Prevotella was enriched in the healthy group whereas Bacteroides were prevalent in the ESRD group (LDA score > 4.5). 11 bacterial taxa were significantly overrepresented in samples from ESRD and 22 bacterial taxa were overrepresented in samples from healthy controls. The butyrate producing bacteria, Roseburia, Faecalibacterium, Clostridium, Coprococcus and Prevotella were reduced in the ESRD group . Canonical correspondence analysis (CCA) indicated that Cystatin C (CysC), creatinine and eGFR appeared to be the most important environmental parameters to influence the overall microbial communities. In qPCR analysis, The butyrate producing species Roseburia spp., Faecalibacterium prausnitzii, Prevotella and Universal bacteria, were negatively related to CRP and CysC. Total bacteria in faeces were reduced in patients with ESRD compared to that in healthy individuals. The enterotypes change from Prevotella to Bacteroides in ESRD patients. The gut microbiota was associated with the inflammatory state and renal function of chronic kidney disease.We evaluated differences in the compositions of faecal microbiota between 52 end stage renal disease (ESRD) patients and 60 healthy controls in southern China using quantitative real-time polymerase chain reaction (qPCR) and high-throughput sequencing  methods. The absolute quantification of total bacteria was significantly reduced in ESRD patients (p < 0.01). In three enterotypes,  This microbial community forms a natural defense barrier and influences nutrition 4, physiology5, immune function )7, and metabolism 8. Various clinical and animal studies suggest that our gut microbial environment plays a critical role in both maintenance of health and disease pathogenesis, such as in obesity9, diabetes11, non-alcoholic fatty liver disease (NAFLD)10, IBD12, cardiovascular disease13, cancers14, and refractory Clostridium difficile infection15.The human gut is immensely populated with microorganisms, predominantly anaerobic bacteria. This internalized ‘Microbial organ’, which are not encoded in the host genome, consists of at least 1017. CKD patients have enteric bacterial overgrowth and harbor. A greatly increased microbial flora comprises both anaerobes (107 bacteria/mL) and aerobes (106 bacteria/mL) in the duodenum and jejunum, which is quantitatively comparable to those in blind loop. However the composition of these does not differ significantly between the two groups18. An fecal analysis revealed a disturbed composition of microbiota characterized by an overgrowth of aerobic bacteria in hemodialysis patients19. Recent studies have revealed profound alterations of gut microbiota in patients and animals with CKD. Vaziri et al. demonstrated via 16S rRNA genePhyloChip analysis that uremia profoundly alters intestinal microbial flora20. Moreover some studies have suggested the pathogenic role of gut microbiota in kidney disease21. Alterations in the composition of the microbiome and accumulation of gut derived uremic toxins , p-cresyl sulphate (PCS), amines, ammonia, and trimethylamine oxide) contribute to the systemic inflammation, cardiovascular disease and numerous other CKD associated complications23. IS and PCS were associated with elevated levels of selected inflammatory markers  and an antioxidant in CKD patients24 and predict progression of CKD25. Butyrate produced from microbial fermentation is important for energy metabolism and normal development of colonic epithelial cells, mainly has a protective role in relation to colonic disease, and appears to decrease the inflammatory response27. Smith et al.28 found that short chain fatty acids (SCFAs) regulate the size and function of the colonic Treg pool, which play a major role in the pathogenesis of systemic inflammation, maintaining immunological self-tolerance, limiting the inflammatory response to foreign antigens and protecting against colitis. Butyrate regulates the differentiation of Treg cells29. ESRD is compounded by the depletion and dysfunction of regulatory T lymphocytes30. CKD impairs the barrier function and alters microbial flora of the intestine. Bacterial translocation and uremic toxicity as possible sources contributed to the chronic inflammation noted in uremia32. The aim of this study was to evaluate and quantify differences in the composition of gut microbiota in ESRD patients in southern China.Chronic kidney disease (CKD) is widespread, afflicting millions of people worldwide. In China, approximately 119.5 million adults have CKD, making it an important public health problem2 for 3 months, irrespective of the presence or absence of kidney damage33. All ESRD patients were diagnosed in accordance with this guideline by professional kidney internal medicine physicians33. All methods, including the collection of blood and faecal samples, were performed in accordance with the relevant guidelines and regulations. All the people have signed the informed consent. The study was reviewed and approved by the Medical Ethics Committee of the Southern Medical University, Guangzhou, China. Fresh faecal samples collected in sterile containers from 52 ESRD patients and 60 healthy volunteers (controls) were used for quantitative PCR (qPCR), of these, samples from 27 ESRD patients and 26 healthy volunteers underwent Pyrosequencing. The underlying cause of 21 ESRD patients was chronic glomerulonephritis, 11 was hypertensive nephropathy, 6 was obstructive nephropathy, 3 was polycystic kidney disease, 2 was systemic lupus erythematosus, 2 was chronic pyelonephritis, 7 was unclear. Only two of the ESRD patients have received hemodialysis therapy through deep venous catheterization for once before the enrollment because of the acute hyperkalemia. The rest patients have never been treated with dialysis. All ESRD inpatients had never been treated with dialysis or without a regular dialysis. Exclusion criteria included treatment with antibiotics, probiotics/prebiotics and other laxatives in the 4 weeks preceding sample collection. We also excluded cholecystectomy, colectomy or intestinal disease and diabetes and hyperlipidemia from our data. Clinical datas of all the subjects were shown in Table CKD definitions and classifications in this study are in accordance with the 2002 clinical practice guideline, end stage renal disease (ESRD) was defined as the estimated glomerular filtration rate (eGFR) less than 15 mL/min/1.73 mFasting venous blood samples were collected in the morning, and centrifuged at 3000 g/min, at 4 °C for 10 min. The recovered supernatants was separated in 200 μL tubes and immediately frozen at −80 °C. We used the enzymatic method  to measure the creatinine. A modified kinetic Jaffé method was used to measure blood urea nitrogen (BUN) and the CKD Epidemiology Collaboration (CKD-EPI) equation was used to measure estimated glomerular filtration rate (eGFR) values. Cystatin C (CysC) and C-reactive protein (CRP) were measured by immunoturbidimetric assays. Lipopolysaccharide (LPS) was detected with the chromogenic end-point Limulus Amebocyte Lysate (LAL) assay. Plasma cholesterol (CHOL); triglycerides (TG); and high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) cholesterol levels were determined using enzymatic methods.Fresh stools were collected one day after enrollment and frozen at −80°C, patients who did not have a bowel movement were excluded. Two tubes were collected and filled at least 1/3. According to the TIANamp Stool DNA Kit  manufacturer steps to extract the faecal DNA. All DNA samples were stored at −80 °C until further processing.2O. The PCR cycle conditions: an initial denaturation at 94 °C for 5 min, 25 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR products were sequenced using Illumina GAII  at the Beijing Genomic Institute . Sequencing results were clustered by lllumina paired barcoded - sequencing (end) (BIPES) (PE) process for preliminary analysis, the rest of the sequence were screened by UCHIME and removed the suspected chimeric sequence. All reads were sorted into different samples according to their barcodes. Then the two stage clustering (TSC) was used for clustering to extract the OUT in order to to distinguish the high abundance and low abundance sequences. Principal coordinates analysis (PcoA) based on UniFrac distance was performed with QIIME. The linear discriminant analysis (LDA) with effect size measurements (LEfSe) were used to identify indicator bacterial groups specialized within the two groups.Isolated fecal DNA was used as a template for amplification of the 16S rRNA V4-6 region using the universal primer V4F (5′-GTGCCAGCMGCCGCGGTAA-3′) and V6R (5′-ACAGCCATGCNCACCT-3′). 20 μl reaction mixture: 10 μl TaKaRa Premix Taq, 2 μl template DNA, 0.5 μl 10 μM barcode forward primer, 0.5 μl 10 μM reverse primer, and 7 μl double-distilled HEscherichia coli (E. coli) belongs to Proteobacteria. Bacteroides fragilis group belong to Bacteroidetes. Bifidobacterium belong to Actinobacteria. Enterococcus spp., Lactobacillus group and Clostridium coccoides group belong to Firmicutes. Based on the sequencing data, Roseburia spp., Faecalibacterium prausnitzii and Prevotella which are typical butyrate producing bacteria were decreased in ESRD patients, so we chose it. All qPCR primer are listed in Table 38. qPCR assays were performed in a 96-well optical plate on a LightCycler® 480 Real-Time PCR System . All assays were carried out in duplicate. The reaction mixtures consisted of 10 μl TaKaRa Premix Taq, 2 μl template DNA, 0.4 μl 10 μM barcode forward primer, 0.4 μl 10 μM reverse primer, and 7.2 μl double-distilled H2O. The copy number of target DNA was determined by serially diluting standards (101 to 107 copies of plasmid DNA containing the respective amplicon for each set of primers) running on the same plate. Bacterial quantity was expressed as log10 bacteria per gram of stool.The bacteria selected for qPCR are wellknown bacteria in gut. Enumeration data are tested by chi-square. Independent-samples T test was used to analyse the quantitative data. Spearman rank correlation were calculated to estimate the linear correlations between variables. Wilcox test, Kruskal-Wallis, PcoA, LEfSe, Monte Carlo test CCA were used to analyse the sequcing datas. Canonical correspondence analysis (CCA) was performed to measure physiological properties that have the most significant influence on microbial communities. Statistical analyses were performed with the statistical software package SPSS13.0 . P < 0.05 was considered indicative of statistical significance.CysC, BUN, and creatinine was significantly higher, and eGFR was reduced in ESRD patients compared to healthy controls. Levels of the plasma inflammatory biomarker CRP differed significantly between ESRD patients and controls (p = 0.005). LPS was increased in ESRD patients (p = 0.033). The ethic background of all the participants were Han nationally Chinese. All the ESRD patients had been treated with phosphate binders, oral iron supplements or intravenous iron compounds, antihypertensive drugs. 12 of the ESRD patients had been treated with calcium supplements and Vitamin D. There were no significant differences in age, sex, body mass index (BMI), glucose, TG, CHOL, VLDL, LDL, HDL, Lpa, Lipoprotein a (Lpa), apolipoprotein E (ApoE), and apolipoprotein A, B  (Table p > 0.05) as assessed by chao1, observed_species, Shannon, simpson diversity indexs. PCoA based on the UniFrac metric did not reveal a separation trend of healthy controls and ESRD patients  analysis, Bacteroidaceae with p-Cresol production enzymes enriched in ESRD patients. Desulfovibrionaceae, Bacteroidaceae, Alcaligenaceae, Pseudomonadaceae, and Pasteurellaceae produced urease, Bacteroidaceae’s relative abundance was higher, the others were much lower in ESRD patients than the controls group in this study. Microbes of the genus Prevotella, Roseburia, Faecalibacterium, Clostridium, Coprococcus can produce butyrate26, Dorea was the other predominant SCFA-producing genera40. All of these species were reduced in ESRD patients, indicating that bacteria producing SCFAs especially butyrate were decreased in ESRD patients. Bacteroides (enterotype 1), Prevotella (enterotype 2) and Ruminococcus (enterotype 3) were three main enterotypes of human gut microbiota41. In this study, from healthy people to ESRD patients, the enterotype changes from Prevotella (enterotype 2) to Bacteroides (enterotype 1).LEfSe showed so much biomarkers for ESRD patients and controls subiects Fig.  . However, the numbers of beneficial microorganisms from the Lactobacillus group were similar between two groups (p = 0.395). In ESRD patients, universal bacteria were decreased, and the butyrate producing species Clostridium coccoides group, Faecalibacterium prausnitzii, Roseburia spp. and Prevotella were also reduced, consistent with the sequencing results.qPCR was used to assess changes in bacterial absolute quantity in faecal samples from the two groups Fig. . BacteriAmong these, the butyrate producing species Roseburia spp., Faecalibacterium prausnitzii, Prevotella and Universal bacteria, were negatively related to inflammation index CRP  , Firmicutes (~40%) and Proteobacteria (~10%) were the predominant phyla in both healthy individuals and CKD patients, consistent with reports from previous studies among cohorts from Western countries, Africa and Asia40 were diminished in ESRD patients. Members of Prevotellaceae possess phosphotransbutyrylase and butyrate kinase39, and Prevotella can produce SCFAs45. In this study, both Prevotella and Prevotellaceae were reduced in ESRD patients. The human colonic butyrate (Short-chain fatty acids) producers are Gram-positive firmicutes, but are phylogenetically diverse. Clostridiales cluster the XIVa (Clostridium coccoides) including Ruminococcus, Coprococcus, Eubacterium hallii , Eubacterium rectale/Roseburia spp. and Clostridiales cluster IV (Clostridium leptum) including Faecalibacterium prausnitzii, and Eubacterium spp. are normally the two most abundant groups of human faecal bacteria that produce butyrate47. Roseburia, Coprococcus, and Faecalibacterium belong to Firmicutes-Clostridiales. All of them are typically producing butyrate bacteria and were particularly and significantly more abundant in healthy controls and decreased in ESRD patients and consistent with previous studies39. The qPCR analysis of Roseburia spp. and Faecalibacterium prausnitzii showed a similar trend in ESRD. Butyrate gets involved in the adjustment of body reaction to inflammation29. Systemic inflammation in patients with end-stage renal disease (ESRD) is mediated by activation of the innate immune system48. The presence of persistent inflammation magnifies the risk of poor outcome, and is a risk factor for cardiovascular disease (CVD), via mechanisms related to exacerbation of both wasting and vascular calcification processes and self-enhancement of the inflammatory cascade49. High dietary total fiber intake is associated with lower risk of inflammation and mortality in kidney disease50. Interestingly, CRP was increased in ESRD compared with that in controls. Spearman rank correlation analysis demonstrated that the absolute abundance of Roseburia spp., Faecalibacterium prausnitzii, Prevotella and Universal bacteria were negatively associated with CRP level and renal function indexes. These data indicate that bacteria producing butyrate as biomarkers may involve in the pathological process of CKD. Recently, Andrade-Oliveira V proved that SCFAs can reduce inflammation in acute kidney injury (AKI), which supports our inferences51.Distribution of a number of genera could be differentiated between ESRD patients and controls. The SCFAs  are a by-product of the fermentation of non-absorbable complex carbohydrates. Firmicutes- Clostridiales- Lachnospiraceae -Dorea producing SCFAsE. coli, Bifidobacterium, Bacteroides fragilis group, Enterococcus spp., Clostridium coccoides group, Faecalibacterium prausnitzii, Roseburia spp. and Prevotella were decreased in ESRD. Bifidobacterium, Roseburia and Clostridium coccoides45 can produce SCFAs. Bacteroides fragilis and Clostridium spp. can protect against dextran sulfate sodium (DSS)-or trinitrobenzenesulfonic acid-induced colitis53. This suggests that CKD status may influence the absolute quantity of the microbiome, which may result from accumulation of uremic toxins, inflammation and malnutrition and needs further investigation. This reduction in beneficial bacteria may play an important role in the pathogenic processes of CKD.Reduced quantity of fecal microbiota were found in ESRD patients on qPCR analysis, This means that the absolute quantity of total faecal microbiota was decreased in CKD patients. In general, Universal bacteria, 54. LPS may accelerate activation of neutrophils and macrophages/monocytes, which further explain the persistent inflammation of ESRD55. Although most CKD patients presented signs of fluid overload that was associated with endotoxaemia, there was no association between endotoxaemia and systemic inflammation, suggesting the endotoxaemia may not be the main determinant of the inflammatory status in CKD patients56. So the correlation between LPS and inflammation is unclear. In this study, LPS was elevated in ESRD patients, but we didn’t find the correlation between LPS and bacterial amounts. To determine the characteristics of gut microbiota based on kidney function, we excluded the influences of body mass index (BMI), blood lipids, and blood glucose. No significant differences in blood lipid and blood glucose levels were found between CKD patients and controls consistent with that reported in previous study by McIntyre, C. W.57. Further research is needed in this area to provide more conclusive evidence while taking into account the relationships of gut flora with human diet, environment and habits.LPS is derived from the cell wall of gram negative bacteria, and the increase of the gamma Proteobacteria is also effective in increasing the LPS level in circulation. The degree of circulating endotoxemia might be related to the severity of systemic inflammation and features of atherosclerosis in peritoneal dialysis (PD) patients


The overlap of morphological feature and mean ADC value restricted clinical application of MRI in the differential diagnosis of orbital lymphoma and idiopathic orbital inflammatory pseudotumor (IOIP). In this paper, we aimed to retrospectively evaluate the combined diagnostic value of conventional magnetic resonance imaging (MRI) and whole-tumor histogram analysis of apparent diffusion coefficient (ADC) maps in the differentiation of the two lesions.mean), median ADC (ADCmedian), skewness, kurtosis, 10th, 25th, 75th and 90th percentiles of ADC  were evaluated and compared between orbital lymphoma and IOIP. Multivariate logistic regression analysis was used to identify the most valuable variables for discriminating. Differential model was built upon the selected variables and receiver operating characteristic (ROC) analysis was also performed to determine the differential ability of the model.In total, 18 patients with orbital lymphoma and 22 patients with IOIP were included, who underwent both conventional MRI and diffusion weighted imaging before treatment. Conventional MRI features and histogram parameters derived from ADC maps, including mean ADC (ADC10 (P = 0.023) and involvement of orbit preseptal space (P = 0.029) were the most promising indexes in the discrimination of orbital lymphoma and IOIP. The logistic model defined by ADC10 and involvement of orbit preseptal space was built, which achieved an AUC of 0.939, with sensitivity of 77.30% and specificity of 94.40%.Multivariate logistic regression showed ADC10 are valuable in differential diagnosis of orbital lymphoma and IOIP.Conventional MRI feature of involvement of orbit preseptal space and ADC histogram parameter of ADC Orbital lymphoma and idiopathic orbital inflammatory pseudotumor (IOIP) represent the most common lymphoproliferative disorders affecting the orbit , accountPrevious studies have indicated some MRI features, such as signal intensity on T2W sequence, presence of flow void sign, and degree of contrast enhancement, have the potential to discriminate between lymphoma and IOIP , 7, 8. NTherefore, the purpose of this study was to investigate the independent and combined value of conventional MRI features and whole-tumor histogram parameters derived from ADC maps in the differentiation of orbital lymphoma from IOIP.n = 17), tumor subtype (n = 3), and diffuse inflammation subtypes (n = 2).The Institutional Review Board of Shanghai Ninth People’s Hospital approved this retrospective study and the requirement for informed consent was waived. The following criteria were adopted for patient selection: 1) The mass was primary orbital tumor; 2) The patients underwent both conventional MRI and DWI scan before treatment; 3) Masses with short axis ≥ 10 mm; 4) The MR images could be acquired and interpreted. Through a comprehensive search of our institutional medical report database from January 2012 to September 2016, we identified 18 patients with orbital lymphoma  and 22 patients with IOIP . The final diagnoses were made upon histopathological results in 36 patients, and upon response to corticosteroid treatment and minimum 1-year follow-up in 4 patients with presumed IOIPs. 16 cases of lymphomas were MALT lymphomas, and the other 2 cases were diffuse large B-cell lymphomas. Myositis and optic perineuritis subtypes of IOIPs were not included in the present study because they demonstrated characteristic findings on conventional MRI, which were easy to be differentiated from lymphoma. In the current study, the patients with IOIPs included dacryoadenitis subtype  with a 12-channel head coil. The conventional MRI protocol for orbital lesions include axial T1-weigthed images , axial fat-saturated T2-weighted images , coronal T2-weighted images , as well as axial, coronal and sagittal contrast enhanced fat-saturated T1-weighted images . For contrast-enhanced T1-weighted image, a standard dosage of 0.1 mmol/kg of gadopentetate dimeglumine  was administrated.b, 0 and 700 s/mm2.Before contrast medium administration, diffusion-weighted images in the axial plane were acquired using a single-shot spin echo echo-planar imaging (SS-SE-EPI) sequence. The parameters were used as followings: TR/TE, 4000/100 ms; section thickness, 3 mm; flip angle (FA), 90°; field of view (FOV), 200 × 200 mm; matrix, 384 × 384; Qualitative MRI analysis was performed by two radiologists with 3 and 7 years of experience in head and neck imaging, who were blinded to the clinical information and diagnosis. Consensus between the 2 readers was reached by virtue of an additional reading session. The following characteristics were evaluated and recorded from conventional MR images: 1) laterality , 2) margin (well-defined or ill-defined), 3) location , 4) signal intensity on T2-weighted image , 5) degree of contrast enhancement , 6) involvement of orbit preseptal space  7) presence of “flow void sign” on T2-weighted image , 8) findings indicative of sinusitis .2R, New York University, NY, USA) by two radiologists independently. For each case, volume of interest (VOI) was outlined on all sections where the tumor can be visualized. T2-weighted and contrast-enhanced images were used for reference to avoid necrotic components and surrounding tissues. In addition, it should be noted that ADC measurements might be affected by image distortions due to susceptibility artifacts. Therefore the marginal part of tumor was left out to avoid the influence of these artifacts. In patient with bilateral lesions, the larger lesion was analyzed. After the VOI of the lesion was determined, voxel-based ADC map was calculated with standard monoexponential fit S = S0*exp.