Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients’ ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease. Cancer is heterogeneous disease; each tumor in different patients has different cancer genomes. Furthermore, another level of heterogeneity exists: even a single tumor harbors multiple genetically distinct subclones. This intratumor heterogeneity is presumably one of causes of therapeutic difficulty, and its understanding is clinically necessary. In this study, we investigated intratumor heterogeneity in colorectal cancer by analyzing sample obtained from geographically separated regions of 9 colorectal tumors. Our integrated data analyses combined with computational simulation strongly suggest that, after clonally shared alterations were accumulated by aging, numerous subclones were generated by neutral evolution. Importantly, this view can explain the robustness and evolvability of cancer: therapeutic action inducing an environmental change would convert some of the numerous neutral mutations to driver genes that confer therapeutic resistance. We believe that this study not only provides insights into colorectal cancer pathogenesis, but also constitutes a new basis for designing therapeutic strategies. Cancer is a heterogeneous disease. Recent cancer genomics studies have revealed extensive genetic diversity among patients. Moreover, even a clonal tumor in one patient often harbors multiple subclones. This phenomenon is called intratumor heterogeneity (ITH) and is presumably generated by branching clonal evolution of cancer cells. Understanding of ITH is clinically important, since the existence of multiple subclones presumably boosts the evolutionary adaptation of tumors against therapies, constituting a source of resistant clones .Recently, a multiregional sequencing approach, which sequences DNA sampled from geographically separated regions of a single tumor, has revealed branched evolution and ITH. Yachida et al.  investigSeveral studies employing multiregional exome sequencing have revealed the occurrence of branched evolution and ITH in several other types of cancers, including clear cell renal cell carcinomas and non-small cell lung cancers. ITH of clear cell renal cell carcinomas is characterized by parallel evolution, in which the same driver gene is independently mutated in different branches of evolutionary trees . In contIn this study, we present genetic and epigenetic analysis of ITH in a series of nine colorectal cancers. Following multiregional sampling, we performed exome sequencing and copy number (CN), methylation, and mRNA expression array profiling. Our integrated analysis revealed not only extensive ITH, but also the evolutionary histories of the nine tumors. Finally, we also performed computational simulation of cancer evolution, which suggested a possible evolutionary principle underlying the extensive ITH.To study ITH in colorectal cancer, we performed genomic analysis of samples from geographically separated regions from nine colorectal tumors . In thisEach of the multiregional mutation profiles harbored founder and progressor mutations; founder mutations are shared by all regions while progressor mutations are not. We further divided progressor mutations into two subcategories: “unique” and “shared” mutations, which are unique to a single specific sample and shared by multiple but not all samples, respectively. Targeted deep sequencing validated 100% (5068/5068), 93.9% (1745/1857) and 95.4% (1362/1427) of founder, shared, and unique mutations, respectively. We can assume that founder, shared, and unique mutations are acquired in this order during cancer evolution. Applying the maximum parsimony method . Then P ∝ Binom)PP(c). For calculating P, we obtained every possible configuration of p from qa, and qb, and assumed that they are equally probable. For example, when qa, = qb = 1, P(p = 1) = 1. When qa, = 1 and qb = 2, P(p = 1) = 2/3 and P(p = 2) = 1/3, because a mutation could occur in one copy of allele a, one copy or two copies of allele b. P(c) is a prior probably of c and we assumed a uniform prior. P was then obtained by calculating these values over a regular grid of 100 c values and reported after normalization. We defined the median of P as CCF.Cancer cell fraction (CCF) in which each mutation occurs was estimated by modifying a previously reported approach . Considem ad n were obtained from WES data while α, qa and qb were obtained by ASCAT analysis on SNP array data. For deducing α > 0.6. CCF is also used for obtaining clonal and subclonal mutations from single sample sequencing data; we assumed mutations of which CCF exceed 0.8 as clonal mutations, and others as subclonal mutations.In this study, https://cghub.ucsc.edu/ with the accession no. 26594–6, which were first converted back to fastq files, where the ‘read1’ and ‘read2’ entries in fastq files were reconstructed based on pair-read information. For reverse strand reads, we generated complemented bases and assigned BaseQuality accordingly. In this process, the reads that had Flag of “not primary alignment”, “read fails platform/vendor quality checks” or “supplementary alignment” were discarded using samtools-0.1.18. Then, the fastq file were input into our WES analysis pipeline to call mutations.TCGA WES data were processed by the same pipeline used for our WES data. Because only BAM files were available for TCGA data, we first convert BAM files to fastq files. BAM files for COADREAD paired tumor/normal samples were downloaded from https://tcga-data.nci.nih.gov/tcga/. The probe-level signal intensities were converted to BAF and LLR as follows: BAF = Ib /(Ia + Ib) and LLR = Ia + Ib where Ia and Ib were signal intensities for A and B alleles. Then, BAF and LLR were input into the ASCAT algorithm to estimate purity and allele-specific absolute CN. In total, 295 COAD and 110 READ TCGA patient samples were available for both the mutation and CN data.SNP 6.0 array data of COADREAD paired tumor/normal samples were also obtained from From these data, CCFs were calculated for each mutation to obtain clonal and subclonal mutations. In the analyses of mutational signatures and correlation between patients’ ages and mutation rates, 53 COAD and 5 READ samples whose mutation rates ≥ 10 Mb were separately analyzed, assuming that they belong to the hypermutated subtype.Genomic DNA was bisulfite treated using the EZ-96 DNA Methylation Kit  and genome-wide DNA methylation were profile with the Illumina HumanMethylation450 BeadChip (Illumina). A methylation level of each probe was then obtained as a β-value. In this study, we focused on only the probes designed for autosomal sequences. In analyses of methylation variance and signature, Positional categories were based on probe information of the HumanMethylation450 BeadChip.For each probes, differential methylation was quantified with each tumor samples and matched normal tissue by the difference of β-values . For each case, we assumed that a probe was subjected to founder hyper or hypomethylation, if Δβ > 0.3 or Δβ < 0.3 in all the samples of the case, respectively. Progressor methylations were identified as done for progressor CN alterations; we searched for differentially methylated probes among every pair of the sample groups divided by mutation-based evolutionary tree. For each probe, we obtained difference of mean methylation between every pair of the sample groups, and the maximum difference as a statistic Δ’β. For a threshold value θ, we obtained probes of which |Δ’β| >θ as probes subjected to Progressor methylations. For each case, θ was adjusted so that FDR was 0.1. The FDR was calculated by permutation of the samples. The progressor methylations were further divided into progressor hyper and hypomethylations based on plus and minus of Δβ averaged across the samples. For making heat maps of multiregional methylation profiles in k groups has nj variables, xij, the total sum of square SST is decomposed into between- and within-group sum of squares, SSw and SSb:For each probe, we calculated inter- and intra-variance (variance among cases and within cases). Our approach was based on partitioning of the sum of squares, which is utilized in ANOVA. Assuming that each of SST = SSw +SSb, where xij. Inter- and intra-variance were measured by SSw and SSb, respectively.In our setting, case 2–8 can be regarded as the k groups while the β-values of each sample can be regarded as http://www.broadinstitute.org/gsea/msigdb/), to obtain module activities of significant expression modules as expression signatures.mRNA expression profiling were performed with oligonucleotide microarrays  as described previously . Multiren genes, each of which is represented as a binary value, 0 (wild) or 