diagnostic value has been long predicted but not realized. Many epitopes have been identified from extensive studies on malaria121314P. falciparum proteins . The iPDMS membrane provides a near “zero” background for serological assays, even without blocking treatment  in length with 15 aa overlapping (abbreviated as 30/15 aa thereafter). A heat map was obtained by converting the resulting SNR value to grayscale . Signifivs. peptides) was converted to a 1/0 matrix , which is in agreement with the previously reported 2% value of B-cell linear epitopeset al. using phage displayFirst, for the ease of algorithm design, SNR values larger than the cutoff (SNR ≥ 2) were converted to 1, otherwise to 0. As a result, the SNR matrix , SAM succeeded in extracting peptides that show different responsive rates for different serum subgroups : a totallustered . ECPs wilustered  were finDi = 0; if SNR ≥ 2, Di = 1, where D represents the digital diagnosis and i represents a peptide. We do not judge whether this response is due to specific interaction. Indeed, an SNR of 2 could be due to low concentrations of specific antibodies  but is more likely due to nonspecific interactions because there are as many as 105 different antibodies in human serum . Instead, we rely on probability to determine if the response is from specific interactions.First, we defined a universal binary cutoff value of SNR = 2 as the indication of being responsive . For more than 10,000 microarrays assayed, the mean SNR of blank dots  was 0.5 ± 0.3, so the value of (blank + 3 std) is approximately 1.4. We chose 2 as a strict and more conservative cutoff. This stable and near zero background value is critical for the binarization treatment: if a peptide has SNR < 2, we assign Dsum is a variable similar to the role of SNR and n is the digit-cutoff value for the digitized diagnostic microarray. For example, if the digit-cutoff n = 2, Dsum = 0 and 1 indicate healthy , and Dsum ≥ 2 indicates P. falciparum infection . A surprising improvement was observed when we set the digit-cutoff to n = 2; both the sensitivity and specificity increased from below 73.2% to 92.7% and from 98.4% to 99.2%, respectively . When we set the digit-cutoff n = 2, any two responsive peptides indicate a positive result. The 8 peptides thus gave 28 combinations  colorization, which removes the need for an instrument. One more critical improvement is the use of chimeric epitopes constructs. For 6 ECPs with identified epitopes, we created 3 chimeric epitope constructs that have improved sensitivity . BLAST results indicated that P. falciparum and P. vivax shared homology for those 5 peptides and for many of the other peptides that we screened (Extended Data Table 5). To differentiate P. falciparum and P. vivax infection, we applied the DEIFS to an additional two proteins with low homology, namely, P. falciparum and P. vivax CSP  and hand-foot-and-mouth disease , as long as the immune system responds to the infection by producing antibodies. Although a large-scale linear B-cell epitope screening is the prerequisite for such digitized diagnostic microarray development and requires a certain cost, the overall cost could be shared because the microarray may also find a use in the discovery of vaccine candidates, the evaluation of vaccine potency, and the study of disease progression. There are still many infectious diseases lacking proper diagnostic tools, especially neglected tropical diseases, because the identification process involves genomics, protein engineering and many other disciplinesDEIFS is readily applicable to other infectious diseases, especially to those neglected tropical diseases that cause significant economic burden and humanitarian crises in less developed countries, by producing a chip that contains 1000 thousand peptides and is able to screen 200 pathogens in one test.Microscopy is still regarded as the gold standard for the diagnosis of all P.falciparum infected serum, 176 samples of P.vivax infected serum were collected from southwest area of China, which were confirmed by the microscope method; 214 (125 + 89) negative samples of healthy serum were collected from local hospitals. Those samples were used as the training group for seroscreening. 244 samples of P.falciparum infected serum, 215 samples of P.vivax infected serum, 1043 negative samples of healthy serum were obtained from Institute of Malaria Control in Yunnan province, China, which were confirmed by the microscope method. Those samples were used as the testing group for seroscreening (Extended data Table 2). Informed consent was obtained from all the subjects.A total number of 924 malaria infected serum and 1257 healthy serum were used in this study.289 (179 + 110) samples of P.falciparum infected serum were collected at ring stage, a few at gametophyte stage. P.vivax infected serum samples were collected at erythrocytic stage . Randomized, double-blind, parallel-controlled trials was conducted for test group study.All malaria infected serum samples were collected after onset. Most of the iPDMS membrane−1 stock solution and then diluted into 200 μg mL-1 with printing buffer  for further printing. iPDMS membranes were first activated with 0.1M EDC and 0.1 M NHS mixtures for 30 min and then rinsed with Milli-Q water and used for printing immediately. For the training group, we used a homemade reaction chamber to conduct a double side screening for repeated experiment . Microarray was prepared using contact printer Smart 48  with about 0.6 nL printing solution for each sample. All the peptide samples were printed in single to form 7 × 7 × 4 array, each sub-array has positive control with H-IgG at the concentration of 100 μg mL−1 and negative control with printing buffer .For the test group, microarray was prepared using non-contact printer sciFLEXARRAYER S1  with 200 drop of 0.4 nL printing solution for each peptide in triplicates. H-IgG was also spotted as the positive control at a concentration of 100 μg mL−1 and printing buffer was spotted as the negative control.Microarray was prepared in a 100,000 grade clean room. Peptides were first dissolved with 30% acetonitrile solution  to 1 mg mL°C). Microarray incubated with serum-dilution buffer was conducted as negative control. The microarray was then rinsed for 3 times with washing buffer and incubated with 200 μL of 1 mg/mL HRP-IgG diluted 1:20000 in Peroxidase Conjugate Stabilizer/Diluent for another 30 min on the shaker , followed by the same washing steps as described above. 15 μL of Chemiluminescence substrate was added onto the microarray and the Images were taken at a wavelength of 635 nm using LAS4000 imaging system . Data analysis were conducted according to the workflow of Extended data Fig. 2d–h. For the RDT experiment, 100 μL serum sample of 1 : 10 dilution were incubated for 15 min on the shaker , rinsed for 3 times with washing buffer and incubated with 100 μL of 0.8 mg/mL HRP-IgG diluted 1 : 8000 in Peroxidase Conjugate Stabilizer/Diluent for 15 min on the shaker , followed by the same washing step. 100 μL TMB chromogenic reagent was added onto the microarray and stand for 3 min to read the results.Serum was first diluted 1 : 200 with serum-dilution buffer  and 200 μL was added into each peptide microarray, incubated for 30 min on the shaker . Significant analysis of microarray (SAM) is performed using “samr” package. Cluster analysis and heat map are performed using “pheatmap” package. Violin plot is performed using “beeswarm” package. Receiver Operator Characteristic curve (ROCC) is performed using “OptimalCutpoints” package. Sensitivity and specificity were calculated according the following formulas: Sensitivity = True positive/ × 100%; Specificity = True negatives/ × 100%. The positive predictive value (PPV) is defined as PPV = True positive/ × 100%; The negative predictive value (NPV) is defined as NPV = True negative/ × 100%. Evaluations of statistically significant differences between the tests were based on sensitivity, specificity, negative and positive predictive values. The area under the curve (AUC) was used to validate diagnostic application of the 8 ECPs (Extended data Table 6). Agreement between categorized indices was assessed by Cohen’s kappa coefficient . (Extended data Table 7)The statistical analysis were all computed by R version 3.0.0 .


Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression.The presence of methyl groups on cytosine nucleotides across an organism’s genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the 2 = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions.We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation  contains supplementary material, which is available to authorized users. DNA cytosine methylation is an epigenetic modification that acts in conjunction with histone modification and small RNAs to regulate gene expression –3 and coArabidopsis thaliana, CG DNA methylation in regulatory sequences is negatively correlated with gene expression [Arabidopsis genes within differentially methylated regions tended to be more highly expressed in individuals with increased CG methylation, but lower in individuals with increased non-CG (CHG and CHH) methylation [Recent work has greatly expanded our knowledge of the mechanisms involved in maintaining and modifying DNA methylation in plants –18, yet pression , 19, pospression , 10, 20,pression . Similarpression , and maipression . Additiohylation . Howeverhylation .Saccharomyces cerevisiae, and [The standard method for characterizing genomic patterns of DNA methylation is to classify genes into methylation quantiles and then compare gene expression across these groups , 26–29. iae, and  for a SaMimulus guttatus have demonstrated transgenerational epigenetic inheritance [M. guttatus methylome. We utilize a novel modeling approach to untangle the complex interactions between methylation and gene expression. We show that non-CG gene body methylation may have a significant effect on gene expression despite occurring at relatively low levels. Utilizing a GO term enrichment approach, we demonstrate that certain functional categories are over-represented in genes with high gene body CG methylation. We provide evidence that there are differences in methylation and gene expression between chromosomes, such that mean gene expression is significantly lower across some chromosomes than others. Finally, we look at transcriptional start sites across the genome, where recent evidence suggests increased recombination in M. guttatus [Previous studies of eritance –35. Herberitance , has maderitance –39. Howeguttatus , and finM. guttatus Iron Mountain inbred line, IM62, the line that was sequenced to establish the M. guttatus reference genome [http://phytozome.jgi.doe.gov). When the second leaf pair of seedlings was just visible we collected leaf tissue from multiple seedlings, flash froze it in liquid nitrogen, and stored it at −80 °C. We preformed DNA extractions using a CTAB protocol [M. guttatus was treated with bisulfite using EZ DNA Methylation kit from Zymo Research. Two rounds of random primer extension for tagging bisulfite treated DNA with adaptors were performed using primers for single-end library construction as described in [We germinated seeds from the e genome  ( http:/protocol . We poolprotocol . From thprotocol . With 1 ribed in . The conhttp://itolab.med.kyushu-u.ac.jp/BMap/index.html) to map bisulifte treated reads to the M. guttatus v2.0 reference genome (http://phytozome.jgi.doe.gov). In short, BMap first searches candidate genomic loci for each read in two duplicated genome sequences, one with every C in the genome converted to a T (C2T), and one with G to A (G2A), using an approach called adaptive seed [We used the software BMap  , exon length , number of exons, and interaction terms up to the third degree. Gene length, intron size, and intron number are all known to be positively correlated with gene expression in plants , oppositM. guttatus genome from the repeat-masked genome assembly [We identified transposable elements across the assembly . GenomicMimulus genomic studies eg. [M. guttatus is higher in all contexts than Oryza sativa [Arabiopsis thaliana [Brachypodium distachyiom [Solanum lycopersicum [Zea mays [Glycine max [M. guttatus than Z. mays and G. max, the opposite is true for CHG methylation. CG methylation is highest in Z. Mays, moderate in M. guttatus, and lowest in G. max . This proportion is typical for dies eg.  given tha sativa , Arabioptachyiom , lower ipersicum , and botcine max  dependinM. guttatus chromosomes are given by the location and density of centromeric repeats [13,17042 = 4.43, CG: F13,17042 = 10.85, CHG: F13,17042 = 19.07, CHH: F13,170423 = 6.10, p < 0.001)). Chromosomes that have on average higher levels of methylation tended to also have lower gene expression  in all three regions is likely due to the involvement of similar enzymatic machinery in the propagation of both types of non-CG methylation [The methylation levels in all contexts  and genic positions  at a given gene were significantly correlated with one another Fig. . These whylation . The exthylation .Fig. 4Co2) 20.1 % of the variation in log transformed expression values , and the minimum calculated R2 calculated in 3-fold cross-validation was 17.9 %. Generally, there is an excess of genes predicted to be expressed at log-transformed values between 1.5 and 2.5, that were actually expressed at levels less than 1.2, as well as genes expressed above 4, which this model never predicts  based on minimum BIC. Out of a possible 454 parameters, the minimum BIC criterion selected a model with 29 factors that explained (RLinear Effects: log(GE) = 2.61 − 0.07mcg = f  1, where mcg is gene body CG methylation and GE is gene expression. Controlling for gene architecture and other forms of methylation, we observe a negative linear effect of gene body CG methylation on gene expression  is −0.07 (Table mcg = − 1 (32 %) is predicted to have 35 % higher expression than one with mcg = 1 (80 %)  on gene expression, and leads to a predicted local mcg maximum for gene expression . As gene methylation increases or decreases relative to a moderate 45 % methylation, gene expression is expected to decrease  , lead to a change in our linear mcg coefficient. For example, at ucg = 1 (82 %), 0.2mcg is subtracted from our earlier model, we are left with:ucg = 1 (82 %) we find that the local maximum for gene expression is at mcg = − 0.62 (40.2 %), while at ucg = − 1 (24.1 %) the local maximum for gene expression is at mcg = − 0.29 (48.8 %). As up-stream CG methylation decreases  = f 5: While only up-stream CG methylation showed a negative interaction with gene body CG methylation, three terms have positive linear interactions: Up-stream CHH methylation, gene body CHH methylation, and exon length. These can be treated in much the same way as our negative interaction term. Depending on the values of these terms, they can offset each other and lead to the removal of any interaction