(−b*ADC), in which S refers to the signal intensity with motion probing gradients applied and S0 refers to the signal intensity with b = 0 s/mm2. Then a histogram of ADC values with a bin width of 10− 3 mm2/s was generated, and concurrently the following histogram parameters were obtained including ADCmean, ADCmedian, skewness, and kurtosis. In addition, four cumulative histogram parameters were calculated including the 10th (ADC10), 25th (ADC25), 75th (ADC75), 90th (ADC90) percentiles of ADC values. To ensure intra-reader reproducibility, the DWI data was analyzed again with a minimum interval of 1 month. The average of the two measurement results was adopted for statistical analysis.Quantitative evaluation of DWI was performed with FireVoxel software (CAIx2 testing (the Fisher exact testing where appropriate) was used to compare the frequency distribution of each qualitative MRI feature. Mann–Whitney U test was used to compare all ADC histogram parameters. Multivariate logistic regression analysis was used to identify significant independent variables in the differentiation of lymphoma and IOIP. Receiver operating characteristic (ROC) curves were used to assess the diagnostic utility of identified variables and diagnostic models. Intra- and inter-observer variability of ADC histogram analysis was tested by calculating intraclass correlation coefficients (ICC). Intra-observer reproducibility was computed from the two measurements of the first reader. Inter-reader reproducibility was computed from the first measurement of reader 1 and the measurement of reader 2. Agreement was interpreted according to ICC as: < 0.40, poor; 0.41–0.60, moderate; 0.61–0.80, good; ≥0.81, excellent [P level of less than 0.05 was considered as a statistical significance. Statistical analysis was performed using SPSS  and MedCalc .Univariate analysis was performed for each qualitative and quantitative variable in an attempt to elucidate the ability of each variable for discriminating between lymphoma and IOIP. The xcellent . A P levP = 0.003), moderate degree of contrast enhancement (P = 0.007) and involvement of orbit preseptal space (P = 0.01) were more frequently seen in orbital lymphomas. Findings suggestive of sinusitis (P = 0.031) were more common in IOIPs. The qualitative MRI features of lymphoma and IOIP are listed and compared in Table Four qualitative variables were found significant in the univariate analysis for differentiating orbital lymphoma from IOIP, including tumor margin, degree of contrast enhancement, involvement of orbit preseptal space and findings suggestive of sinusitis. The characteristics of well-defined margin , whereas kurtosis was significantly higher in lymphoma (P = 0.023), when compared to IOIP. There was no significant difference in the skewness between the two groups. The representative cases are shown in Figs. 10 achieved the highest area under the curve (AUC) of 0.899 in the differentiation of orbital lymphoma from IOIP. Considering of collinearity between the ADC histogram parameters, only the parameter with the best differential performance was adopted for further multivariate logistic regression analysis. The diagnostic performance of histogram parameters is shown in Table Based on the intraclass correlation coefficient (ICC), assessment of intra- and interobserver variability in ADC measurements demonstrated good to excellent agreement with intraobserver ICC values ranging from 0.774 to 0.854 and interobserver ICC values ranging from 0.782 to 0.870, respectively. The comparison of the ADC histogram parameters between lymphoma and IOIP is shown in Table  P = 0.02, when co10, margin, degree of contrast enhancement, involvement of orbit preseptal space and findings suggestive of sinusitis, were entered into a multivariate logistic regression model. Multivariate logistic regression identified ADC10 (P = 0.023) and involvement of orbit preseptal space (P = 0.029) as significant independent variables for discrimination. By performing the ROC analysis, we found the combined model  defined by these two parameters achieved a significantly higher AUC (P = 0.0017) than morphological feature of involvement of orbit preseptal space alone . However, no significant difference of AUC was detected between combined model and ADC10 (P = 0.294). ROC curves of ADC10, involvement of orbit preseptal space and combined model are shown in Fig. Histogram and conventional MR parameters, including ADC P = 0.02 and invo10 were the most significant independent variables for discriminating orbital lymphoma from IOIP. To our knowledge, our study was the first to evaluate the combined diagnostic value of conventional MRI and ADC histogram for the differentiation of the two entities.Our primary results demonstrated that involvement of orbit preseptal space and ADCWith respect to conventional MRI features, we found involvement of orbit preseptal space was more frequently observed in orbital lymphomas 13/18) than IOIPs (6/22). Akansel et al.  reported/18 than Besides morphological MR findings, significantly lower mean ADC value in orbital lymphoma than IOIP has been demonstrated in prior investigations , 10, 20.10 had a better performance than ADCmean in the differentiation. Previous studies have reported low percentiles of ADC performed better in classification and grading of tumor than high percentiles [0–10 metric achieved greatly superior performance in the differentiation of low- from high-Gleason score prostate tumors. Kierans et al. [10 allowed for the accurate prediction of malignant endometrial lesions, while ADCmean did not. Recently, Xu et al. [10 could predict malignant orbital tumors with a higher AUC than ADCmean. Given our similar results in the differentiation of orbital lymphoma and IOIP, it is possible that low percentiles of ADC truly better reflect the presence of densely packed solid components within tumor tissues. The high percentile of ADC value may be easily vulnerable to the cystic or necrotic components. In clinical, small areas of microcystic changes are usually failed to be excluded from region of interests because of limitation of visual detection, which may affect the accuracy of ADC measurement. Therefore, low percentiles of ADC seemed to be more effective in discriminating the two lesions with distinct compactness.Being capable to measure the distribution frequency of ADC values as a marker of structural heterogeneity and complexity, ADC histogram analysis has been gaining increasing clinical adoption for differential diagnosis of benign and malignant abnormalities , 21, andcentiles . Rozenbecentiles  suggestes et al.  revealedu et al. , 16 repoIn addition, we found significantly greater kurtosis in orbital lymphomas than IOIPs. This might be attributed to the homogeneity of orbital lymphomas. Pathologically, orbital lymphoma consists of monomorphous sheets of lymphocyte, which would appear as a steep peak in histogram . In cont10, diagnostic model achieved a higher AUC and specificity than using either alone in the differentiation. The optimal specificity would enhance our diagnostic confidence, which is necessary for the prompt determination of a therapy plan and doctor-patient communication. However, significant difference of AUC between combined model and ADC10 was not observed. Further studies with a larger sample size would be needed to validate our results.With the combined qualitative MRI variable and ADCMost quantitative DWI studies of orbital tumors have been based on 2-dimensional region of interest (ROI) placed on a representative slice of the tumor , 26. In 10. Secondly, although cases with severe susceptibility artifacts have been excluded, slight image distortion might still exists in some images imperceptible with naked eye. Further improvement of the imaging quality of DWI would be essential for the study of orbital lesions. Turbo field echo with diffusion-sensitised driven-equilibrium preparation (DSDE-TFE) technique and half-Fourier acquired single-shot turbo spin-echo (HASTE) DWI sequence would be feasible to reduce these artifacts [b value of 700 s/mm2 for DWI. In fact, b values of 800 or 1000s/mm2 was more commonly used in previous orbit studies. The threshold values of ADC histogram parameters obtained in the present study might not be applicable for other studies that used b = 800 or 1000s/mm2. Moreover, we did not discuss dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in the current study, which is approved to be helpful for differential diagnosis of orbital lesions [Our study had several limitations. First, it was a retrospective study with relatively small amount of patients, which may contribute to the inconspicuous difference in the differential utility between combined model and ADCrtifacts , 28. Thi lesions . Only co10 and involvement of orbit preseptal space were significant independent variables for differentiating orbital lymphoma from IOIP. Histogram analysis of ADC maps demonstrates the heterogeneity of the lesions, which can provide additional and valuable information for the diagnosis of the two entities.ADC


RIPC significantly shortened mechanical ventilation (MV) duration [weighted mean difference (WMD) = −0.79 hours; P = 0.002; I2 = 53%), and reduced intensive care unit (ICU) stay . Univariate meta-regression analyses showed that the major sources of heterogeneity for AKI stage I were age  and proportion of complex surgery . Subsequent multivariate regression and subgroup analyses also confirmed these results. The present meta-analysis suggests that RIPC reduces the incidence of AKI in adults undergoing cardiac surgery and this benefit was more pronounced in younger patients undergoing non-complex cardiac surgery. RIPC may also shorten MV duration and ICU stay. Future RCTs tailored for those most likely to benefit from RIPC warrants further investigation.We aimed to conduct an up-to-date meta-analysis to comprehensively assess the renoprotective effect of remote ischemic preconditioning (RIPC) in patients undergoing adult cardiac surgery. 21 randomized controlled trials (RCTs) with a total of 6302 patients were selected and identified. Compared with controls, RIPC significantly reduced the incidence of acute kidney injury (AKI) [odds ratio (OR) = 0.79; P = 0.02; I Moreover, with increasing morbidity  in this population, postoperative AKI is becoming an important issue in adult patients undergoing cardiac surgery7.Acute kidney injury (AKI) occurs in up to 30%8. RIPC has proven to be beneficial to protect against I/R injury of various organs9 including the kidney13 in numerous animal studies. In human, RIPC has also been shown to prevent reperfusion-induced endothelial dysfunction16, and offers a promising strategy for reducing the burden associated with AKI in patients undergoing cardiac surgery.Remote ischemic preconditioning (RIPC) is a noninvasive, feasible and low-cost approach elicited by several brief episodes of ischemia and reperfusion (I/R) in a remote organ (a limb using a blood pressure cuff in this study) to offer protection from subsequent ischemic injury22 have reported on the impact of RIPC on preventing AKI, but the results are mixed. Recently, several striking large-scale RCTs27 with mixed findings have added to the available evidence for the renoprotective effect of RIPC in adult cardiac surgery. Therefore, we aimed to conduct an up-to-date meta-analysis to comprehensively evaluate the effect of RIPC on the incidence of AKI and identify the related potential influential factors in adults undergoing cardiac surgery.Several randomized controlled trials (RCTs)37 with a total of 6302 patients met the inclusion criteria. 