1 (mutated). Namely, the genome is represented as a binary vector g of length n. In a unit time step, each cell in the simulated tumor dies with a probability q. If the cell does not die, the cell then divides with a probability p. Before the cell division, we mutate the genome vector g: each of 0 elements of g is set to 1 with a probability r. The first d genes in g are assumed as driver genes, whose mutations accelerate the division speed. A normal cell without any mutations has a division probability p0, and acquisition of one driver mutation increases p by 10f-fold in the next time step; i.e., p = p0 · 10fk, where q = q0. Let c and t denote the size of the simulated cell population and the number of the time steps, respectively. We started a simulation with c0 normal cells and repeated the unit time step while population size c ≤ cmax and time step t ≤ tmax. A flowchart of the simulation was shown as n = 300, p0 = 0.001, q0 = 10−7, c0 = 10, cmax = 106 and tmax = 5×106. r, f, and d were subjected to parameter fitting as described below.We computationally simulated BEP employing a cellular automaton model, which assumes each cell in a tumor as a cellular automaton . A cell c0 cells are initialized as close as possible to the center of the lattice. When a cell dies, the occupied point is cleared and becomes empty. When a cell divides, we place the daughter cell in the neighborhood of the parent cell, assuming the Moore neighborhood . If empty neighbor points exist, we randomly select one point from them. Otherwise, we create an empty point in any of the eight neighboring points by the following procedure. First, for each of the eight directions, we count the number of the consecutive occupied points that range from each neighboring points to immediately before the nearest empty cell as indicated in li, where li (1 ≤ i ≤ 8) is the count of the consecutive occupied points for each direction. The consecutive occupied points in the selected direction are then shifted by one point so that an empty neighboring point appears as shown in We designed a simulated tumor to grow in a two-dimensional square lattice where each cell occupies one lattice point. In the beginning, s samples, s small square lattices were randomly selected from the small square lattices where at least a half of lattice points were occupied by cells  fall within 1 standard deviation from the mean of the observed values was calculated and visualized as heat maps in We next performed the simulation with different parameter settings to evaluate which parameter setting leads to summary statistic values similar to the observed ones. Here, d (the number of driver genes), f (strength of driver genes) and r (mutation rate) were subjected to parameter fitting analysis. We prepared 10 integers from 1 to 10 as m cells  to obtain an n×m single-cell mutation profile matrix, each of whose m columns is the n-dimensional genome vector g for each cell. We obtained color labels for each cell, as done for the sample color labels in the real data. Namely, we applied principal component analysis to the single-cell mutation profile matrix and mixed RGB colors according to dimension-reduced genome vectors. Each cell on the two-dimensional square lattice was colored with the corresponding color label  Mean depth of each sample. (B) Fractions of the target region covered with at least 10x, 20x and 30x depth.(PDF)Click here for additional data file.S2 FigWe searched for progressor mutations that were shared by distant branches in the evolutionary trees of the 9 cases, and found clear examples of such singular mutations in case3 and case7. The heat maps show VAFs of the singular mutations from exome and targeted deep sequencing for case3 and case7, respectively. Timings of subclonal mixing events were inferred and indicated by red arrows on the evolutional trees.(PDF)Click here for additional data file.S3 Fig(A) The color table shows whether any member of the 5 driver pathways was disrupted by founder and progressor genomic alterations  in the 9 cases. The driver pathways and their members were obtained from the TCGA paper . (B) Gen(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigIn addition to our data, multiregional mutation data from previous clear cell renal cell carcinoma  and panc(PDF)Click here for additional data file.S6 FigA) The Sciclone analysis [B) For each of the clonal and subclonal mutations estimated based on cancer cell fraction, proportions of founder, shared and unique mutations were presented. This analysis was performed for samples whose purities exceed 0.6. The result demonstrated that, when we focused on each single sample, founder and progressor mutations tended to exist as clonal and subclonal mutations, respectively.Click here for additional data file.S7 FigA) Correlations between mutation rates and patients’ ages. P-values were calculated by The Wilcoxon rank-sum test. (B) Mutational signature analysis. (C) For each base substitution type, the number of patients having the larger number of clonal or subclonal mutations was shown. P-values were calculated by the binomial test.((PDF)Click here for additional data file.S8 Fig(A) Founder and progresor mutations in the 9 cases were divided into 96 substitution patterns based on mutated bases and their 5’ and 3’ flanking bases, and the percentages of each substitution pattern were plotted as a bar plot. (B) Clonal and subclonal mutations in the TCGA samples were analyzed as in A.(PDF)Click here for additional data file.S9 FigThe heat maps show LRR across chromosomes in each sample.(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fighttps://tcga-data.nci.nih.gov/tcga/. For the 2000 probes showing the highest variance in the TCGA samples, we made clustered heat maps of β values for only the TCGA samples (A) and the TCGA samples mixed with our samples (B). Based on this result, we classified cases 5 and 9 into the CIMP subtype.Methylation datasets of 529 TCGA COADREAD samples were obtained from (PDF)Click here for additional data file.S12 FigEnrichment scores for intratumor (A) and intertumor (B) variance were calculated as in (PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S14 FigThe heat map shows Δβ values of probes contained by the CpG island promoters of the epigenetic gatekeepers  and CIMP(PDF)Click here for additional data file.S15 FigWe assumed that hyper- and hypomethylated probes have Δβ> 0.3 and Δβ< -0.3, respectively. CpG island hypermethylation was significantly correlated with patients’ ages. r’s are Pearson’s correlation coefficients and p-values were calculated by the Pearson's correlation test.(PDF)Click here for additional data file.S16 Figf -fold.A cell has n genes, d out of which are driver genes. In this schema, n = 10 and d = 4, and red and blue boxes denote driver and non-drive genes, respectively. In a unit time step, a cell divides or dies with probabilities p and q, respectively. During each cell division, each gene is randomly mutated with a probability r, and one driver mutation, which is denoted by a red cross, increases p by 10(PDF)Click here for additional data file.S17 FigA) Observed values of summary statistics in the real data. The proportion of founder and unique mutations were obtained for 9 cases. For each of the cases except case4 and case9, downsamplings were performed to obtain 10 multiregional profiles of 5 samples and the statistics were averaged over the downsampling trials. The error bars indicate standard deviations for the downsampling trials. Finally, an “average” over the 9 cases was obtained as an estimate of the observed value of each summary statistic. The error bars at “average” indicate standard deviations over the 9 cases. (B) Multiregional mutation profiles from the real experiments. For the cases except case4 and case9, representative samples from the 10 downsampling trials were presented as in ((PDF)Click here for additional data file.S18 FigA) The proportion of simulation instances fitted to the real data. The proportion of simulation instances whose statistics fall within 1 standard deviation from the mean of the observed values was calculated for each parameter settings and visualized as heat maps (B) Multiregional mutation profiles from the simulations. Representative instances from simulation with indicated parameter settings were presented as in ((PDF)Click here for additional data file.S19 FigSimulated tumors, simulated single-cell and multiregional mutation profile matrix from four simulation trials are shown as in (PDF)Click here for additional data file.S20 FigA) Snap shots of growing tumors in a simulation. Differently colored cell populations represent each clone. (B) A growth curve of the simulated tumor. The snap shots were obtained at each plotted point. (C) Single-cell mutation profiles of the simulated tumor during growth. Timing at which the mutation profiles were obtained are indicated by red rectangles in (A) and red plotted points in (B). Top colored bars represent each clone while left blue bars represent driver genes.