effect. For example if exon length (lexon) = − 1 and up-stream (uchh) and gene body (mchh) CHH methylation = 1 these positive interaction terms cancel out (−.4 + .2 + .2 = 0). However, if we consider them varying in the same direction, they can have a striking effect on the relationship between gene body CG methylation and gene expression. At uchh = mchh = lexon = 1 (and the negative interaction term ucg = 0), we see the local maximum is at a methylation level of mcg = − 0.05 (55.1 %). If our negative interaction term ucg = − 1 , this increases to mcg = + 0.05 (57.3 %)  the local maxima for gene expression does so as well). When uchh = mchh = lexon = − ucg < 0, gene body CG methylation is almost purely repressive. At a summed interaction value less than −0.7 there is no longer a local maximum, and CG methylation has a purely negative effect on gene expression.Quadratic Interaction Terms: log(GE) = f 5+.03mcg2lexon = f 6. Finally the interaction between the quadratic gene body CG methylation term and exon length is included in this model. As our quadratic term increases, not only does the position of the local gene expression maximum increase, so to does the inflection point (the point at which the function changes from concave to convex). Now, at the same linear interaction values tested above (uchh = mchh = lexon = − ucg = 1), our local maximum occurs at mcg = 0.07 (57.8 %) , we find that the local maximum for gene expression occurs at mcg = 0.90 (78.1 %) and at lexon = 4 (6kb) there is no longer a local maximum for mcg, and the highest expected gene expression occurs at mcg approaching 100 %  Fig. . As exonIndividual effects of interaction terms: log(GE) = f 5 + .08lexon − .02lexon2 = f 6: Finally, we consider the effect of multiple terms simultaneously. Up until this point we have only included gene body CG methylation effects, and its interaction terms, while not including the independent effects of the term with which it interacts. Independent of gene body CG methylation, we find that gene expression tends to increase as the standardized exon length increases from −1 (500bp) to 2 (2kb), and beyond this point we expect a decline. In the absence of interaction terms, only considering independent effects of gene body CG methylation and exon length, we would estimate that peak gene expression occurs at an exon length of 2kb, and methylation of 45 %. Here we show that the effect of gene body CG methylation on expression is extremely size dependent, and that gene expression is expected to be highest for large highly gene body CG methylated genes, but lowest for small highly gene body CG methylated genes  in this model the results quickly become difficult to conceptualize or visualize, yet through full-model construction, followed by simplification methods as presented above it is possible to decipher complex higher order regulatory interactions. We briefly discuss the effects of the other significant gene size and methylation terms in this model.Intron length shows significant first, second, and third order effects with a gene expression peak at an intron size of approximately 1700 base pairs. Additionally, a positive interaction term with both exon length and number of introns suggests that generally, longer genes with more introns tend to be more highly expressed. Although relatively large genes do tend to be most highly expressed, there are negative quadratic terms for both exon and intron length that suggest after a certain point, increasing exon and intron length should be associated with decreased gene expression.Gene body CHG methylation had significant linear, quadratic, and cubic independent terms, and an exon length interaction term. Gene body CHG methylation has a negative effect on gene expression across nearly its full range of possible values Fig. , and it Gene body CHH methylation was found to have a negative effect on gene expression Fig. , but a pWhile it has been suggested that non-CG gene body methylation may be misattributed to genomic regions that are actually pseudogenes or paralogs , 56, herAlong with a negative interaction with gene body CG methylation, up-stream CG methylation also has a direct negative effect on gene expression Fig.  and a neArabidopsis similarly found that there was a positive correlation between gene expression and regulatory CHH methylation  [A previous study in amework) . They poWhile the traditional method of looking for simple associations between methylation state and gene expression has provided some insight into epigenetic regulation, here we demonstrate that modeling approaches can provide additional insight into these systems. We explain a surprisingly high (20.1 %) amount of the variation in log(gene expression) simply through methylation and gene architecture variation. We considered a potential 454 parameters in our model before settling on 29, but it is important to note that many other factors such as presence of enhancers within the gene body and distance to transposable elements, likely also modify the role of methylation on expression. By considering exon and intron length within this model we take the first steps to account for these potential confounding factors of methylation on expression. It is worth stressing that the gene expression and methylome data were not only collected from different individuals, but also different genetic lines, using different vegetative tissue types, and grown under slightly different greenhouse conditions. It is certainly possible that a similar model, tuned across multiple paired methylome and gene expression samples, could predict gene expression with greater precision. This portion of gene expression variation explained represents that which is at least relatively stable across individual genotypes, tissue, and conditions. While here we apply this model to gene expression on a gene-by-gene basis, through altering the response variable to another parameter of a gene, such as it’s mutation rate, gene expression variance, or the tissues in which it is expressed, this model could be extended to look for other roles of DNA methylation on gene function and evolution.Results from this and other , 29, 59 Brachypodium, rice [Arabidopsis [Comparing genes in the top 10 % genome wide for gene body CG methylation with the remainder of the genome, we found numerous gene categories that are either enriched or depleted in our set of highly CG methylated genes. Genes coding for proteins with kinase activity, involved in signal transduction, and nucleotide binding were among those which tended to be highly methylated, while proteins functioning in the thylakoid, plastid, and ribosome, as well as proteins