6 RCTs were conducted for isolated coronary artery bypass graft (CABG)31, 5 RCTs were done in isolated valve surgery37, and 10 RCTs included a combination of CABG and valve surgery36. The ischemic protocol (cycles × I/R) was 3 × 5 min/5 min in 11 RCTs37, 4 × 5 min/5 min in 7 RCTs34, 3 × 10 min/10 min in 2 RCTs21, and 2 × 5 min/5 min in 1 RCT23. The upper limb was used in 11 RCTs35, the lower limb in 3 RCTs37, the thigh in 6 RCTs36, the combined of upper limb and thigh in 1 RCT23. The incidence of AKI was reported in 17 RCTs36 (AKI stage I in 11 RCTs36), the need for RRT in 19 RCTs37, mortality in 19 RCTs36, MV duration in 13 RCTs35, ICU stay in 16 RCTs37, and hospital length of stay (LOS) in 16 RCTs37. 19 RCTs37 had a Jadad score of more than 3. Further details of RCTs characteristics and the RIPC protocol used in each RCT are provided in Tables Figure 2 = 38%; Fig. AKI was reported in 6054 study subjects, and the overall incidence was 25% . Postoperative incidence of AKI was significantly reduced by RIPC  = 0.79; 95% CI, 0.65 to 0.96; P = 0.02; I2 = 55%; Fig. For AKI, stage I the overall incidence was 19% . RIPC significantly reduced the risk of AKI stage I . The risk of postoperative RRT was not lowered in the RIPC group , and there was a trend towards reduced ICU stay by 0.23 days  , hypertension , additive ischemia , and complex surgery  as shown in Table Age, male, previous myocardial infarction (MI), diabetes, hypertension, dyslipidemia, renal dysfunction, cardiopulmonary bypass duration, baseline left ventricular ejection fraction, complex surgery, CABG, use of volatile anesthesia, aspirin, angiotensin-converting enzyme inhibitors, beta-blockers, and statins, cumulative duration of preconditioned ischemia, and additive ischemia were included in the random-effect univariate meta-regression analyses for AKI stage I. The major sources of heterogeneity were age  and in those RCTs with less complex cardiac surgery (<25%). However, RIPC did not affect AKI stage II and III/requirement for RRT, hospital length of stay, and mortality.39. Even minor increase in postoperative serum creatinine level following cardiac surgery has been shown to be associated with increased MV duration40, prolonged ICU stay41, and the risk of short-term mortality42. Although AKI can occur due to numerous reasons and the underlying mechanisms remain unclear, acute tubular necrosis has been implicated as being the predominant pathology43. There is currently no effective renoprotective strategy to reduce the burden of AKI in this setting44. Several RCTs have investigated the renoprotective effect of RIPC in patients undergoing cardiac surgery but with conflicting results. To minimize heterogeneity due to trial design and patient selection, our study only included RCTs involving adult cardiac surgery, but not in combination with major vascular surgery46, pediatric cardiac surgery47, percutaneous coronary intervention48, or organ transplantation47.Post-operative AKI in adult cardiac surgery is a common complication, occurring in up to a third of surgical caseset al.26, which was specifically designed and powered to look at the effect of RIPC on AKI as the primary endpoint in 240 patients. Of note, they only included patients at high risk of AKI. Furthermore, they used volatile anesthesia instead of propofol, the latter of which may potentially attenuate the effect of RIPC50. They showed a 15% and 10% absolute risk reduction in the incidence of AKI and the need for RRT, respectively. RIPC also reduced the duration of stay in ICU but there was no difference in overall hospital length of stay26.We found that RIPC reduced the incidence of AKI stage I and MV duration, and there was a trend towards shorter ICU stay. Our findings are consistent with the RCT by Zarbock 52. In our analysis, the incidence of RRT was 3.1% and the 30-day mortality was only 1.5% (4152 patients), many of whom presented with normal preoperative serum creatinine level. RIPC did not affect the need for RRT or mortality in our analysis. Thielmann et al.53 randomized 329 patients undergoing CABG and obtained similar findings to our study for 30-day mortality. However, they found that RIPC reduced 1-year mortality and the result remained significant after 4-year follow-up. Therefore, longer follow-up duration should be considered in future RCTs to see a benefit in mortality.The risk of death is proportional to the severity of AKI, with the highest rate occurring in patients requiring RRT following adult cardiac surgery55, surgical complexity56, and previous MI57) has proven to be challenging. Our meta-regression analysis showed that age was negatively correlated with the reduction in AKI stage I by RIPC. Likewise, the proportion of complex surgery was negatively correlated with the reduction in AKI stage I. Based on the findings from our study and that of Zarbock et al.26, whether pre-selecting a younger cohort of patients who are at risk of AKI, undergoing non-complex surgery using volatile anesthesia may more likely show a significant reduction in all stages of AKI by RIPC and eventually improve clinical outcomes, remain to be assessed in future, adequately powered RCTs.Translating renoprotective strategies that have shown promise in young and healthy animals into the clinical population with various co-morbidities and/or confounders  and cardiovascular medications (such as volatile anesthetics59 and statins60) may have been underestimated. Secondly, AKI was based on different definitions such as AKI Network classification (AKIN), Risk/Injury/Failure/Loss/End-stage (RIFLE) criteria or the Kidney Disease: Improving global Outcomes (KDIGO) classification62, and the patient selection, type of surgery and RIPC protocol used were different and may have contributed to the heterogeneity. Thirdly, although we included several recently published large RCTs, the sample size was still relatively small to be adequately powered for hard clinical outcomes. Last but not least, only 11 RCTs qualified for the meta-regression analysis and therefore the conclusions may not be robust but hypothesis generating.There are several limitations in our study. Firstly, we were unable to access the patient-level data. Therefore, the potential influences of co-morbidities , usage of volatile anesthesia, aspirin, angiotensin converting enzyme inhibitors, beta-blockers and statins. Cumulative duration of preconditioned ischemia was calculated multiplying the number of cycles by the ischemic duration . Additive ischemia65 was calculated using cumulative duration of preconditioned ischemia relative to the CPB duration.The literature review and data extraction were independently completed by two investigators (J.G. and Y.Z.). Any disagreements were resolved by consensus. Quality assessment was performed according to Jadad score: randomization; blinding; withdrawals and dropouts (a possible score between 0 and 5). Trials with a score of more than 3 were considered as being of high-quality61) was used for this study.The primary endpoints were incidence of AKI as a whole and AKI stage I–III individually, and the definition used by each RCT , mechanic ventilation (MV) duration, intensive care unit (ICU) stay, and hospital length of stay (LOS).et al.66 was used to convert to mean and standard deviation. Random-effect model was used in view of differences in patient selection and the RIPC protocol used among the RCTs. Publication bias was assessed by Begg’s test and Egger’s test. Heterogeneity among RCTs was quantified using I2 statistics with I2 of 0–40%, 30–60%, 50–90% and 75–100% considered as low, moderate, substantial and considerable heterogeneity, respectively, as defined by the Cochrane handbook of systematic reviews67 and moderate heterogeneity was considered acceptable. Meta-regression (P < 0.1) and subgroup analysis were conducted for positive results to explore the potential sources of heterogeneity68. To reduce the possibility of over-fitting in the multivariate regression model, at least four studies or sub-studies were set for the identification of each influential factor70. P < 0.05 (2-sided) was considered to be statistically significant. All statistical analyses were performed in Stata .For dichotomous outcomes (reported as incidence), we calculated OR with 95% CI. For continuous outcomes  reported as mean and standard deviation, the WMD for the pooled estimates with 95% CI were calculated. For RCTs reporting median and interquartile range, or median and range, the method described by Hozo


Markers of inflammation, HOMA, body composition, and GI/GL (estimated from 3-day food records) were assessed at baseline and 18 months. By 18 months, the GL in the CHO group increased by 34%, 88.4 ± 5.2 → 118.5 ± 4.9 and did not change in the PRO group, 86.5 ± 4.1 → 82.0 ± 3.6 (p < 0.0001). Despite this change there were no differences in serum CRP, IL-6, or HOMA at 18 months between the two groups, nor were there significant associations between GL and inflammatory markers. However, trunk lean mass (p = 0.0375) and total lean mass (p = 0.038) were higher in the PRO group compared to the CHO group at 18 months There were also significant associations for GL and change in total fat mass , change in BMI , and change in the lean-to-fat mass ratio . Our data suggest that as dietary GL increases within the moderate range, there is no detectable change in markers of inflammation or insulin resistance, despite which there is a negative effect on body composition.Controversy exists as to whether high glycemic index/glycemic load (GI/GL) diets increase the risk of chronic inflammation, which has been postulated as a pathogenic intermediary between such diets and age-related alterations in body composition and insulin resistance. We conducted an ancillary study to a randomized, double-blind trial comparing the effects of a whey protein supplement (PRO,  A current widely held view is that chronic low-grade inflammation is a major contributor to common chronic diseases, such as cardiovascular diseases , diabeteThe GI is the measure of the glycemic response obtained after the ingestion of a carbohydrate-containing food, compared with a standard  . The GL Cross-sectional studies in women have reported a positive association between GI/GL and C-reactive protein (CRP) ,11, a wiData from studies examining the relationship between GI/GL and insulin resistance are also mixed. Results from the Framingham Offspring Cohort suggests a positive association between GI/GL and homeostatic model assessment of insulin resistance (HOMA-IR) , while LWe recently completed a double-blind, randomized, controlled trial (RCT) comparing the effects of a whey protein supplement (PRO) and an isocaloric maltodextrin supplement (CHO) on bone density and body composition in older adults, the SPOON Study . The st® 290, Glanbia Nutritionals, Twin Falls, ID, USA) or isocaloric maltodextrin control supplement  on bone mineral density and body composition in postmenopausal women and older men. Subjects were recruited from two study centers  between May 2007 and November 2010. Subjects who consumed a minimum of 20 g of either the protein  or isocaloric maltodextrin  supplement for 18 months were included in this ancillary study.Eighty-four post-menopausal women were selected for this ancillary study from SPOON . As noteMethods used to quantify the subjects’ dietary intake in SPOON have been previously described . In brieThe GL of individual food items was assigned according to a publically available database maintained by the University of Sydney  and pubFasting blood samples were collected by routine venipuncture at baseline and 18 months of SPOON, processed, and stored at −70 °C. Fasting fingerstick glucose measurements were made during SPOON using the Novastrip glucose meter  or the Bayer Contour glucometer . For this ancillary study, all other metabolites were analyzed using previously frozen samples. IL-6 and insulin were measured by the Yale Bone Center