((PDF)Click here for additional data file.S21 FigA) A flowchart of our simulation.  illustration of division operation. See the simulation section in Materials and Methods.((PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Vernonia amygdalina are often utilized as vegetable and for medicinal purpose compared to the old leaves. This study was designed to evaluate and compare the antidiabetic effects between ethanolic leaf extracts of old and young V. amygdalina on streptozotocin (STZ) induced diabetic rat for four weeks. Preliminary screening of both young and old ethanolic extracts revealed the presence of the same phytochemicals except flavonoids which was only present in the old V. amygdalina. Difference in antioxidant power between the young and old leaf extracts was statistically significant (p < 0.05). Both leaf extracts produced a significant (p < 0.05) antihyperglycaemic effect. Also results from treated rats revealed increasing effect in some haematological parameters. Similarly, the higher dose (300 mg/kg) of both extracts significantly (p < 0.05) reduced serum ALT, AST, and ALP levels as compared to the diabetic control rats. Results also showed significant (p < 0.05) decrease in LDL-C and VLDL-C in the extract-treated rats with a corresponding increase in HDL-C, as compared to the diabetic control rats. Moreover histopathological analysis revealed ameliorative effect of pathological insults induced by the STZ in the pancreas, liver, and spleen, most significantly the regeneration of the beta cells of the islets of Langerhans in treated rats.The young leaves of Radical scavenging activity was measured by method described earlier , 17, witAfter one week of acclimatization period, DM was induced experimentally in rats by multiple intraperitoneal injections of 40 mg/kg STZ dissolved in citrate buffer daily for three days. Control rats were intraperitoneally injected with the citrate buffer alone. After the three days of STZ treatment, blood samples were taken from tail vein using a tail clip and glucose levels measured with a glucometer (Accu-CHEK). Rats with nonfasting glucose levels higher than 15 mmol/dL were considered as being diabetic and were included in the study. Group 1 : non-STZ induced rats. Group 2 (negative control), STZ induced diabetic rats: normal saline. Group 3 (positive control), STZ induced diabetic rats: standard drug (glibenclamide 10 mg/kg).  V. amygdalina (10 mg/kg).Group 4, STZ induced diabetic rats: extract of old  V. amygdalina (30 mg/kg).Group 5, STZ induced diabetic rats: extract of old  V. amygdalina (300 mg/kg).Group 6, STZ induced diabetic rats: extract of old  V. amygdalina (10 mg/kg).Group 7, STZ induced diabetic rats: extract of young  V. amygdalina (30 mg/kg).Group 8, STZ induced diabetic rats: extract of young  V. amygdalina (300 mg/kg).Group 9, STZ induced diabetic rats: extract of youngA total of 56 experimental rats were used to assess the effect of the leaf extracts on the experimental rats: 49 STZ induced diabetic rats plus 7 normal control rats. Animals were divided into eight (8) major groups and housed under controlled environmental conditions. Rats were divided into the following groups:The body weight measurement and fasting plasma glucose levels were assessed before and during administration of the extracts weekly, till the end of the study.Experimental rats were sacrificed and blood was dispensed into EDTA tubes and then analyzed for haematological indices such as red blood cells (RBCs), haemoglobin (Hb), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), monocytes (MON), neutrophils (NEU), and lymphocytes (LYM) using an automated haematology analyzer . Another portion of blood was collected in empty vacutainer tubes and centrifuged at 13000 rpm for 5 mins. The serum was then retrieved and biochemical parameters such as AST, ALT, ALP, triglycerides, TB, TP, TC, triglycerides, HDL-C, LDL-C, and VLDL-C in serum were analyzed using commercially available kits and a standard BS-120 Mindray Chemistry Analyzer.μm using the rotary microtome. The sections were deparaffinized and subsequently stained with haematoxylin and eosin (H&E) as described earlier [The rats were dissected and the liver, spleen, and pancreas were removed and observed for evidence of gross pathology. For light microscopic examination, tissue samples were immediately fixed in 10% phosphate buffered formalin, processed using an automated tissue processor  and finally embedded in paraffin wax. The tissue blocks were then cut into serial sections with thickness of 6  earlier . The pant-tests for significance of differences were used. To compare biological effects of the treatment, analysis of variance (ANOVA) was used. Post hoc analysis was also used to identify the source of the variation. p values less than 0.05 were considered statistically significant.GraphPad Prism Version 5.0 for Windows  was used for data analysis. Data were presented as mean ± SEM on line graphs and tables. For comparing control and treatment group, independent samples  V. amygdalina leaves revealed the presence of alkaloids, tannins, saponins, cardiac glycosides, terpenes, and steroids  (p < 0.05), the ratios showed that the extracts have high free radical scavenging properties at higher concentrations, with YL having a higher degree of radical scavenging activity than OL.The crude extract of YL and OL ofide (NO) . At highp > 0.05), there was appreciable gain of weight  reduced the elevated levels of the liver enzymes  compared to the negative control. Though not significant for the other liver function tests (TP and TB), decreased TP was elevated while elevated levels of TB were appreciably reduced in the treated groups as compared to the diabetic control group. Similarly, the diabetic control group showed elevated levels of TC, triglycerides, LDL-C, and VDL-C with lowered levels of HDL-C (p < 0.05) with a corresponding increase in HDL-C in treated rats. Comparing the different doses of the two groups (YL and OL), though not significant, the normalization of the biochemical indices was higher in the YL (10–300 mg/kg) than in the OL (10–300 mg/kg) as compared to the normal control group, except for ALP, where the high dose of OL (300 mg/kg) showed a better curative effect (532.21 ± 2.11 U/L) than the 300 mg/kg YL (528.25 ± 6.59 U/L).All doses of YL and OL, including the positive control, significantly (of HDL-C . Serum lp < 0.05) antihyperglycaemic effect on the 14th, 21st, and 28th day of the experiment compared to the negative control group as shown in A general increase in the blood glucose level was observed in the rat following the administration of STZ, with mean glucose levels ranging within 15.0–15.60 mmol/dL. Treatment of the hyperglycemic rats with the different doses  of both YL and OL produced a significant  showed hypercellularity of resident cells but no haemosiderosis Figures . The posβ) cells and non-β-cells). There is also compact arrangement of cells of the islets. Section from the diabetic control experimental rats  in the YL may probably exhibit the corresponding increase in the antioxidant power realized, and this is demonstrated in the high DPPH scavenging activity of the young. The cumulative effect of this may be seen in the efficient scavenging of free radicals produced in the animals; thus this compound effectively augments the endogenous antioxidant defense mechanisms as reported elsewhere , produci V. amygdalina in rat. STZ is a diabetogenic agent which induces diabetes by damaging the pancreatic β-cells of the islets leading to hyperglycemia [The positive control group and the extract-treated rats all manifested a significant reduction in blood glucose levels as compared to the negative control. The highest reduction (4.10 ± 2.60 mmol/dL) was observed in the positive control group (treated with glibenclamide), followed by treated rats that received 30 mg/kg YL (4.13 ± 0.46 mmol/dL) of the plant extract. However, the diabetic control group manifested continuous increase in glucose levels from day 0 to the 28th day. The result of this study confirms earlier report by other researchers , 12 on tglycemia . This stglycemia , 27, whe V. amygdalina together with 10 mg/kg glibenclamide brought parameters to near normal as compared to the normal control group. This confirms reports by researchers elsewhere [ V. amygdalina, shared similar effect, but with the 30 mg/kg YL, demonstrating high