involved in primary metabolism, photosynthesis, and RNA binding tended to be lowly or moderately methylated Fig. . Similarum, rice , and Arabidopsis .Fig. 7GeM. guttatus genetic recombination occurs at higher frequency near gene start sites. In other systems it has been shown that DNA methylation is negatively correlated with recombination [We looked for changes in methylation near gene transcription start sites. We found that CG, CHG, and CHH methylation all were significantly depleted at and around gene start sites Fig. . This debination , and it M. guttatus [Arabidopsis [Mimulus. Here, evidence of depleted methylation near TSS (Table Decreased methylation near transcription start sites (TSS) was one of the earliest discovered phenomena of gene methylation . Howeverguttatus  providesguttatus  identifibidopsis  and ricebidopsis . At the SS Table ; Fig. 8 M. guttatus genome was estimated to be of transposable element sequence, and methylation levels within transposable elements were significantly higher than that of genes, and at similar levels to inter-genic regions . Percent methylation was calculated in each of three sequence contexts. In total, 34 % of the ns Table .Table 5TM. guttatus as is this case in other angiosperms. Found at 2,380 copies, the helB8c family of helitron elements is far and away the most common transposon in the Mimulus genome , is promising and has numerous potential applications. Recent efforts have shown that the plant methylome is relatively stable throughout development , unlike


Background: Greater adiposity is associated with higher blood pressure. Substantial uncertainty remains, however, about which measures of adiposity most strongly predict blood pressure and whether these associations differ materially between populations.Methods: We examined cross-sectional data on 500 000 adults recruited from 10 diverse localities across China during 2004–08. Multiple linear regression was used to estimate the effects on systolic blood pressure (SBP) of general adiposity  vs central adiposity , before and after adjustment for each other. The main analyses excluded those reported taking any antihypertensive medication, and were adjusted for age, region and education.Results: The overall mean [standard deviation (SD)] BMI was 23.6 (3.3) kg/m2 and mean WC was 80.0 (9.5) cm. The differences in SBP  per 1SD higher general adiposity  were greater than for central adiposity , with a 10 kg/m2 greater BMI being associated on average with 16 (men/women: 17/14) mmHg higher SBP. The associations of blood pressure with measures of general adiposity were not materially altered by adjusting for WC and HC, but those for central adiposity were significantly attenuated after adjusting for BMI .Conclusion: In adult Chinese, blood pressure is more strongly associated with general adiposity than with central adiposity, and the associations with BMI were about 50% stronger than those observed in Western populations. Among the relatively lean Chinese adults who were not taking any blood pressure-lowering drugs, greater adiposity, irrespective how it was measured, was associated with higher blood pressure.Measures of general adiposity  provided better prediction of blood pressure than measures of central adiposity .2 greater BMI was associated on average with 16 mmHg higher SBP, which was ∼50% stronger than observed in Western populations.In this Chinese population, a 10 kg/mAmong a subset of individuals who were treated with antihypertensive therapies, the association of SBP with BMI was only ∼1/3 as strong as among those without any such treatments.,,2 higher body mass index (BMI) is associated with ∼10 mmHg higher systolic blood pressure (SBP), throughout the range typically found in Western populations.Controlled trials of weight loss interventions,,,,,,,,,,,,,,,,,,,,Furthermore, there is conflicting evidence—both in China,We examine the associations of blood pressure with several different measures of adiposity among 500 000 adults in the China Kadoorie Biobank (CKB) study.,Details of the CKB design, procedures and ethics approval have been reported elsewhere.2). WC and HC were measured to the nearest 0.1 cm using a soft nonstretchable tape. WC was measured midway between the lowest rib and the iliac crest or, when this was not practicable, 1 cm above the umbilicus . HC was measured at the maximum circumference around the buttocks . WHR was the ratio of WC to HC. Body fat percentage was the fraction of total weight that was estimated to be fat weight by the Tanita body composition analyser using proprietary algorithms. In addition, other derived measures of adiposity were also considered separately, including fat mass (the product of body fat percentage and body weight), fat mass index , and waist-height ratio (WC divided by height).In this report six main adiposity variables, either directly measured or derived, were assessed, including height-adjusted weight, BMI, WC, hip circumference (HC), WHR and body fat percentage. All measurements were made once by trained technicians while participants were wearing light clothes (appropriate for the season) and no shoes. Standing height was measured to the nearest 0.1 cm using a stadiometer. Weight was measured to the nearest 0.1 kg using a body composition analyser , with subtraction of weight of clothing by 0.5 kg in summer, 1.0 kg in spring/autumn and 2.0–2.5 kg in winter. BMI was calculated as the weight in kilograms divided by the square of the height in metres (kg/mwww.bhsoc.org/bp-monitors), after participants had rested in the seated position for at least 5 min. Standard cuff size was used and if the difference between the two measurements was >10 mmHg, then a third measurement was taken withthe last two readings recorded. The mean values of the two recorded measurements were used for the analyses. The chief analyses reported are for SBP, as SBP is a better predictor of vascular mortality than diastolic blood pressure (DBP).supplementary material (available as Supplementary data at IJE online).Blood pressure was measured twice on the unclothed right upper arm using an automated A&D UA-779 digital monitor, recommended by the British Hypertension Society  and blood pressures  or missing data on body fat percentage (n = 241) were excluded. In addition, individuals who reported current use of ACE-inhibitors (n = 7133), beta-blockers (n = 6512), diuretics (n = 1452) or calcium antagonists (n = 14 132) were also excluded, except as shown in To limit effects of any possible measurement error, participants with extreme values of any adiposity measures , adjusted for baseline age (8 categories) and area (10 categories).For ‘categorical’ analyses , 