Mineral Metabolism Laboratory using R&D Systems High Sensitivity Quantikine ELISA kit  and Millipore Human Insulin Specific Radio-immunoassay kit , respectively. The lower limit of detection for the IL-6 assay was 0.156 pg/mL and for the insulin assay, 2.715 µU/mL. In addition to fingerstick glucose determinations available from SPOON, serum glucose was also measured from stored samples as was high sensitivity-CRP. Both these latter measurements were done in the Yale University Center for Clinical Investigation (YCCI) Core Laboratory using an Alfa Wasserman autoanalyzer .The HOMA2 calculator, published by the University of Oxford, was used to calculate insulin resistance from fasting insulin and serum glucose. As noted above, serum glucose was analyzed from frozen samples. Glucose is known to be stable in thawed serum samples , nonetheBody composition in SPOON was measured by DXA, using either a Hologic 4500 W machine  or a Lunar Prodigy DPX-IQ  at baseline and 18 months. DXA measurements took place in the morning after an overnight fast.The study was conducted in accordance with the ethical standards of the Investigational Review Boards (IRB#0610001951) at Yale University School of Medicine and University of Connecticut Health Center. All SPOON participants gave their written informed consent.t-test  was used to assess within group differences at baseline vs. 18 months. Between group comparisons were performed using a Mann–Whitney test (for nonparametric data) or unpaired t-test (for parametric data). Spearman correlation  or Pearson correlation  analysis was used to examine the relation with dietary GL and change in markers of inflammation, insulin resistance, BMI, and body composition. Data were analyzed using GraphPad Prism 6 . A probability level of p < 0.05 was considered statistically significant.Data are reported as means ± SEM. The D’Agostino-Pearson omnibus test was used to test data for normality. A Wilcoxon signed-rank test  or paired Baseline demographics, anthropometric measures, and dietary and supplement intakes of the study population are summarized in p = 0.0002) and GL . In contrast, GI (45.7 ± 1.4 → 46.6 ± 1.1) and GL (86.5 ± 4.1 → 82.0 ± 3.6) did not change with the addition of the whey protein supplement. The maltodextrin-induced change in dietary GI and GL resulted in significant differences in dietary glycemic parameters between the two study groups  or insulin resistance .Although maltodextrin supplementation increased the mean dietary GL of the CHO group by 34%, while protein supplementation resulted in a 5% decline in GL in the PRO group, there were no within or between group differences in markers of inflammation or measures of insulin resistance at 18 months . There wp = 0.033). These changes resulted in a significant difference in the absolute value for lean body mass at the end of the study (p = 0.038). Similar and significant changes were also observed for truncal lean mass over the 18-month study period . Furthermore, fat mass increased by 4.7% in the CHO group, although this change did not reach statistical significance (p = 0.451). Alterations in body fat were not observed in the PRO group . At the end of the study, there was a trend towards a significant difference in change in fat mass between the CHO and PRO groups (p = 0.08). The change in the lean-to-fat mass ratio was significantly different between the two study groups at 18 months (p = 0.045), with unfavorable alterations in body composition being observed in the CHO group. There were significant associations for GL and change in total fat mass , change in BMI , and change in the lean-to-fat mass ratio .Although the addition of a fixed GL supplement to the usual diets of postmenopausal women for 18 months did not result in changes in markers of inflammation or insulin resistance , there wThis study evaluated the long-term effects of the addition of a fixed GL supplement to the usual diets of postmenopausal women on markers of inflammation, insulin resistance, and body composition. On average, the BMI of our study population was within the healthy range for their age . MaltodeThe relationship between dietary GI/GL and markers of inflammation has also been examined in older adults experiencing, or at higher risk for, chronic diseases thought to be due in part to chronic low-grade inflammation. Bullo et al. conducted a cross-sectional study in 511 older men and women with type 2 diabetes or multiple CVD risk factors that were recruited for the PREDIMED trial  and founFive studies have reported positive associations between dietary GI/GL and markers of inflammation, particularly for IL-6 and CRP, in young and older adults with and without chronic diseases ,26,27,28One potential explanation for these varying results is that baseline levels of inflammation vary depending on the study population and, if high, could potentially mask the effect of a change in dietary GI/GL. However, our baseline levels of IL-6 and CRP were similar to the baseline values observed by Bullo  and by oAlthough markers of inflammation did not change in our study, we observed favorable changes in body composition with lower GI/GL intakes. Previous studies have reported similar findings ,32. HareThe current study had a number of strengths. Supplement adherence and diet were carefully monitored by dietitians during the primary RCT . DetailsIn summary, we found that the addition of a fixed glycemic load supplement to the diets of otherwise reasonably healthy individuals did not impact markers of inflammation or insulin resistance. However, higher dietary glycemic parameters in healthy older women appear to have unfavorable effects on body composition, and thus, lower GI/GL diets that moderately exceed the RDA for dietary protein should be further explored as a means to help preserve muscle mass and reduce fat mass with aging.


In order to explore the molecular mechanism of fruit coloring regulated by hematin, transcriptomes in the hematin- and non-hematin-treated fruit were analyzed. A large number of differentially expressed genes (DEGs) were identified in regulating anthocyanin synthesis, including the DEGs involved in anthocyanin biosynthesis, hormone signaling transduction, phytochrome signaling, starch and sucrose degradation, and transcriptional pathways. These regulatory networks may play an important role in regulating the color process of strawberries treated with hematin. In summary, exogenous hematin could promote fruit coloring by increasing anthocyanin content in the white stage of strawberries. Furthermore, transcriptome analysis suggests that hematin-promoted fruit coloring occurs through multiple related metabolic pathways, which provides valuable information for regulating fruit color via anthocyanin biosynthesis in strawberries.Anthocyanin in strawberries has a positive effect on fruit coloration. In this study, the role of exogenous hematin on anthocyanin biosynthesis was investigated. Our result showed that the white stage of strawberries treated with exogenous hematin had higher anthocyanin content, compared to the control group. Among all treatments, 5  Fragaria × ananassa Duch.) is one of the most popular fruits with global economic importance [Strawberry  in plants [ Arabidopsis [ HO1 deficient mutants, which can not synthesize the phytochrome chromophore, have a major reduction in anthocyanin accumulation.Light is one of the most important environmental factors regulating anthocyanin biosynthesis . Phytochn plants . Addition plants , 19 and bidopsis –22 HO1 d34H33O5N4Fe), a protoporphyrin complex, is an inducer and substrate of HO1 in animals and plants [ Medicago sativa [ Brassica nigra seed germination under nanosilver stress, and relieve etiolation in the leaves of wheat seedlings under complete darkness [Hematin , an octoploid (2n = 8X = 56) species, was planted under standard culture conditions in a greenhouse . The maximum light intensity inside the greenhouse was 55,300 lux. The developmental stage of the strawberry fruit was divided into seven visual stages: small green (SG), big green (BG), degreening (DG), white (Wt), initial red (IR), partial red (PR), and full red (FR) [n ≥ 50 for each treatment) were sprayed with 0, 1, 10, or 100 μM hematin , respectively [n ≥ 30 for each treatment) were harvested when the fruit entered the PR stage (48 h after treatment). The fruits were immediately frozen in liquid nitrogen and stored at −80°C for further analysis. We found the 10 μM hematin-treated strawberries accumulated most anthocyanin among all treatments . By using the same method to treat the fruits with 0, 5, 10, or 15-μM hematin, we found the treatment by 5 μM hematin was the optimal condition for increasing anthocyanin production in strawberry (p < 0.01). The treatment with 5 μM hematin was used for subsequent analysis. Three independent replicates were prepared for each treatment.Strawberry (red (FR) . Strawbeectively . Strawberawberry  . Three independent replicates were conducted for each treatment.Strawberry fruit (1 g) was homogenized in a Potter-Elvehjem homogenizer using 1.2 mL ice-cold 0.25 M sucrose solution containing 1 mM phenylmethyl sulfonyl fluoride, 0.2 mM EDTA, and 50 mM potassium phosphate buffer (pH 7.4). Homogenates were centrifuged at 20,000 ×g for 20 min and chloroplasts were used for activity determination. BV was assayed as previously described . The conμL 10 mg/mL proteinase K  was added to remove contaminating proteins. Total RNA was extracted using phenol/chloroform/isoamyl alcohol (25 : 24 : 1) and precipitated in a sodium acetate and ethanol mixture. The mixture was resuspended in an appropriate volume of DEPC-treated distilled water and then stored at −80°C for the next step. The quality and quantity of the total RNA were measured using a NanoDrop ND-1000 Spectrophotometer . Only samples that met the criteria of 1.8 ≤ OD260/280 ≤ D2.0 and OD260/230 ≥ 1.8 and concentration ≥ 200 ng/μL were used for sequencing. RNA samples of 30 strawberry fruits harvested in the same treatment group were pooled together for subsequent experiments.Total RNA isolation was carried out as previously described . The proRNA samples from two biological replicates were used for cDNA library construction and RNA-Seq at the Beijing Genomics Institute . Total RNA samples were treated with DNase I  to remove any possible DNA contamination. The mRNA was enriched by using oligo (dT) magnetic beads  and cut into short fragments (about 200 bp). The first-strand cDNA was synthesized using a Superscript Preamplification System Kit  as described in the manufacturer's instructions. The double stranded cDNA was purified with the oligo (dT) magnetic beads following the manufacturer's instructions. End repair was performed and adaptors were ligated to the ends of these fragments. Ligation products were purified using TAE-agarose gel electrophoresis. The fragments were then enriched by PCR amplification with an initial denaturing step at 98°C for 30 s, followed by 15 cycles of amplification  and a final extension at 72°C for 5 min. The PCR products were then purified using the oligo (dT) magnetic beads. DNA size, purity, and concentration were checked on an Agilent 2100 Bioanalyzer .http://strawberry-garden.kazusa.or.jp/) using Ion Torrent's mapping program . No more than two mismatches were allowed in the sequence alignment. Quality assessment of reads, statistics of alignment, sequencing saturation analysis, and randomness assessments were carried out subsequently to assess the quality of sequencing.RNA sequencing was performed using the ion proton platform at the Beijing Genomics Institute. The original sequence data were filtered to obtain clean reads for further analyses by removing short reads (less than 30 bp) and trimming adapters. Adapter reads were trimmed by first calculating the average quality of the first 