ameliorative effect, compared to the other doses. The high dose (300 mg/kg) of the YL showed the least in normalization of the haematological indices. This adverse effect may probably be as a result of the high antioxidant capacity in this dose and is in line with reports elsewhere [ V. amygdalina had the potential of adversely affecting haematological indices. The observed significant difference between the negative control and treated rats in some of the enzyme level associated with liver function test  as well as HDL-C, LDL-C, and VLDL-C levels, and this is in agreement with reports from previous research works [ V. amygdalina and glibenclamide significantly reduced the ALT, ALP, and AST levels in blood of the STZ induced diabetic rats, indicating the ameliorative effect of the extract on the hepatocytes of the liver after action of the injurious agent (STZ) in the diabetic state, which is in agreement with research work carried elsewhere [Furthermore, the study revealed a decrease in a number of haematological parameters especially RBCs, neutrophil, and monocyte count in the negative control group, confirming work done by Ajagbonna et al. , who replsewhere , 30. Moslsewhere , 31, whelsewhere  which such works , 32 wherlsewhere , 27. Thelsewhere . V. amygdalina leaves with their respective doses (10–300 mg/kg) together with the positive control.The observed weight gain, though the differences were not statistically significant, was exhibited by the ethanol extract of both YL and OL ofHence, the ability of the extracts to significantly reduce the high blood glucose levels and effectively increase the body weight of the diabetic rats may be attributed to its antihyperglycaemic effect. Other recent scientific findings , 27 atteHistopathological results showed normal pancreatic acini and islets of Langerhans in the normal control group, with the negative control group showing evidence of nuclei degeneration and cytoplasmic changes in the cells of the islets, specifically within the middle portion of the islets where there was reduced cellularity. This concurs with previous reports where STZ induced diabetic rats showed atrophy and focal necrosis within islet cells of Langerhans , 27.β-cells of the pancreas with respect to the doses, and this fact is supported by the decrease in blood glucose levels. These changes were consistently observed in all animals in these groups. However 30 mg/kg YL and 300 mg/kg OL of the extract manifested the greatest pancreas regenerative ability compared to the other groups. This study affirms the work done by Ojiako and Nwanjo [ V. amygdalina has protective effect as evidenced by minimal necrotic changes seen in injured tissues after extract administration.Both extracts (young and old) showed regeneration of the The increase in lipid profile parameters  and liver enzyme parameters  in the diabetic control rats in our results could be explained by the fatty changes (cytoplasmic vacuolation) and hepatocytic degeneration observed in the liver sections. This may be as a result of the STZ conversion to metabolites in the liver that caused catalytic membrane phospholipid peroxidation, breaking down the endoplasmic reticulum, which ultimately caused reduced lipid export from the liver cells and thus accumulation of lipid in the hepatocytes, as described elsewhere , 36. ThiHaemosiderosis observed in spleen sections of the negative control group may be as a result of the toxic effect of the STZ on the RBCs as mentioned earlier. This pathological effect manifested as an accumulation of iron pigments, depicting increased destruction of RBCs , which cGross anatomical inspection showed hypertrophy of spleen of rats that received 300 mg/kg of both YL and OL of the extracts. Research work from other authors , 16 usin V. amygdalina have the ability to protect against STZ induced tissue degeneration in diabetes and clearly justifies the use of the younger leaves as potent antidiabetic agents.In summary, for the lower concentrations, 10 and 30 mg/kg YL of the extracts were more efficacious than the OL (10 and 30 mg/kg OL) as compared to the normal control group for blood and tissue parameters that were analyzed. For the high concentrations, 300 mg/kg OL exhibited a better curative ability for haematological parameters than the other doses, but with reduced ameliorative effect for the biochemical parameters as compared to 300 mg/kg YL. The observations taken together add credence to the assertion that leaf extracts of V. amygdalina possess antidiabetic properties which may explain the traditional use of this plant for management of diabetes mellitus and its complications but differ in their phytochemical compositions, antioxidant capacity, and pancreatic and hepatic regenerational abilities.From the results obtained in this study, it can be concluded that both YL and OL extract of


Objectives. This study analyzed the long-term hearing loss after treatment of primary nasopharyngeal carcinoma to elucidate its causal factors. Methods. Ninety-two nasopharyngeal carcinoma patients were treated with radiotherapy or chemoradiotherapy. Pure tone audiometry was performed before the therapy and annually up to 9 years after completing treatment. The hearing thresholds were corrected for age-related deterioration and compared to the results without adjusting for age. Results. The mean air and bone conduction threshold with and without correction for age-related deterioration differed significantly 2–9 years after completing radiotherapy (p < 0.05). The audiometry results with age correction showed a flattened configuration compared to the results without age correction. The total radiation dose and radiation modality showed a causal relationship with a greater incidence of hearing loss after therapy (p < 0.05). There was more deterioration in the air and bone hearing thresholds with conformal radiotherapy than intensity-modulated radiotherapy (p < 0.001). A radiation dose >72 cGy resulted in more severe hearing loss than <72 cGy (p < 0.05). Conclusion. Hearing loss after completing therapy should be corrected for age-related hearing deterioration to reveal the true extent to which the loss is a therapeutic complication. Both the radiation modality used and the dose were significantly associated with hearing loss. Nasopharyngeal carcinoma (NPC) is often located in the fossa of Rosenmüller near the opening of the Eustachian tube into the nasopharynx. Of these tumors, 95% are undifferentiated squamous cell carcinoma, which is very sensitive to radiotherapy (RT) . ChemoraWhile RT or chemoradiotherapy is the standard approach for NPC, the radiation and cisplatin ototoxicity inevitably damage the auditory apparatus –8. This Any study of HL may be erroneous if the influence of patient age on hearing status is not considered because age can affect HL . We reviNinety-two newly diagnosed cases of NPC were treated in a single medical center between June 1994 and July 2010. Audiograms were obtained before RT and at least 12 months of follow-up after completing RT. We enrolled NPC patients who were older than 18 years and had no significant preexisting HL; that is, the average bone conduction hearing threshold levels (0.5–4 kHz) were below 70 dB before RT. Patients who received additional RT for metastatic or recurrent disease, who were exposed to noise, or who received ototoxic medications other than cisplatin were excluded from the study. This study protocol was approved by the institutional review board of Chang Gung Memorial Hospital.2.Patients with stage I/IIa were treated with RT alone, while the locally advanced stage IIb-IV patients were treated with chemoradiation therapy. With updates at our institution, the radiation technique has changed from two-dimensional conformal RT (2D CRT) to three-dimensional conformal RT (3D CRT) to intensity-modulated RT (IMRT). The total radiation dose was 59.4–79.2 Gy with a mean dose of 70.86 Gy. Patients with locally advanced disease were given concurrent intravenous cisplatin chemotherapy at a mean dose of 365.57 (range 120–670) mg/mAudiometry was performed before and after RT at 1-year intervals for up to 9 years. European Committee for Standardization (CEN) data were used to obtain corrected thresholds by subtracting the loss corresponding to age and sex from the auditory threshold obtained at each frequency . For exat-test was used to determine the correlation between the possible predisposing factors and average hearing threshold deterioration. Univariate and multivariate analyses were performed with a Cox stepwise logistic regression model to identify independent prognostic indicators. A paired t-test was used to assess the serial changes in hearing with or without age adjustment. A