7a, adjFor continuous analyses , 7b, SBP2, with only 4% having a BMI ≥ 30 kg/m2. The mean BMI was about 1 kg/m2 higher for individuals living in urban than in rural areas (24.2 vs 23.1 kg/m2), and slightly higher among women than men (23.7 vs 23.4 kg/m2). Among men, mean BMI was highest at 40–49 years, whereas among women it was highest at 50–59 years  age was 51 (11) years, and the mean BMI was 23.6 (3.3) kg/m59 years . Body faIn contrast, mean (SD) WC was greater in men than in women [81.8 (9.7) cm vs 78.8 (9.4) cm], but it decreased somewhat with age in men and increased strongly with age in women, such that mean WC in old age was approximately similar between men and women . Mean  than in women (129/76 mmHg).BMI, WC and HC were all highly intercorrelated, with all correlation coefficients for pairwise comparisons in the range 0.76–0.86 . Body fa2 higher BMI was associated with 17 mmHg higher SBP in men and 14–15 mmHg higher SBP in women. After additional adjustment (dashed lines) for WC, the strengths of these associations were attenuated by 13–14% in men and by 8% in women.In both sexes, the next strongest associations after basic adjustment were for other measures of general adiposity: BMI, body fat percentage, fat mass, fat mass index and height-adjusted fat mass , and the associations were only slightly attenuated by additional adjustment for WC and HC. After just basic adjustment, the associations for some measures of central or pelvic adiposity were nearly as strong as those for measures of general adiposity: WC, waist-height ratio and HC (4.8–5.0 mmHg per SD in men and 4.1–4.4 mmHg per SD in women). The association was substantially weaker for WHR . However, all of these associations with WC, waist-height ratio, HC and WHR were again largely or wholly obliterated by additional adjustment for BMI.2 in more detail. There was only modest variation in the strength of the association across various population subgroups .Height-adjusted weight was the strongest predictor of SBP, but this variable is not easy to apply in clinical practice. Of the next strongest predictors, BMI and body fat percentage may be clinically the most applicable, but BMI is the simpler of the two and does not require any technically advanced equipment. So, 2 higher BMI . Mean adult SBP is substantially higher in China during winter than summer,Supplementary Figures 2–7, available as Supplementary data at IJE online).The associations of SBP with BMI and body fat percentage were somewhat, but not entirely, independent of each other, with adjustment of each association for the other factor generally attenuating the associations by 40–60% , Chinese Ministry of Science and Technology (2011BAI09B01), Chinese National Natural Science Foundation (81390541). The British Heart Foundation, UK Medical Research Council and Cancer Research UK provide core funding to the Oxford CTSU.Baseline survey:


To quantitate key parameters of the platelet-rich plasma (PRP) product from five commercial canine PRP systems in healthy, adult canines.–t-test with significance established at p = 0.05.A prospective study was performed from January 2013 to April 2014. Five commercial systems were analyzed using 10 healthy dogs per system.p = 0.0088) and System 3 (p < 0.0001), and was significantly decreased for System 2 (p < 0.0001). All five systems significantly decreased the red blood cell concentration (p < 0.0001 for each system comparison). Neutrophil concentration was significantly decreased for System 2, System 3, and System 4 (p < 0.0001 for each system comparison). Neutrophil concentration was significantly increased for System 5 (p = 0.0089).Platelet concentration was significantly increased for System 1 (The systems with the highest platelet yield were System 1 and System 3. System 3 increased platelet concentration while significantly reducing the RBC and neutrophil concentrations. Further study is indicated to assess the efficacy of PRP therapy in canines, the efficacy of canine PRP systems, and the clinical applications for PRP therapy in dogs. Platelet-rich plasma (PRP) is an autogenous fluid concentrate composed primarily of platelets and growth factors. Recent studies have shown PRP to mediate healing by supplying growth factors, cytokines, chemokines, and other bioactive compounds –7. InitiPlatelets play roles in both hemostasis and wound healing. Platelets contain granules that release growth factors to stimulate other cells of the body to migrate to the area of trauma, thus facilitating tissue healing. It is the growth factors contained within the platelets that are of significance for tissue healing. These growth factors include platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), transforming growth factor-β2 (TGF-β2), vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), and epidermal growth factor (EGF) , 6. ManyMultiple formulations of PRP have been developed and studied. Previous studies in humans have reported that the ideal PRP product should have anywhere from a four- to sevenfold increase in platelets –4, 6. HoMultiple commercial PRP separation systems have been developed for both human and equine use. PRP products from different commercially available PRP separation systems often have variations in their concentrations of platelets, WBC, and growth factors , 43. FurA prospective study involving two small animal surgery specialty centers was performed from January 2013 to April 2014. In accordance with AAALAC International Rules of Accreditation, this study was performed with the approval of the VOSM Research Committee and with owner consent. In this study, all dogs who participated were client-owned dogs deemed healthy by a veterinarian. All clients volunteered their dog for the study and provided written consent as required by Veterinary Orthopedic and Sports Medicine Group for every study participant. All dogs that participated in the study were directly overseen by a veterinarian to ensure no harm was incurred during study participation.p = 0.01. All data sets were found to be normally distributed. Data were then analyzed using a paired t-test. These values were analyzed using statistical software.p = 0.05.The following five commercial systems were prospectively analyzed and labeled as follows: System 1,n = 7), Border Collie (n = 5), German Shepherd Dog (n = 4), Golden Retriever (n = 4), Rottweiler (n = 3), Boxer Dog (n = 2), Doberman (n = 2), English Bulldog (n = 2), Pit Bull Terrier (n = 2), American Bulldog (n = 1), American Staffordshire Terrier (n = 1), Belgian Malinois (n = 1), Collie (n = 1), French Bulldog (n = 1), Great Dane (n = 1), Keeshond (n = 1), Mastiff (n = 1), Newfoundland (n = 