15 bases from 3′-end until the average quality was larger than 10 and then removing the bases that were counted. The high-quality clean reads were mapped against the strawberry reference genome | > 1 were set as the threshold to identify significant differentially expressed genes. In statistics, correction for false positive errors was carried out using the FDR statistic [Gene expression levels were quantified by the software Sailfish . Raw coutatistic .https://www.blast2go.com/). Gene function was annotated using the following databases: NCBI nonredundant protein sequences, NCBI nonredundant nucleotide sequences, protein family, Clusters of Orthologous Groups of proteins, the manually annotated and reviewed protein sequence database, KEGG Ortholog database, and Gene Ontology. After GO annotation for DEGs, we performed GO functional classification for DEGs by the WEGO software [http://www.yeastgenome.org/help/analyze/go-term-finder) to search for significant shared GO terms.N is the number of all genes with GO annotation; n is the number of DEGs in N; M is the number of all genes that are annotated to certain GO terms; and m is the number of DEGs in M. The calculated p value was subjected to Bonferroni Correction [p value < 0.05 was set as the threshold. GO terms fulfilling this condition are defined as significantly enriched GO terms in DEGs. The pathway enrichment analyses using the KEGG database (http://www.genome.jp/kegg/) were conducted subsequently to study the functions of differentially expressed genes identified between the control and the hematin-treated strawberry groups. The formula is the same as that in GO analysis. Here N is the number of all genes with KEGG annotation, n is the number of DEGs in N, M is the number of all genes annotated to specific pathways, and m is the number of DEGs in M.Gene functions of strawberry were annotated according to the Gene Ontology (GO) standardized terms for molecular function, cellular component, and biological process using Blast2GO .Quantitative real-time PCR (qRT-PCR) experiments were conducted to assess the reliability of the RNA-Seq data . Eleven t method , 36. TheμM (control), 5, 10, or 15 μM hematin. The results indicated that the anthocyanin content in the hematin-treated strawberries was more than the control after 48 h. The treatment by 5 μM hematin was found to be the optimal condition for increasing anthocyanin production (p < 0.01). The anthocyanin content in the 5 μM hematin-treated strawberries was 2.5 times higher than that in the control. This is the first report that hematin could increase the anthocyanin production in fruit. We measured the expression of FaHO-1 and the content of biliverdin (BV) which is the metabolite of heme oxygenase. We found that hematin could also significantly increase the expression of FaHO-1 in strawberry fruit . This indicates that the obtained reads are sufficient for complete transcriptome coverage. The randomness of RNA fragmentation in the four libraries was assessed for subsequent bioinformatics analysis. The results showed that the reads in each position of the reference gene were distributed evenly and demonstrated highly similar tendencies in all libraries .R2) were high . Genes expressed in both replicates were screened. A total of 28,713, 28,999, 27,976, and 28,211 genes  were detected in each library. Additionally, correlations among genes based on RPKM between the two biological replicates were analyzed; the correlation coefficients  genes were differentially expressed in the 5 p < 0.05 were represented among genes with significant changes in expression over a given interval. In this study, only three terms including anatomical structure arrangement, meristem structural organization, radial pattern formation were significantly enriched in biological process (Supplementary Table S2). Although most Go terms were not significantly enriched, they could provide a reference for further study. According to the GO cellular components, a large number of the DEGs were classified into main cell organelles, such as vacuole, nucleolus, mitochondrion, apoplast, chloroplast, plastid, cytoplasm, and membrane. In particular, many DEGs were assigned to the plastid and chloroplast  enrichment analysis of the DEGs in the control and hematin-treated groups was performed to reveal the possible mechanisms under which hematin promotes anthocyanin biosynthesis. GO terms with oroplast , indicatoroplast . It is pQ-value < 0.05)  . Althoug FaMYB1 and FaMYB9 and grapevine VvMYB4, which can significantly suppress the biosynthesis of anthocyanins and flavonols [ MYB family of R2R3 MYB transcription factors was significantly upregulated. Moreover, increasing evidence indicates that the expression of the bHLHs promotes anthocyanin accumulation in fruit [Transcription factors (TFs) are proteins that regulate the expression of downstream target genes. Many TFs, such as v-myb avian myeloblastosis viral oncogene homolog (MYB), basic helix-loop-helix (bHLH), WD40-repeats protein (WD40), and MADS-box (MADS), directly regulating the expression of the structural genes in anthocyanin biosynthesis have been identified from many species . Therefoin fruit . In our  DFR genes and one LAR gene were significantly upregulated . In addition, all six genes encoding UDP-glycosyltransferase were significantly upregulated. Among these, the expression of one gene (Fanhy_icon15742070o.1.g00001.1) was upregulated 18305-fold. The results suggest that exogenous hematin may have an important role in promoting anthocyanin biosynthesis.The anthocyanin biosynthetic pathway has been studied intensively. The anthocyanin biosynthetic pathway via the phenylpropanoid pathway is well known . In this Zea mays and regulate anthocyanin production and composition in suspension cultures of strawberry cells [Auxin has been shown to negatively regulate the expression of the anthocyanin biosynthesis genes . In thisry cells . In this FaNCED1 gene result in colorless strawberry fruit, which can become colored through the application of exogenous ABA [Extensive researches have shown that ABA plays an important role in the regulation of anthocyanin biosynthesis in nonclimacteric fruit . In thisnous ABA . Anothernous ABA , 49. Our Arabidopsis, JAs can affect anthocyanin accumulation via the interaction of negative regulators with the MBW complex of transcription factors involved in anthocyanin biosynthesis [Fuji” apples enhances red coloration [Inynthesis . Preharvloration . JA vapoloration . In thisloration , 54. The HO-1 expression in many plants [ FaHO1 , transcription factor genes (MYB and bHLH), and phytochrome chromophore-related gene (HO-1). The primers used for qRT-PCR are listed in Supplementary Table S1. The qRT-PCR results were consistent with the RNA-Seq data (2 ratio (hematin-treated/control) in the transcriptome data than in qRT-PCR. These results indicate the RNA-Seq data from the strawberry transcriptome is reproducible and accurate.To validate the expression of the DEGs obtained from RNA-Seq, 11 DEGs were selected for qRT-PCR, including structural genes . Vertical bars represent standard errors; Values with different letter are significantly different at p<0.01. Figure S2. Saturation analysis of control 1, control 2, hematin 1, and hematin 2 mRNA libraries. (TIFF); Figure S3. Randomness assessments of control 1, control 2, hematin 1, and hematin 2 mRNA libraries. (TIFF); Table S1. Eleven pairs of gene specific primers of the DEGs in strawberry fruit. Table S2. Gene classification based on gene ontology and pathway terms for DEGs treated by hematin.


Percutaneous ablation has quickly arisen as one of the important alternative treatments for hepatocellular carcinoma (HCC). We aimed to compare the therapeutic effects of radiofrequency ablation (RFA) and other ablative techniques on HCCs.Databases were searched to identify literature on complete tumor ablation (CTA), overall survival (OS), local tumor recurrence (LTR), and complications of RFA in the treatment of HCC, compared with those of microwave ablation (MWA), percutaneous ethanol injection (PEI), PEI plus RFA, cryoablation (CRA), laser ablation (LSA), and high-intensity focused ultrasound. Randomized controlled trials and high-quality cohort studies were included in the assessment.P < 0.05). CTA rates were lower in patients treated with PEI , and higher in those treated with PEI plus RFA , with an increased incidence of fever (P < 0.05). LSA resulted in lower CTA rates  and OS , with a lower incidence of complications.The effects of MWA and CRA appeared to be similar to those of RFA, but lower rates of LTR and higher rates of CTA in large tumors compared with RFA were reported (Compared with RFA, identical effects were found in MWA and CRA groups. Fewer complications were observed in PEI and LSA group. PEI plus RFA appeared more effective, with a higher rate of complications. Well-designed randomized controlled trials are further needed to confirm above results. Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, with a poor prognosis. Currently, nearly 78% of cases occur in Asia, and the global incidence of HCC is increasing steadily , 2. AlthIn the past 20 years, with the development of imaging techniques, such as ultrasonographic guidance, percutaneous ablation has become an important alternative treatment for small HCC and cases deemed unresectable by surgery . Many diPEI was among the first ablative treatments for HCC; RFA has been recently employed and widely accepted for early-stage and unresectable HCC . InternaThe aim of the present systematic review and meta-analysis was to compare the outcomes of various ablation methods in HCC treatment, with summarization of the evidence supporting the selection of RFA and other ablative techniques. The rates of complete tumor ablation (CTA), local tumor recurrence (LTR), overall survival (OS), and complications of RFA were assessed relative to those of other percutaneous ablative techniques.A computerized literature review was conducted to identify articles published between January 1995 and September 2016 using the PubMed, Embase, Cochrane Library, and China Biology Medicine databases. The initial year of 1995 was chosen because the first report on the use of RFA on a patient’s liver was published in this year. The search terms used were: “carcinoma, hepatocellular” (MeSH term) OR “hepatocellular carcinoma” (text) AND “radiofrequency ablation” (text) OR “high-intensity focused ultrasound ablation” (MeSH term) OR “high-intensity focused ultrasound” (text) OR “laser coagulation” (MeSH term) OR “laser ablation” (text) OR “microwave ablation” (text) OR “percutaneous ethanol injection” (text) OR “cryosurgery” (MeSH term) OR “cryoablation” (text) OR “ablation techniques” (MeSH term). The reference list of identified publications and review articles were checked manually to identify additional related articles. The last search was performed in September 2015.The studies meeting all the following criteria were included: (1) full text available in English or Chinese, due to language limit; (2) the results providing data relative to CTA, LTR, OS, or complications, and compared between the outcome of RFA with that of other percutaneously ablative techniques on HCCs, such as MWA, PEI, LSA, CRA, HIFU, or a combination; (3) randomized controlled clinical trials; (4) high quality cohort studies after assessed by Newcastle-Ottawa scale (NOS).The following forms of publications were excluded: (1) literature in the form of case reports, editorials, reviews, and conference abstracts; (2) the full text not written in English or Chinese; (3) lack of the required postoperative data in the results, and no response after attempts to connect with the author; (4) duplicate data if recent studies were already included; (5) the animal or in vitro research; (6) ablative techniques