p value less than 0.05 was considered to be statistically significant. All statistical tests were two-sided.The data were analyzed using SPSS ver. 19.0 . An independent This study included 182 ears from 92 patients. The patient characteristics and therapeutic modalities are summarized in The average hearing thresholds (not considering age-related HL or presbycusis) are shown in p < 0.05), especially at 4–8 kHz (p < 0.05). The levels of hearing deterioration were less than those without adjusting (p < 0.05). The deterioration at all frequencies tended to decrease after adjusting for age, especially at 4 kHz , while the differences were not significant at 1 year after completing RT (p = 0.16) . We found that adjusting for age decreased the deterioration of HL after completing treatment.The air conduction hearing deterioration at 0.5–4 kHz with and without age-related correction differed significantly at the 2- to 9-year follow-ups ( = 0.16) . The patp < 0.001; 14.01 dB versus 6.85 dB p < 0.001), radiation dose , and OME  were important determinants of hearing deterioration via both air and bone conduction. Radiation modality (p < 0.001) and radiation dose  remained significant in multivariate analyses. We found that deterioration in both the air and bone conduction thresholds was less in the IMRT group than in the 2D/3D CRT group 5 years after completing RT. The deterioration in hearing threshold was also greater when the total radiation dose to the primary tumor was >72 Gy.The mean hearing deterioration after adjusting for age at 0.5–4 kHz in the first 5 years was analyzed in terms of gender, age, radiation modality (2D-3D CRT and IMRT), concurrent chemotherapy, radiation dose (<72 Gy and >72 Gy), and post-RT otitis media with effusion (OME) . The radp < 0.001; 16.19 dB versus 5.91 dB p < 0.001), radiation dose , and OME  were important determinants of hearing deterioration via both air and bone conduction in the chemoradiotherapy group. Radiation modality (p < 0.001 for both air and bone) was significant in multivariate analyses. Only radiation dose (12.07 dB versus 22.67 dB p = 0.036 for air conduction) remained significant in the RT group.In order to evaluate the effect of cisplatin, we further analyzed the hearing deterioration in both RT (89 ears without cisplatin) and chemoradiotherapy (93 ears with cisplatin) groups. The radiation modality  5 years after completing RT  and chemoradiotherapy (93 ears with cisplatin) groups. The radiation modality (CRT and IMRT), radiation dose, and OME were significant via both air and bone conduction in the chemoradiotherapy group. Radiation modality remained significant in multivariate analyses. Only radiation dose was significant in the RT group. Cisplatin appeared to increase the hearing deterioration in NPC patients using a CRT technique.Chemotherapy using cisplatin did not predict HL, in agreement with previous reports  24, 26], 2624, 22 , 28. In Post-RT OME was not an independent predictor of hearing threshold deterioration. The characteristics of post-RT OME differed with the radiation dose and modality. The post-RT OME group treated with IMRT and a radiation dose <72 Gy might have had less severe hearing threshold deterioration. With IMRT, the dose to the Eustachian tube was <52 Gy, reducing the incidence of post-RT OME and severity of air and bone conduction threshold (0.5–4 kHz) deterioration , 30. YouSome studies have observed a persistent association between the presence of post-RT OME and HL , 32. The per se on hearing status were not considered; again, these factors affect HL.We found no significant correlation between age and gender and post-RT HL data adjusted or not for age-related threshold deterioration. The effects of age and gender on the development of RT-induced HL were inconsistent in previous reports . Some auThis study was retrospective in nature, and several potential limitations should be mentioned. First, follow-up was performed after various intervals by different physicians, and it is likely that the follow-up protocols differed greatly. For example, the instruments used for measurement will have varied . In addition, the RT techniques will have varied. NPC patients may have died or become lost to follow-up. Second, data incompleteness and differences in the way data were annotated in databases and medical charts render it difficult to define the cause of HL after RT. Moreover, all the evaluation of long-term HL after treatment of primary NPC was performed at a single center. We believe that these factors have affected all cases similarly, merely rendering our estimations imprecise; we do not believe that we have exaggerated or minimized such estimations. Patients with possible confounding factors, such as significant preexisting HL, use of ototoxic medications other than cisplatin, or exposure to noise, were excluded from the study.There is a concern that the issue of cisplatin, an ototoxic drug, could confound the data collected. Since late stage NPC patients treated with cisplatin were included in this study, it was difficult to separate an aging and radiation in patients with cisplatin versus no cisplatin. Cisplatin interferes with the overall findings and conclusions reached.After adjusting for age-related hearing threshold deterioration, HL owing to the treatment of NPC was present clinically at least 12 months after RT and involved all frequencies. Therefore, it is essential to adjust for patient age when determining the true change in hearing following RT or chemoradiotherapy. IMRT and total radiation dose less than 72 Gy tended to result in less deterioration in hearing.The hearing thresholds of air conduction at 0.25, 0.5, 1, 2, 4, and 8 kHz were measured and adjusted (Supplementary Table 1). The air (0.25–8 kHz) and bone (0.5–4 kHz) conduction hearing deterioration with and without age-related correction at the 1- to 9-year follow-up after RT were listed in Supplementary Table 2 and 3, respectively. The mean air and bone conduction hearing deterioration at 0.5–4 kHz with and without age-related correction were measured (Supplementary Table 4).


Background. Epidemiological and randomized clinical trials indicate that marine polyunsaturated n-3 fatty acids (n-3 PUFAs) may have cardioprotective effects. Aim. Evaluate the associations between serum fatty acid profile, traditional risk factors, the presence of cardiovascular diseases (CVD), and peak Troponin T (TnT) levels in elderly patients with an acute myocardial infarction (AMI). Materials and Methods. Patients (n = 299) consecutively included in the ongoing Omega-3 fatty acids in elderly patients with myocardial infarction (OMEMI) trial were investigated. Peak TnT was registered during the hospital stay. Serum fatty acid analysis was performed 2–8 weeks later. Results. No significant correlations between peak TnT levels and any of the n-3 PUFAs were observed. However, patients with a history of atrial fibrillation had significantly lower docosahexaenoic acid levels than patients without. Significantly lower peak TnT levels were observed in patients with a history of hyperlipidemia, angina, MI, atrial fibrillation, intermittent claudication, and previous revascularization . Conclusions. In an elderly population with AMI, no association between individual serum fatty acids and estimated myocardial infarct size could be demonstrated. However, a history of hyperlipidemia and the presence of CVD were associated with lower peak TnT levels, possibly because of treatment with cardioprotective medications. Coronary heart disease (CHD) including myocardial infarction (MI) is one of the leading causes of mortality in the Western world and the incidence increases with advancing age . The carMozaffarian et al. examined a large cohort of elderly subjects without prevalent CHD and found that higher levels of very long chain marine n-3 PUFAs in plasma were associated with a lower total mortality and especially fewer cardiac deaths . There aStudies on mechanisms have revealed n-3 PUFAs triglyceride lowering and anti-inflammatory and antiarrhythmic and platelet inhibiting and blood pressure lowering effects, including improved endothelial function and plaque stability . The TroThe aim of our study was to evaluate the serum fatty acid profile, traditional risk factors, and relevant cardiovascular disease states as related to myocardial injury estimated by peak TnT in an elderly population of patients that have experienced an acute MI. Our hypothesis was that the pattern of serum fatty acids as well as traditional risk factors and previous cardiovascular disease states would influence peak TnT levels. To our knowledge this has not been previously examined.The present study is a substudy of the OMEMI trial with a design that has previously been described in detail . In shorNCT01841944).Hypertension (HT) was defined as previously known systolic blood pressure >140 and/or diastolic blood pressure >90 mmHg or treatment for HT. Diabetes was defined as being insulin dependent or noninsulin dependent. Hyperlipidemia was defined as being previously treated for/or diagnosed with dyslipidemia. Atrial fibrillation (AF) was defined as a history of ECG-documented paroxystic, persistent, or permanent AF and smokers were defined as current smokers. The study was carried out in compliance with the Helsinki Declaration and approved by the Regional Ethics Committee. All subjects gave their written informed consent to participate  by standard venipuncture between 08:00 and 11:00 am, before daily intake of medication. Serum was prepared by centrifugation within one hour at 2500 g for 10 min and kept frozen (−80°C) until analyzed. Routine blood samples were determined with conventional methods. Electrochemiluminescence technology for quantitative measurement was used for repeated measures of TnT  with interassay coefficient of variation (CV) of 7%. NT-proBNP was measured in serum using Elecsys proBNP sandwich immunoassay on Elecsys 2010  with CV of 7%.