1), Standard Poodle (n = 1), Vizsla (n = 1), and mixed canine (n = 6). There were 6 intact males, 17 neutered males, 8 intact females, and 17 spayed females. The mean weight was 31.6 kg (range 11.3–55.5 kg). The mean age was 6.2 years old (range 1–13 years old).Blood was obtained from a total of 48 different healthy, adult canines. The following breeds were represented: Labrador Retriever (p = 0.0088), which was a 219% increase, and for System 3 (p < 0.0001), a 550% increase. Mean platelet concentration was significantly decreased for System 2 (p < 0.0001), which was a 91% decrease. There was no statistically significant change in mean platelet concentration for System 4 (p = 0.6107) and System 5 (p = 0.0897).A statistically significant difference was found between the mean whole blood and mean PRP product platelet concentrations of System 1, System 2, and System 3 , System 2 (p < 0.0001), System 3 (p < 0.0001), System 4 (p < 0.0001), and System 5 (p < 0.0001). System 2 was found to have the greatest decrease in RBC concentration (98%), followed by System 4 (96%), System 3 (95%), System 1 (85%), and System 5 (37%).A statistically significant difference was found between the mean whole blood and mean PRP product RBC concentrations of all five PRP systems Figure . Mean RBp < 0.0001) and System 4 (p < 0.0001), an 89% decrease for both systems. Mean WBC concentration was significantly increased for System 5 (p = 0.0005), which is a 110% increase. There was no statistically significant change in mean WBC concentration for System 1 (p = 0.2161) and System 3 (p = 0.3439).A statistically significant difference was found between the mean whole blood and mean PRP product WBC concentrations of System 2, System 4, and System 5 Figure . Mean WBp < 0.0001), System 3 (p < 0.0001), and System 4 (p < 0.0001). System 2 yielded a 90% decrease, System 3 yielded an 85% decrease, and System 4 yielded a 98% decrease in mean neutrophil concentration compared to mean whole blood values. Mean neutrophil concentration was significantly increased for System 5 (p = 0.0089), a 59% increase. There was no statistically significant change in mean neutrophil concentration for System 1 (p = 0.9300).A statistically significant difference was found between the mean whole blood and mean PRP product neutrophil concentrations of System 2, System 3, System 4, and System 5 , which was a 62% decrease, and for System 4 (p < 0.0001), a 79% decrease. Mean monocyte concentration was significantly increased for System 5 (p = 0.0032), a 114% increase. There was no statistically significant change in mean neutrophil concentration for System 1 (p = 0.1245) and System 3 (p = 0.7759).A statistically significant difference was found between the mean whole blood and mean PRP product monocyte concentrations of System 2, System 4, and System 5 , which is a 74% decrease, and System 4 (p = 0.0004), a 67% decrease. Mean lymphocyte concentration was significantly increased for System 1 (p = 0.0494), System 3 (p = 0.0260), and System 5 (p = 0.0013). System 1 yielded a 191% increase, System 3 a 220% increase, and System 5 a 267% increase in mean lymphocyte concentration compared to mean whole blood values.A statistically significant difference was found between the mean whole blood and mean PRP product lymphocyte concentrations of all five PRP systems Figure . Mean lyPlatelet-rich plasma concentration results varied among systems. The systems with the highest platelet yield were System 1 and System 3. However, while System 1 yielded a 219% mean increase in platelets and 85% decrease in RBC from baseline, this system failed to reduce neutrophil concentrations. System 3 yields a 550% mean increase in platelets while removing >95% of the RBC and 85% of neutrophils.Multiple formulations of PRP have been developed and studied. Previous studies in humans have reported that the ideal PRP product should have anywhere from a four- to sevenfold increase in platelets –4, 6. HoReducing RBC concentration is thought to be important when developing the ideal PRP product . A recenThe effect of leukocyte concentration in PRP products has also been investigated. Both LR-PRP and LP-PRP have been studied and compared. Recent studies have shown LR-PRP is associated with increased pro-inflammatory mediators, including IL-1β, IL-6, IL-8 IFN-γ, and TNF-α , 40, 41.While an increased neutrophil concentration in PRP is known to have negative effects, the effect of monocytes and lymphocytes remains largely unknown. There was great variation in this study of monocyte and lymphocyte concentrations in the final PRP product. Recent studies suggest that monocytes are associated with an increase in cellular metabolism and collagen production in fibroblasts and a decrease in release of anti-angiogenic cytokines interferon-γ and IL-12 , 48. PlaWhen compared to the previous study evaluating platelet concentration of PRP products from various canine commercial systems, our study found similar results . Three oLimitations of this study include small sample size and limitations inherent to its design. Different dogs were used for each PRP system, and while all dogs were confirmed to be healthy, one cannot assume that the dogs used are representative of the entire canine population. Ideally, the same dog would be used for all systems and all blood samples would be drawn at the same time to eliminate variation in hematological composition. We attribute the variation in results to this as well as inherent patient factors. However, it was not possible to use the same dogs for simultaneous sample collection and processing as the study was conducted at two separate centers. Furthermore, it was also not feasible to obtain the large blood sample required for all systems to be tested simultaneously.The goal of this study was to compare key parameters of the PRP product from five commercial canine PRP systems in healthy, adult canines. No claims regarding the efficacy of PRP therapy in canines or the efficacy of the PRP formulations evaluated can be deduced from this study. Further study is indicated to assess the concentrations of growth factors and cytokines in the commercial canine PRP products and to determine the concentration of platelets and growth factors required for therapeutic effect. Further study is also needed to evaluate the efficacy of PRP therapy and further define its clinical applications in canines.All authors assisted with study development, procedures, data analysis, and manuscript preparation.Regenerative medicine products and systems were received from the companies below for system validation and clinical testing: Arthrex, EmCyte, Harvest, PulseVet, and Canine Regenerative Therapies.