combined with transhepatic artery chemoembolism; (7) ablation under laparoscope or during surgical operation.NOS and Cochrane collaboration’s tool for bias risk assessment were used for quality assessment of cohort studies and RCTs, respectively, by two reviewers . Different opinions were solved through consultation.For observational cohort studies, assessment of quality was performed in eight items described in Table For RCTs, low, unclear or high risk of bias in the items including random sequence generation, allocation concealment, blinding of outcome assessment, incomplete outcome data, and selective reporting, was assessed for each study.Two of the authors  independently extracted data, including: (1) country and publication year; (2) number of patients; (3) age; (4) number and size of the tumor; (5) number of patients in CTA; (6) rate of LTR; (7) data about OS or from the OS curve; (8) number of patients with complications. Engauge Digitizer 4.1 was used to extract survival data from OS curves.CTA was defined as no enhancement in ablated areas on enhanced CT, MRI, or US within 4 weeks after ablation, while LTR was considered as the tumor enhancement near the ablated areas on enhanced CT or MRI during follow-up period. Major complications were mentioned as the events leading to substantial morbidity and disability increase the duration of stay in hospitals or the caring rank, such as subcapsular/intrahepatic hematoma, biliary system injury, hemathorax, liver infarction, liver compensation, symptoms of breath holding and incomplete intestinal obstruction. The other complications were considered minor, such as pain, fever, skin burns, pleural effusion, liver transaminase change, and tumor seeding.The comparison of MWA versus RFA, PEI versus RFA, PEI plus RFA versus RFA, CRA versus RFA, LA versus RFA, and HIFU versus RFA were conducted. Observational studies and RCTs were conducted through meta-analysis, respectively. Reviewer Manager  was used for pooling data.I2. I2 > 50% was considered as exist of significant heterogeneity. Fixed-effect model was conducted in statistical analyses if there was no obvious heterogeneity; otherwise the random-effect model was used. Inverse variance was used for evaluation of continuous variables, such as the diameter of tumor. Publication bias was evaluated by the funnel plot and Egger’s regression. In all analyses, P < 0.05 was regarded as statistically significant.In the meta-analysis, hazard ratio (HR) with 95% CI was calculated for comparison of survival data. Results about CTA, LTR, and complications were compared by calculating odds ratio (OR) with 95% CI. Peto OR was used for the low incidence of events. Heterogeneity was assessed by calculating The initial database search yielded 2675 publications Fig. , 2633 ofAccording to the Cochrane Collaboration tool, 13 of the RCTs were affected by performance bias Fig. . BecauseThe 16 cohort studies were given 7–9 stars upon NOS scale evaluation Table . One of Six cohort studies –14 and tNo significant difference in the CTA rate, OS, 1- or 3-year survival rate was detected between the MWA and RFA groups Fig. . In the P = 0.05) in the 4 cohort studies  of patients in the MWA group and 4.39% 13 of 296) of patients in the RFA group (Table  of 342 o of 296 oP = 0.018) (10).Subgroup analysis of tumors with diameters >3.0 cm revealed no difference in CTA in 2 cohort studies , 13 and Ten studies  for RCTs –26 and 1In the meta-analysis comparing OS in the PEI and RFA groups, no significant difference was detected for the RCTs Table  22–26].–26.22–26P < 0.05) [P < 0.05] and 13, 24, and 24% vs. 31, 52, and 52% [P < 0.05], respectively). One-, 2-, and 3-year OS rates were higher in the RFA group  than in the PEI group  [Brunello et al.  reported < 0.05) . For tum < 0.05)  reportedTwo RCTs , 29 and P = 0.06) for LTR in the PEI plus RFA group compared with the RFA group. OS rates did not differ between groups , whereas the incidence of pain did not differ significantly .P < 0.05).Tumor diameter ≤3 cm was reported as a significant predictor of higher CTA in two studies , 28. ForFour cohort studies –33 and oP > 0.05).The cohort studies reported on CTA after CRA and RFA .In the cohort studies, LTR occurred in 27.4% (43 of 157) patients in the CRA group and 16.7% (33 of 198) patients in the RFA group . In the RCT, the incidence of LTR was 5.6% in the CRA group and 10% in the RFA group . Only two studies supplied OS data . Wang etThe incidence of patient-related complications ranged from 3.8 to 40.7% in the CRA group and from 3.3 to 24% in the RFA group –20. A raP = 0.006) [A lower LTR rate in the CRA group than in the RFA/MWA group for tumors with diameters >2 cm (= 0.006) . AlthougThree RCTs were included in this assessment , 34, 35.CTA rates ranged from 66.7 to 96.2% in the LSA group and from 86.7 to 97.4% in the RFA group .The Peto OR for the incidence of complications was 0.57 (95% CI 0.31–1.07). Ferrari et al.  reportedP > 0.05). Procedure-related complications occurred in the RFA group comparable with HIFU group (P = 0.06) (Table Only one cohort study referring to HIFU versus RFA was included . No obvi6) Table .P value over 0.05 were detected, which suggested there was no evidence of publication bias  than in the RFA group . The amount of ethanol injected into the tumor was significantly and positively correlated with the volume of coagulated necrosis, but not with the energy requirement. Therefore, in the application of PEI-RFA in high-risk areas, such as near vessels or other important organs, ethanol injection helps to decrease the RFA energy, thereby protecting the surrounding tissues. However, in this study, a higher incidence of fever was found after PEI-RFA than after RFA.As a non-thermal ablation, the use of PEI for the treatment of HCC was popular before the widespread use of RFA. In this study, we observed lower CTA rates and higher LTR rates in the PEI groups than in the RFA groups. The intratumor fibrous septum may interfere with the injection of ethanol . Thus, tCRA caused the tumor cell death through ice crystal formation during rapid freezing. We observed no obvious difference between CRA and RFA, while in two studies the tumor size in CRA was noted to be obviously larger than that in the RFA group , 33. On P < 0.05). The tendency of fewer complications was indeed detected in the LSA group. Thin needle for LSA may improve the ablative effects on the nodules with irregular shape or in high-risk location. In addition, in an Italian study, a lower cost of LSA was mentioned but had no statistical analysis [In the current study, it was shown that lower CTA rates and higher LTR rates were observed in the LSA patients, whereas higher OS rates in RFA patients were seen, particularly among larger HCCs (analysis .There were some limitations in this study. Many studies have demonstrated that tumor size, number of single lesion, and Child-Pugh class were important prognosis factors , 35. ButAbove all, the availability of multiple ablative techniques is the reason for optimism. It was pleasing that there have been many kinds of ablative techniques for treating HCCs. The combination of PEI and RFA apparently yielded a better prognosis than a single RFA. When the outcome of MWA appeared identical to that of RFA, a potential role of CRA used in larger tumors needs to be investigated. Less complications in PEI and LSA implied their application in high-risk areas for protecting the important organs. Indeed, well-designed randomized controlled trials are further needed to confirm the above results. With the different characteristics, multiple ablative techniques may be combined in one treatment procedure, so as to achieve better effects and avoid adverse events.


Cyprinus carpio L. aquaculture. So far, it is unknown how the different variants of KHV have developed and how they spread in the fish, but also in the environmental water bodies. Therefore, a phylogenetic method based on variable number of tandem repeats (VNTR) was improved to gain deeper insights into the phylogeny of KHV and its possible worldwide distribution. Moreover, a VNTR-3 qPCR was designed which allows fast virus typing. This study presents a useful method for molecular tracing of diverse KHV types, variants, and lineages.Worldwide koi herpesvirus (KHV) causes high mortalities in  Moreover, its ornamental variety, the koi, is one of the most expensive ornamental fish in the world. A severe infection that leads to high mortalities of Cyprinus carpio stocks has been reported repeatedly since 1997. This disease is associated with skin lesions and gill necrosis , 1% non-essential amino acids (Biochrom), and 0.12 g/l pyruvic acids sodium salt (Merck). Different KHV isolates  according to the manufacturer's instructions. Samples were obtained either from cell cultures (500 μl each) or from gill swabs of infected fish. They were incubated overnight at 56°C with 180 μl ATL buffer containing 20 μl proteinase K. Afterwards 200 μl Buffer AL were added and incubated at 70°C for 10 min, followed by addition of 200 μl ethanol. This mixture was applied to the spin columns and centrifuged for 1 min at 800 × g. The flow-through was discarded and the column was washed with 500 μl buffer AW1. After centrifugation for 1 min, the column was washed with 500 μl buffer AW2. The column was spun down and placed in a new tube. DNA was eluted in 50 μl DEPC treated water.Beside the published primer pairs , AK129 (unpublished), KHV-GZ11 (GenBank: KJ627438.1), KHV-U (GenBank: DQ657948.1), NC_009127 (NCBI: NC_009127.1), FL_BAC (GenBank: KP343683.1), KHV-J (GenBank: AP008984.1), and KHV-T (unpublished) were used.Sequence information was imported into Geneious , for a sample from Poland and one diagnostic sample from koi in Germany (F11/16). These three samples showed positive signals for both, the Asian and the European lineage. Most interestingly, diagnostic sample F34/13-2, identified as a so-called atypically reacting KHV, did not react positive with the diagnostic qPCR . Only with the primers of VNTR 2 a signal was detected from CyHV-1 and CyHV-2 DNA  were found in the same isolate or specimen. Moreover, the results obtained from VNTR sequences are comparable and confirmed the results from genome sequences or the results obtained with the duplex PCR groups within the two major lineages which cannot be identified by duplex PCR.In order to investigate possible mutations of the virus over time in cell cultures, two samples of different origin (KHV-T and KHV-E) were monitored over 100 passages onto CCB cells at 20°C. For this purpose, samples were analyzed periodically with the three. The results revealed a constant evolutionary pressure even within a consistent environment. Plotting the VNTR frequencies over time Figure  reveals in vivo replication of the infecting agent that generates different variations or types, thereby increasing the likeliness of infection of a fish or a population at the same time. Mixed infections with differential KHV have already been reported, although further investigations are required to elucidate their ecological significance .Finally, this method does not only permit to monitor KHV in the wild and in carp or koi pond cultures but also to use it in cases of KHVD in these fish. Moreover, this technique has the potential to trace back introduced KHV inducing KHVD to their source. This might become useful, if Australia realizes its plan to eradicate or control the common carp population by using KHV (SK and SB planned and performed experimental work. SK, QW, WZ, YW, YL, SZ, JK, PL, MM, and SB discussed the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We aimed to investigate the effects of creatine (Cr) supplementation on the plasma lipid profile in sedentary male subjects undergoing aerobic training.