μL was mixed with 5 mL chloroform-methanol 2 : 1 containing 50 μL/mL butylated hydroxytoluene as antioxidants and shaken. After addition of 750 μL 0.9% sodium chloride, the tubes were mixed and centrifuged at 3220 g for 10 minutes. The lower organic phase containing total lipids was collected and a second extraction was performed on the remaining protein disc. The organic phase was then dried under nitrogen for 45 min at 40°C and dissolved in 1 mL chloroform. Separation of phospholipids fatty acid fraction from total lipids was performed by the method of Burdge et al. [Serum phospholipids fatty acid composition was analyzed by gas chromatography at the Lipid Research Laboratory, Aalborg University Hospital, Denmark. Briefly, serum lipids were extracted by the Folch procedure . Serum 5e et al. , with thThe serum content of linoleic acid (LA) , arachidonic acid (AA) , eicosapentaenoic acid (EPA) , and docosahexaenoic acid (DHA) (22:6 n-3) was expressed as a percent of total fatty acids (wt%) and the CVs were 0.4%, 0.6%, 1.1%, and 1.8%, respectively.U test was used for continuous variables. Analyses of correlations were performed with Spearman rho. A two-tailed value of p ≤ 0.05 was considered statistically significant. The statistical analyses were performed with IBM SPSS Statistics, version 21.0.0.2 .As most data had a skewed distribution, the results are presented as median values (25 and 75 percentiles) unless otherwise stated. Nonparametric statistics were used throughout. For group comparison, Mann-Whitney Flowchart for the inclusion of patients in the present cohort is shown in We focused primarily on the levels of AA and LA n-6 and marine EPA and DHA n-3 fatty acids, as well as the AA + LA/EPA + DHA (n-6/n-3) ratio, as these are the most relevant for CHD. Median values are shown in p > 0.7). There was also no correlation between any of the fatty acids or the n-6/n-3 ratio and NT-proBNP, an indicator of heart failure.We found no significant correlations between peak TnT levels and any of the fatty acids or the n-6/n-3 ratio . We alson = 135) compared to those who did not (n = 161) were observed, resulting in a significant difference in the n-6/n-3 ratio . However, no difference in peak TnT levels between these two groups  was revealed. Furthermore, there were no significant differences in levels of different fatty acids between patients with ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction.Significantly higher serum levels of EPA and DHA in participants who reported previous intake of n-3 PUFA supplements (n = 38), significantly lower DHA levels  and a higher n-6/n-3 ratio  were observed.In patients with known atrial fibrillation (p < 0.001).There were no significant differences in any of the n-6 or n-3 fatty acid levels between genders or in patients with or without a previous MI or smokers and nonsmokers. Lower levels of AA were found in diabetics versus nondiabetics  levels during index hospitalization and CVD risk factors and relevant disease entities are shown in p = 0.018), angina pectoris (p < 0.001), previous MI (p = 0.003), atrial fibrillation (p = 0.007), and claudication (p = 0.015) and also for participants that have previously undergone revascularization procedures (p = 0.003). However, no significant differences were observed between genders or in patients with or without hypertension or diabetes or smokers versus nonsmokers. We found no correlations between infarct size estimated by peak TnT and age or body mass index (BMI). However, a strong positive correlation between infarct size and NT-proBNP  measured at inclusion was found.Significantly lower TnT levels were observed in patients with a history of hyperlipidemia , we found that, for previous angina, 77% were on aspirin, 67% were on statins, and 72% on were beta blockers; for previous MI, 76% were on aspirin, 79% were on statins, and 79% were on beta blockers; and, for previous AF, 59% were treated with oral anticoagulants.The main finding in the present study was that no significant association could be demonstrated between estimated infarct size by peak TnT and the relative content of long chained marine n-3 PUFAs in serum phospholipids in elderly subjects with an acute MI. There was, however, a significant inverse correlation between serum levels of DHA and the history of atrial fibrillation, and patients with a history of hyperlipidemia and relevant CVD entities presented with significantly lower peak TnT levels. Finally, a strong correlation was observed between peak TnT during the index infarction and NT-proBNP at inclusion.Long chain marine n-3 PUFAs have several different effects that together are considered cardioprotective , 24. TheAn earlier study revealed that EPA significantly reduced infarct size in rabbit hearts, mainly dependent on its ability to increase nitric oxide (NO) production and opening of calcium-activated potassium channels . It has Interestingly, significantly lower levels of DHA in patients with a history of atrial fibrillation were found. In the landmark GISSI-Prevenzione-trial, it was shown that n-3 fatty acid supplementation led to a reduction in sudden cardiac death, pointing to n-3 PUFAs antiarrhythmic potential . Our finWe also found significantly lower TnT levels in those with a history of hyperlipidemia, especially for angina and other relevant CVD states. Kloner et al. did show that patients experiencing an AMI with a history of angina were more likely to have smaller creatine kinase-determined infarcts compared to those without . The autFinally, the rather strong correlation between peak TnT during the index infarction and NT-proBNP at inclusion 2–8 weeks later is to be expected as TnT as a marker of infarct size should be expected to associate with NT-proBNP as a predictor of myocardial injury and long-term prognosis .The strengths of the study are attributed to the rather large population and the serum phospholipids analysis of n-3 fatty acids which is considered an important addition to dietary questionnaires. It could be argued that our measure of serum phospholipids, 2–8 weeks after index hospitalization, would not provide a correct reflection of the situation during the AMI. Although we did find significantly higher levels in patients previously reporting intake of n-3 PUFA supplements versus those who did not, confirming our belief that elderly patients would not largely change their dietary habits in a relatively short time, therefore our results should be considered representative. Our population also has a previously high intake of marine n-3 fatty acids. Also, we determined the relative fatty acid composition of phospholipids in serum and not absolute concentrations of individual fatty acids. Lastly, although peak TnT has shown strong correlation with infarct size assessed by radionuclide imaging, it is not considered the gold standard for this estimation. Also, peak TnT levels may not be the absolute peak as the blood samples have been taken according to clinical guidelines during the MI, and not through a standardized method in the protocol.In an elderly population with AMI, no significant association between serum fatty acids and estimated infarct size could be demonstrated. However, an inverse correlation was noted between serum levels of DHA and a history of atrial fibrillation. Hyperlipidemia and the presence of CVD were associated with lower peak TnT levels, probably because of treatment with cardioprotective medication at the time of the AMI.