CH), cardiac dysfunction and explore the possible protective mechanisms. We surgically created abdominal aortic constrictions (AAC) in rats to induce CH. Rats with CH symptoms at 4 weeks after surgery were treated with celecoxib [2 mg/100 g body‐weight(BW)] daily for either 2 or 4 weeks. Survival rate, blood pressure and cardiac function were evaluated after celecoxib treatment. Animals were killed, and cardiac tissue was examined for morphological changes, cardiomyocyte apoptosis, fibrosis, inflammation and oxidative stress. Four weeks after AAC, rats had significantly higher systolic, diastolic and mean blood pressure, greater heart weight and enlarged cardiomyocytes, which were associated with cardiac dysfunction. Thus, the CH model was successfully established. Two weeks later, animals had impaired cardiac function and histopathological abnormalities including enlarged cardiomyocytes and cardiac fibrosis, which were exacerbated 2 weeks later. However, these pathological changes were remarkably prevented by the treatment of celecoxib, independent of preventing hypertension. Mechanistic studies revealed that celecoxib‐induced cardiac protection against CH and cardiac dysfunction was due to inhibition of apoptosis via the murine double mimute 2/P53 pathway, inhibition of inflammation via the AKT/mTOR/NF‐κB pathway and inhibition of oxidative stress via increases in nuclear factor E2‐related factor‐2‐mediated gene expression of multiple antioxidants. Celecoxib suppresses pressure overload‐induced CH by reducing apoptosis, inflammation and oxidative stress.To explore the effects of celecoxib on pressure overload‐induced cardiac hypertrophy ( Hypertrophic remodelling characterized by enlarged cardiomyocytes and increase in heart size is an adaptation to stress, especially due to pressure overload. Hypertrophy is the leading cause of various cardiovascular diseases, including hypertension, myocardial infarction, valvular disease and cardiomyopathy Pressure overload‐induced cardiac apoptosis is the initial pathogenic characteristic of CH and remodelling Research suggests that cardiac inflammatory mediators expressed in response to pressure overload induces deleterious changes in the cardiac ECM structure and promotes CH in vitro and shown to have anti‐inflammatory effects in the vascular endothelium. In addition, strong evidence indicates that celecoxib also played an important role in relieving oxidative stress induced by smoking or ischemia/reperfusion via upregulating the expression of multiple antioxidants Celecoxib, a cyclooxygenase‐2(COX‐2)‐selective non‐steroidal anti‐inflammatory drug, approved to treat inflammation associated with rheumatoid arthritis and osteoarthritis in vitro study indicated that celecoxib not only was anti‐inflammatory in the vascular endothelium Research recently confirmed that celecoxib can have therapeutic effects on the heart. Specifically, celecoxib prevented cardiac remodelling in mice with inherited dilated cardiomyopathy The protocol was approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University, Zhejiang, China. All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering of the experimental animals.Male Wistar rats , were obtained from the Experimental Animal Center of Beijing University of Medical Science  and housed in the Experimental Animal Center of Wenzhou Medical University at 22°C with a 12 hrs/12 hrs light/dark cycle, with free access to rodent chow and tap water.After 2 weeks of acclimation, AAC surgery was performed to induce pressure overload‐induced CH via an intragastic tube for 2 or 4 weeks Rats were divided into three treatment groups: sham rats (SO: Group 1); CH control rats (Group 2) and CH/celecoxib‐treated rats (Group 3). Group 3 received celecoxib [2 mg/100 g body‐weight(BW)] daily BP readings  were measured in all animals using tail‐cuff manometry and a BP‐300A non‐invasive BPs monitoring system  at each time‐point. Rats were kept warm on a 37°C heating pad to ensure sufficient tail blood flow tail, and then animals were restrained in a plastic tube restrainer where occlusion and volume‐pressure recording cuffs were placed over their tails. Each rat was allowed to adapt to the restrainer for 5 min. prior to BP measurement. Rats were trained for BP measurement over 3 days with 10 acclimation cycles followed by 20 measurement cycles Transthoracic echocardiography (Echo) with a high‐resolution imaging system for small animals , equipped with a high‐frequency ultrasound probe  was performed on all rats after they were anaesthetized with 1.2% 2,2,2‐Tribromoethanol (ip). Rat hair was removed from the chest region using a chemical hair remover, and bubble‐free aquasonic clear ultrasound gel  was applied to the surface of the thorax to optimize cardiac chambers. Parasternal long‐axis and short‐axis views were acquired. Ejection fraction (EF) and fractional shortening (FS) percentages and, LV anterior wall thickness in end‐diastole (LVAWd), LV posterior wall thickness in end‐diastole (LVPWd), LV end‐diastolic diameter (LVIDd) were calculated by Vevo 770 software. The final data represent the average values of 10 cardiac cycles After killed, hearts were removed and washed in cold saline. The entire LV tissue was isolated and fixed with 10% formalin for 2 days at room temperature. After dehydration in ethanol, the tissue blocks were embedded in paraffin and, 3 μm thick sections were made and stained with haematoxylin and eosin for general morphological examination in situ Apoptosis Detection Kit  For terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) staining, slides were stained with the ApopTag Peroxidase via 10% SDS‐PAGE and then transferred to nitrocellulose membranes. After blocking with non‐fat milk for 1 hr at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: atrial natriuretic peptide , brain natriuretic peptide , β‐myosin heavy chain , connective tissue growth factor , murine double mimute 2 , intercellular adhesion