-1 for one week followed by ~10 g·day-1 for a further eleven weeks) or placebo (PL) (dextrose) in a double blind fashion. All subjects undertook moderate intensity aerobic training during three 40-minute sessions per week, over 3 months. High-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), very low-density lipoprotein cholesterol (VLDL), total cholesterol (TC), triglyceride (TAG), fasting insulin and fasting glycemia were analyzed in plasma. Thereafter, the homeostasis model assessment (HOMA) was calculated. Tests were performed at baseline (Pre) and after four (Post 4), eight (Post 8) and twelve (Post 12) weeks.Subjects (n = 22) were randomly divided into two groups and were allocated to receive treatment with either creatine monohydrate (CR) (~20 g·dayP = 0.01), TAG and VLDL . However, no between group differences were noted in HDL, LDL, CT, VLDL and TAG. Additionally, fasting insulin, fasting glycemia and HOMA did not change significantly.We observed main time effects in both groups for HDL (Post 4 versus Post 8; These findings suggest that Cr supplementation does not exert any additional effect on the improvement in the plasma lipid profile than aerobic training alone. Several recent studies have indicated that creatine (Cr) supplementation may reduce the symptoms and progression of certain diseases, including mitochondrial cytopathies , neurodeP = 0.051) brought about by Cr supplementation. Reduced blood glucose was interpreted to indicate an enhancement in insulin sensitivity, which simultaneously could improve the lipid profile if followed by a reduction in fasting insulin levels [To our knowledge, only Earnest et al.  reportedn levels . It is kn levels -10.in vitro a regulatory effect of creatine (Cr) and phosphorylcreatine (PCr) on oxidative phosphorylation in mitochondria which is consistent with a model first proposed by Wyss et al [Pedersen and Saltin  have repss et al , as wellss et al . These ass et al . Severalss et al ,16. Thisss et al  noted thss et al , Arcieross et al  and Earnss et al   for at least the past seven years, without pre-existing renal or cardiovascular diseases, eutrophic, non-smoker, and non-vegetarian. Subject details are given in table The study was approved by the University's ethics committee and all subjects signed an informed consent form before participation.homeostasis model assessment (HOMA) index [A 12-wk, double-blind, randomized, placebo-controlled trial was conducted between 1 March and 1 June 2006. Subjects were randomly assigned to receive either Cr  or placebo . Both groups were submitted to moderate intensity aerobic training for three months. Blood draws were performed prior to training (Pre), after 4 (Post 4), 8 (Post 8) and 12 weeks (Post 12). Samples were centrifuged at 4000 rpm, 4°C, for 5 minutes and stored in a -70°C freezer. We analyzed total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol. Fasting plasma insulin and fasting plasma glucose were also determined and used to estimate A) index . All anaPossible differences between groups and changes during treatment in dietary intake were assessed by three-day 24-hour dietary recall at baseline and Post 12.-1·kg -1 of body weight during the first week (loading phase) and 0.15 g·day-1·kg -1 of body weight for the next 11 weeks (maintenance phase); the PL group was given dextrose in place of Cr, at the same dose. Body weights were those measured at the start of the study. This resulted in absolute doses in high body weight subjects slightly higher that those usually used for loading and maintenance . During the loading phase supplements were presented in four packages, with equal content, and subjects were instructed to ingest one supplement package at breakfast, lunch and dinner and the fourth at 10 pm. During the maintenance phase the dose was divided into two with subjects consuming the supplement during lunch and dinner. The packages were coded so that neither the investigators nor the participants were aware of the contents until completion of the analyses. All subjects were instructed to dissolve the supplements, preferably in juice, in order to mask both the low solubility of Cr and the taste of dextrose. To evaluate subject blinding, the questionnaire asked subjects to indicate which treatment they believed they have received (Cr or placebo) at end of study. The compliance to Cr supplementation was monitored weekly by personal communication. The subjects completed a weekly questionnaire regarding possible adverse effects of Cr supplementation ·dayk et al. ).In order to verify the purity of the Cr used, a sample was analysed by HPLC  and 99.92peak was determined using an incremental treadmill exercise test conducted according to the Bruce protocol. This test is conducted in three minutes stages. The Bruce protocol begins with a speed of 2.74 km·hour -1 and a slope of 10% corresponding to a work rate of approximately 4.6 METS. Each 3 minutes the workload is increased by a combination of increasing the speed and the grade of the treadmill. Attainment of VO2max was accepted when two of three criteria were met: a plateau in VO2, a respiratory exchange ratio (RER)> 1.1 and volitional exhaustion. Ventilatory gas exchange threshold (VT) was determined by the V-slope method[VOpe method.2peak determined by an incremental VO2peak test. Subjects were requested to run at the pre-set heart rate during the whole training period. Thus, an increase in both running distance and running intensity were expected as training progressed. All sessions were monitored by a fitness-professional. From the outset it was ruled that subjects would be dropped from the study if they missed three non-consecutive training sessions, or two consecutive sessions. One subject from PL group was excluded for this reason.Both groups were submitted to aerobic training, three times a week, for 40 minutes per session, for 3 months. Running intensity was set at the corresponding heart rate of 70% of VO®. Additional information was obtained from the Brazilian Table of Food Composition  When data from particular foods were lacking, data from a related food was used.Food intake was assessed by three 24-hour dietary recalls, using visual-aid photographs of real foods. Energy and macronutrient intakes were examined by the Brazilian software Virtual Nutri®, Bioclin, Belo Horizonte).Fasting plasma glucose was determined by colorimetric enzymatic assay, with a commercial kit Fasting plasma insulin was determined by radioimmunoassay technique .Total serum cholesterol concentration was determined as previously described by Kaplan and Pedersen , with a ® – HDL-colesterol, Sao Paulo).HDL-cholesterol was determined according to Finley et al.  using a ® – Triglicerídeos, Sao Paulo). From this VLDL-cholesterol (VLDL-cholesterol = tryglicerides/5) and LDL-cholesterol ) concentrations were calculated.Triglycerides were assayed according to Kaplan and Pedersen , by colo® proc Mixed was used to analyze repeated measures and, when applicable, Tukey Post hoc was used for multiple comparisons. The groups (CR and PL) and periods  were considered fixed factors and the subjects were assigned as random factors.SASFor baseline comparisons between the groups two-sample independent t-test was used. All data is expressed as mean ± SD. The significance level adopted to reject the null hypothesis was p ≤ 0.05.® proc IML was used to determine the power of the statistical tests using a simulation model [Also, SASon model  as suggeon model . ExpecteOf the 103 people who responded to the initial request for volunteers, 79 were excluded as they performed regular physical activity or expressed self-reported chronic disease. The remaining 24 subjects were randomly assigned to the CR or PL groups. Two subjects were subsequently lost in the PL group: one withdrew for personal reasons and one was excluded because they missed 2 consecutive training sessions. Consequently 12 subjects remained in the Cr group and 10 in the PL group.P = 0.89).There were no differences between or within groups in both nutrient and energy intake Table . The bliP = 0.05 time main effect). There was also reduction in body fat content measured by skinfold thickness  and resting heart rate .Twelve weeks of aerobic training significantly increased the running distance per session , there P = 0.01) and decreases in TAG and VLDL-cholesterol  in both groups (main time effect). No variable was significantly changed with Cr supplementation (interaction effect).Lipid profile values can be found in Figure No significant main effects or differences between groups were found in fasting glucose, fasting insulin and HOMA index results Table .-1, with 50% less during the following eleven weeks. The mean body weight of the CR group was 83.1 ± 16.3 kg. The dose of 0.3 g·day-1·kg -1 was chosen to ensure that subjects even at the lowest body weights received a loading dose close to 20 g per day, although the same relative dose resulted in a higher loading dose in subjects at the upper end of the weight range. However, we were cognisant that some authors have suggested that use of a low Cr dose may have been responsible for the lack of change in lipid profile in the study of Volek et al. [-1 used by Arciero et al. [-1 over the next 3 weeks.As body weights in the CR group ranged from 64.3 to 110.0 kg, absolute doses of Cr given in the first week ranged from 19.3 to 33.0 g·dayk et al. . The dosper se on lipid profile. We tested for a possible additive effect of Cr supplementation on the previously described improvement in lipid profile after aerobic training [Following the earlier work by Earnest et al.  our aim training ,27.Our findings do not agree with those of Earnest et al. . The difP = 0.051). According to the authors, such a decrease indicated a possible improvement in insulin sensitivity, which would cause a better lipid profile particularly if followed by a reduction in the fasting insulin levels. However, our results and others [Earnest et al.  also repd others ,29 have d others  and othed others ,30 have d others  verified-1) during 28 days of high intensity training. The supplemented group presented a significant increase in HDL-cholesterol (13%), and a significant decrease in both triglycerides and VLDL-cholesterol (-13%), compared to the placebo group, showing an additive effect of Cr supplementation. Accordingly, Arciero et al. [-1 for 7 days followed by 5 g·day-1 for 11 weeks) significantly decreased total cholesterol values (-9.9%) when compared to strength training only. Their results reinforced the hypothesis that Cr supplementation had an additive effect to training effects on the lipid profile. A possible explanation for this phenomenon was that Cr supplementation enhanced training quality indirectly (i.e. by optimizing volume and intensity). In a review manuscript, Durstine et al. [per se.Kreider et al.  also repo et al.  showed te et al.  suggesteCr supplementation is reported to be effective mainly in the improvement of performance during sustained or intermittent high intensity exercise and not in aerobic training . Whilst -1). However, in our study we provided ~10 g·day-1 and again no difference between the placebo and the Cr group was observed. Thus, whilst exercise is clearly important in determining the plasma lipid profile, Cr itself does not appear to have any additional effect in healthy males.Volek et al.  similarlCr supplementation did not have any additive effect on the plasma lipid profile of healthy sedentary males undergoing moderate intensity aerobic training for 3 months. These results may appear contradictory to those of Earnest et al.  althoughThe authors declare that they have no competing interests.BG is the principal investigator of the project. BG and AHLJ designed the study. BG, CU, GGA, FBB and FBS collected the data. BG, CU, GGA, FBB, FBS and RCH conducted data analysis. All authors wrote the manuscript.