Genome-wide association studies (GWAS) have identified two CHRNA3 polymorphisms (rs578776 and rs938682) associated with lung cancer risk. Furthermore, these polymorphisms were investigated and genotyped by PCR analysis. All eligible case-control studies published up to Mar 1st 2015 were identified by searching Pubmed and Embase database. Negative association between rs578776-T allele and risk of lung cancer was obtained without obvious heterogeneity . Rs938682-C allele carriers had a 12% to 28% decreased risk. Genotype model analysis showed results of dominant model for rs578776 (OR with 95% CI: 0.839(0.718–0.981)), dominant model for rs938682 (OR with 95% CI: 0.778(0.663–0.912)) and homozygous model for rs938682 (OR with 95% CI: 0.767(0.708–0.831)) were statistically significant. Subgroup analysis indicated rs578776-T variant had protective effect in Smokers, Caucasians, two histology subgroups, and two match subgroups. Meanwhile, rs938682-C allele was associated with decreased risk in Smokers, Caucasians, Lung cancer, and two match subgroups. Meta-regression suggested ethnicity might be the major source of heterogeneity in allele model and homozygous model for rs938682. Moreover, smoking status might contribute to part of heterogeneity under allele model. In summary, this meta-analysis suggested both rs578776 and rs938682 were significantly associated with the susceptibility of lung cancer. Approximately, there are estimated 1.3 billion smokers all over the worldIn 2008, three Genome-wide association studies (GWAS) revealed that CHRNA5-A3 cluster situated on chromosome 15q24-25.1 might be potential loci relevant to both nicotine dependence and smoking related cancer67910However, the association between both CHRNA3 single nucleotide polymorphisms  and susceptibility of lung cancer remains inconsistent. To improve statistical power, we conducted the meta-analysis based on case-control studies to assess the effect of two SNPs on the susceptibility of lung cancer.st 2015 using combinations of following terms: (CHRNA3 OR CHRNA5-3 cluster OR CHRNA3-CHRNA5-CHRNB4 cluster OR rs578776 OR rs938682 OR 15q25) AND (lung carcinoma OR lung cancer OR lung neoplasm) AND . The title and abstract of each potential paper was reviewed by two co-authors independently and any irrelevant one was excluded. Process map for the searching details was presented in PUBMED and EMBASE database were searched by two co-authors separately before March 1Articles which met the following criteria were included in this meta-analysis: 1) case-control studies focusing on the association between rs578776, rs938682 and susceptibility of lung cancer; 2) providing the odds risk (OR) and its 95% confidence interval (95% CI) of allele or genotype, or sufficient information to calculate them; 3) Human study and Full-length articles in English. Reasons for exclusion were: 1) no sufficient data; 2) same cohort or duplicated case/control groups; 3) twin or family based studies.Data was extracted by two co-authors separately and any difference was dissolved by discussion participated in by more than two authors. Information extracted were: last name of the first author; published year; histology; study design; case and control match; country and ethnicity; smoking status; sample size, gender and age information; frequency, OR and 95% CI of allele and genotype. Data was not presented in primary publication was marked ‘not applicable (NA)’.et al.We assessed the quality of included studies by using a quality score called ‘Extended-Quality Score’ to limit the bias in the meta-analysis. As the primary designers classify the studies into ‘high, median, or poor’ quality, the quality of our included studies was assessed all above 5 points score. The details of this quality assessment version were previous described by Li, 2 ranging from 0 to 100%)19Odds risk (OR) with associated 95% confidence interval (CI) was used to assess the association strength. The pooled OR were calculated for estimating the outcome of five genetic models: Allele model, heterozygous model, homozygous mode, dominant model and recessive model. Z test was applied to test the significance of pooled OR and value of p less than 0.05 was considered as significant. Between-study heterogeneity was calculated through Cochran’s chi-square-based Q statistic test and assessed by value of inconsistency index  compared with rs578776-C allele. Meanwhile, there were no evidence of between-study heterogeneity (I2 = 0.0%). However, no significant association were detected in the other three models  without obvious between-study heterogeneity .Of all studies, the genotype information was available in Li, Tekpli and Sakoda’s articles . The inc2 = 47.9%, p = 0.088) under random effect model . Moreover, there were evidence of moderate between-study heterogeneity (Ict model .2 = 0.0%) and heterozygous model CT versus TT (0.767(0.708–0.831), p < 0.01, I2 = 0.0%) without statistical heterogeneity. However, the pooled OR was of edge significance with moderate heterogeneity (OR with 95% CI: 0.724(0.520–1.007), p = 0.055, I2 = 60.8%) under homozygous model CC versus TT. However, there were no evidence of the association polymorphism rs928682 with the susceptibility of lung cancer (OR with 95% CI: 0.989(0.756–1.296), p = 0.938, I2 = 0.0%) under recessive model TT + CT versus CC.Four case-control studies included  yielded a total of 5664 lung cancer cases and 6438 controls for genotype analysis. However, Broderick reported pooled OR with 95% CI under homozygous and heterozygous model, and only pooled OR under dominant and recessive model. Thus both heterozygous and homozygous model analysis included Broderick’s research but neither dominant nor recessive model could. Significant decreased risks were observed under both dominant model CC + CT versus TT (pooled OR with 95% CI: 0.778(0.663–0.912), p = 0.002, I2: 36.7; p = 0.176). However, I2 under homozygous model fell to 0.0% accompanied with p value of pooled OR 0.311. It was worth noting that the sample size of Broderick was much larger than the sum of the other studies . That needs to add further relevant studies to verify homozygous model.Thus for the analysis of heterogeneity of pooled OR of allele model C versus T and homozygous model CC versus TT, we conducted the Galbraith plot  to test To seek for the potential sources of heterogeneity and evaluate the impact of polymorphisms on specific subgroups, we performed subgroup analysis on smoking status, ethnicity, match and histology . Rs57877Publication bias analysis was shown in 2 reflected the existence of moderate heterogeneity (I2 = 49.6%) and p value was 0.094. Forest plot and funnel plot were shown in Additionally when screening the published GWAS researches including CHRNA3 gene, we found another independent locus rs8040868 (C > T) might be related to risk of lung cancer. The searching strategy was the same to strategy reported in method part (processing map not shown). Ultimately 1983 cases and 2235 controls for testing rs8040848 were included in 5 articles22293132To our knowledge, this is the first meta-analysis evaluating the association between rs938682 and lung cancer susceptibility. As a whole, we found these SNPs  could reduce the risk of lung cancer (pooled OR with 95% CI: 0.83(0.79–0.86) for 578776; 0.80(0.72–0.88) for rs938682). Although potential sources of heterogeneity between researches can’t be easily evaluated or eliminated, we could assume ethnicity and smoking status might be sources through the subgroup analysis especially for rs938682. For Chinese, the association between rs938682 and susceptibility may not be significant . However, the remarkable significance can be detected in the results of GWAS from Caucasians . Through meta-regression, we evaluated the contribution of ethnicity to heterogeneity. Meta-regression indicated that ethnicity contributed to 100% heterogeneity of rs938682 allele model and 59.67% heterogeneity of rs938682 homozygous model. Meanwhile we observe that difference in the association of SNP rs938682 with lung cancer risk may exist in not only ethnicity but also smoking status subgroups. We calculated the total HR and 95% CI in smokers and nonsmokers, and found significance in smokers  and no significance in nonsmokers . However, the nonsmokers were all Asians. To analyze the effect of smoking status and ethnicity on heterogeneity separately, we further did the meta-regression. For overall studies, ethnicity contributed to 100% heterogeneity. For all smoking status subgroups, smoking status contributed to part of heterogeneity (45.41%). That indicated that both ethnicity and smoking status influence the association rs938682-C allele with susceptibility of lung cancer. Additionally when performing the subgroup analysis of histology, we found that heterogeneity within Adenocarcinoma group as a possible result of ethnicity .in Vivo and Vitro.After identifying the impact of chromosome 15q25.1 and CHRNA3 gene on the susceptibility of lung cancer by three GWAS in 200867et al.CHRNA3 encoding α3 nAChR submit was reported related to cell apoptosis and depleting or restoring its expression could induce or resist cell apoptosisFor the reason that nAChRs play a vital role in nervous system and nicotine conduction pathway, recent researches revealed that polymorphisms rs578776 and rs938682 were relevant to neuropsychiatric disease, nicotine addiction and lung cancer363739et al. emphasized that CHRNA3 might impact indirectly on lung cancer rather than direct effect mainly due to two arguments listed below: variants carriers in CHRNA3 SNPs increase CPD (cigarettes per day); for never smokers the association between SNPs and lung cancer susceptibility may not be significantet al.44et al.However, through the research in rs12914385 and rs8042374 neither rs938682 nor rs578776, Wang, Although not the emphasis in this meta-analysis, rs8040868 allele T mutation was associated with an increased risk of lung cancer . The mutation in rs8040868 is a synonymous mutation (do not change the encoding amino acids and the reading frame)In summary, these two SNPs (rs578776 and rs938682) may reduce the risk of lung cancer due to the difference in central nervous system function and nicotine dependence; nicotine metabolism and exposure; effect on other genes expression and function; and direct effect on lung cancer.However, there are some limitations in our meta-analysis. Firstly, heterogeneity may be the most important problem for the distinct baseline information of cases and controls; study design and implement; lab technique; statistic processing; paper writing and so on. We spared no effort to minimize the heterogeneity by selecting the included paper critically by inclusion and exclusion criteria; extracting and analyzing data strictly by two different authors. Secondly, concrete data of three SNPs can’t be obtained from all GWAS and replication studies. Details needed to perform subgroup analysis can’t be obtained from all articles. We took full advantage of tables, figures and In summary, rs578776 and rs938682 are related to susceptibility of lung cancer, and variants in these two SNPs play a protective role. Rs938682-C allele may be significant in Chinese nonsmokers. And rs578776-T allele and rs938682-C allele may be effective for smokers. This research may contribute to screening high risk groups in healthy people and have vital significance for Public Health. Moreover, this may predict further onset of lung cancer in family with cancer history. More researches may be needed to confirm the association between these SNPs with susceptibility of lung cancer and answer new questions whether these SNPs were related to prognosis and response to chemotherapy.How to cite this article: Qu, X. et al. Association between two CHRNA3 variants and susceptibility of lung cancer: a meta-analysis. Sci. Rep.6, 20149; doi: 10.1038/srep20149 (2016).