molecule‐1 , plasminogen activator inhibitor‐1 , tumour necrosis factor‐α , nuclear factor E2‐related factor‐2 , haeme oxygenase‐1 , NAD(P)H:quinone oxidoreductase‐1 , nuclear factor kappa B , inhibitor of NF‐κB  and β‐actin (1:1000), which were purchased from Abcam . Phosphorylated‐P53 , total‐P53 , cleaved‐caspase3 , kelch‐like ECH‐associated protein‐1 , mammalian target of rapamycin , protein kinase B , phosphorylated‐AKT at ser 473 (1:500), phosphatase and tensin homologue deleted on chromosome ten  and phosphorylated‐PTEN (1:500) were purchased from Cell Signaling Technology . After three washes in Tris‐buffered saline containing 0.05% Tween 20 (TBST), membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hr at room temperature. Antigen–antibody complexes were then visualized using an enhanced chemiluminescence kit , and the intensity of the protein bands was quantified using Quantity one software .Cardiac tissues were homogenized in lysis buffer  and supernatants were collected by centrifugation at 12,000 × g at 4°C. Equal amounts of protein from each sample was separated NQO‐1: forward, 5′‐GAGAAGAGCCCTGATTGTACTG‐3′; and reverse, 5′‐ACCTCCCATCCTCTCTTCTT‐3′; HO‐1: forward, 5′‐CTCCCT GTGTTTCCTTTCTCTC‐3′; and reverse, 5′‐CTGCTGGTTTCAAAGTTCAG‐3′; β‐actin: forward, 5′‐AGGTATCCTGACCCTGAAGTA‐3′; and reverse, 5′‐CACACGCAGCTCATTGTAGA‐3′. RT‐PCR was carried out in triplicate using the SYBR GREEN PCR master mix (Invitrogen) on a Stratagene MX3000p thermocycler . The amount of mRNA was calculated by the comparative CT method, which depends on the ratio of the amount of target genes to reference gene β‐actin.Total RNA was isolated from heart tissue using TRIzol reagent according to the manufacturer's protocol . The total RNA in each sample was quantified with a Nanodrop 2000 . RNA samples were reverse transcribed into cDNA using a High‐Capacity cDNA Reverse Transcription Kit . The following primers were used for RT‐PCR. n = 8/group) and presented as means ± S.D. One‐way anova was used to assess general differences, followed by a post‐hoc Tukey's test for difference between groups, using Origin 7.5 software for laboratory data analysis and graphing. Statistical significance was considered P < 0.05. The Kaplan–Meier method was used for survival analysis.Data were collected from all rats . Impaired cardiac function in the CH/celecoxib group was comparable to the CH group just after celecoxib treatment. However, the protection on cardiac function of celecoxib was observed after celecoxib treatment at both 2 and 4 weeks (Fig. Next, using the M mode of echocardiography, we studied celecoxib's effect on CH. CH rats after AAC surgery had impaired cardiac function characterized by progressively elevated LVAWd Fig. A and F, eks Fig. . StatistTo assess whether the protective effects of celecoxib on cardiac function were attributed to the prevention of CH. We found at 2‐week time‐point, the ratios of HW/BW Fig. A, HW/TL Because CH is associated with cardiac fibrosis, we examined the fibrotic effect of CH on the heart by Sirius‐red staining for collagen Fig. A and B. Cardiac apoptosis is an initial pathogenic signal of CH. Thus, we measured cardiac apoptosis using TUNEL staining Fig. A and B aCardiac inflammation was determined by measuring the expressions of the classic inflammatory factors including ICAM‐1 Fig. A and B, Oxidative stress is a key contributor to the development of CH, which was confirmed in our study. Malondialdehyde, marker of oxidative stress, significantly increased in hypertrophic hearts at the 2‐week time‐point, which was further enhanced at the 4‐week time‐point Fig. A. HoweveCardiac hypertrophy is beneficial to the stressed heart during the early stage, and is characterized by the enlargement of cardiomyocytes size to ensure adequate cardiac function Cardiac hypertrophy can be induced by various stresses, and pressure overload is one of the most common causes The pathogenesis of pressure overload‐induced CH is complex and is generally considered as the consequence of hypertension‐induced inflammation, apoptosis and oxidative stress. Therefore, inhibition of the above pathogeneses may significantly prevent CH onset and the subsequent heart failure.Celecoxib, an anti‐inflammatory agent indicated for osteoarthritis and rheumatoid arthritis therapy, is the first COX inhibitor specific for COX‐2 Because CH remodelling is the leading cause of cardiac dysfunction, via upregulating the expression of MDM2, P53 antagonist (Fig. Although hypertension is an initial cause of pressure overload‐induced CH ist Fig. D and E.via inhibition of the AKT/‐mTOR/NF‐κB signalling pathway in a time‐dependent manner (Fig. Pro‐inflammatory cytokines are involved in the development of CH because they activate the MAPK pathway, decreasing cardiac contractility, and inducing myocardial interstitial fibrosis ner Fig. . In addiner Fig. .via inhibition of apoptosis, inflammation and oxidative stress.In summary, CH is the early response of heart to stresses, which if left unchecked, is the leading cause of subsequent cardiac fibrosis and dysfunction. Inflammation and the associated apoptosis and oxidative stress are considered the main pathogeneses of CH. In this study, we attempted to identify the cardiac effect of celecoxib, which is currently used as an anti‐inflammatory drug for a variety of non‐cardiac conditions in clinical practice. Our findings demonstrated that celecoxib significantly prevented pressure overload‐induced CH development and cardiac dysfunction No potential conflicts of interest relevant to this article were reported.C.Z., F.W., Y.Z., Y.K., H.W., M.S., L.S., X.X., F.X., F.H., L.Y. and J.X. substantially contributed to research design, or the acquisition, analysis or interpretation of data; C.Z., Y.L. and M.X. drafted the paper or revising it critically; C.Z., F.W., Y.Z., Y.K., H.W., M.S., L.S., X.X., F.X., F.H., L.Y., J.X. Y.L. and M.X. approved the final version of manuscript for submission.