Sus scrofa, is an extant wild ancestor of the domestic pig as an agro-economically important mammal. Wild boar has a worldwide distribution with its geographic origin in Southeast Asia, but genetic diversity and genetic structure of wild boar in East Asia are poorly understood. To characterize the pattern and amount of genetic variation and population structure of wild boar in East Asia, we genotyped and analyzed microsatellite loci for a total of 238 wild boar specimens from ten locations across six countries in East and Southeast Asia.Wild boar, Our data indicated that wild boar populations in East Asia are genetically diverse and structured, showing a significant correlation of genetic distance with geographic distance and implying a low level of gene flow at a regional scale. Bayesian-based clustering analysis was indicative of seven inferred genetic clusters in which wild boars in East Asia are geographically structured. The level of genetic diversity was relatively high in wild boars from Southeast Asia, compared with those from Northeast Asia. This gradient pattern of genetic diversity is consistent with an assumed ancestral population of wild boar in Southeast Asia. Genetic evidences from a relationship tree and structure analysis suggest that wild boar in Jeju Island, South Korea have a distinct genetic background from those in mainland Korea.Our results reveal a diverse pattern of genetic diversity and the existence of genetic differentiation among wild boar populations inhabiting East Asia. This study highlights the potential contribution of genetic variation of wild boar to the high genetic diversity of local domestic pigs during domestication in East Asia. Sus scrofa, is one of the most widely distributed mammalian species, native throughout Europe, North Africa, and much of Asia as far south as Indonesia. Wild boar populations have also been artificially introduced in some areas of the world including the Americas and Australasia, principally for hunting, or through escapes from captivity. Sus scrofa is the most common wild ancestor of the domestic pig, with which it freely hybridizes[Wild boar, bridizes. The Fambridizes.th and 19th centuries after independent domestication[Since wild boar is a co-existing wild ancestor of domesticated pig, the patterns and origins of pig domestication worldwide are of increasing interest, not only in economic contexts, but also academically. Previous phylogenetic studies based on the mtDNA D-loop sequence revealed that continental wild boars and domestic pigs are clearly divided into eastern and western clades-5. Thesetication. A recentication.et al.[It has been well known that the cosmopolitan wild boar originated and spread from Islands of Southeast Asia. Knowledet al. ascertaiIn this study, we aimed to characterize genetic relationships and genetic structure of wild boars from East Asia by examining genetic variation at microsatellite loci for a total of 238 wild boar individuals from six countries. Our results shed light on the genetic relationships among populations and help define population boundaries of wild boar in East Asia.FIS, ranged from 0.017 to 0.279 with a mean of 0.091. Most of the populations except for two sample locations, Japan and Vietnam, showed non-significant FIS values, implying no signature of significant inbreeding , Yunnan, China , Vietnam , and Indonesia . The highest number of alleles  was found in wild boars from Indonesia, of which 33 were private alleles. Inbreeding coefficients, HE and ≥ 7.3 in allelic richness), followed by the Russian Far East (Primorsky Krai) and mainland Korea (except Jeolla-do). The lowest level of genetic diversity was found in the samples from Jeolla-do, Korea , and Jeju Island  , are presented in Table FST values ranged from 0.020  to 0.314 . Gene flow estimates (Nm) derived from FST ranged from 0.546 to 12.250. All wild boar population pairs were significantly differentiated from one another except population pairs from the north-central region of Korea. The wild boar population on Jeju Island showed the highest degree of genetic differentiation from other populations (mean FST = 0.253). The lowest mean FST value was found in southeastern Korea (Gyeongsang-do) vs. other populations (mean FST = 0.123).Between population genetic differences, as indicated by pairwise A genetic distance showed wild boars from Vietnam and Indonesia grouped together, forming a basal cluster to all other populations  they represent. Model-based clustering analysis revealed that wild boars in Eastern Asia had the highest ΔK when K was set to 3, assuming three inferred populations: 1. mainland Korea ; 2. Jeju Island (KJIW); and 3. Southeast Asia   and southern region (KGSW and KJLW), although they displayed a genetically admixed pattern  and 11.4% among populations within region (FSR = 0.114), to explain the proportion of genetic variance among populations to the total (FST = 0.198)  Table .2 = 0.078; P = 0.140). However, when the Jeju Island population was excluded, a significant regression was detected   exhibited the lowest genetic diversity among all populations sampled from East Asia. Negligible gene flow from the Korean mainland  were reintroduced to the mainland a decade ago[Archaeological evidence suggests that wild boars appeared on the Korean peninsula in the mid-Pleistocene, ca. 780,000 to 130,000 years before present. Howevercade ago. Archaeocade ago,24. ModeK = 7) showed that wild boars from mainland Korea are represented by two genetic clusters  support a gradual cline in genetic structure in mainland Korea , Japan (JPNW), China , Vietnam (VIEW), Indonesia (INDW) and South Korea. This experimental work was conducted with permission by the Conservation Genome Resource Bank for Korean Wildlife (CGRB) that provided wild boar samples for this study. All samples were legally collected and deposited into CGRB. The procedures involving animal samples followed the guidelines by Seoul National University Institutional Animal Care and Use Committee (SNUIACUC). Wild boars in South Korea were divided into five regional groups according to the province of collection and other geographic considerations: Gyeonggi-do (KGGW), Gangwon-do (KGWW), Gyeongsang-do (KGSW), Jeolla-do (KJLW) and Jeju Island (KJIW)  according the manufacturer’s instructions. The 16 microsatellite loci were amplified using the Multiplex PCR Kit (QIAGEN). Touchdown PCR was carried out under the following conditions: initial denaturation for 15 min at 95°C, followed by seven touchdown cycles starting at 94°C for 30s, 67°C for 90s, and 72°C for 60s, with annealing temperature decreasing by 2°C per cycle to 53°C. The touchdown cycles were followed by an additional 25 cycles at 94°C for 30s, 53°C for 90s, 72°C for 60s, and a final extension at 60°C for 30 min. Individuals were genotyped using a DNA Sequencer .Ad) per locus, observed heterozygosity (HO), and expected heterozygosity (HE) under Hardy-Weinberg assumptions, were estimated using the Microsatellite Tool Kit[Ar)[FIS, and the level of genetic differentiation between each pair of populations, pairwise FST estimates, and their significance values were calculated using a permutation approach with FSTAT v. 2.9.3[Nm, effective number of migrants per generation) were calculated from FST using the equation of Wright[FST/(1-FST), and geographic distance among locations using Mantel’s test in GenAlEx v.6.0, and significance was determined based on 999 permutations. The DISPAN computer program[A genetic distance by the neighbor-joining (NJ) method[Measures of genetic diversity, including mean number of alleles (Tool Kit. AllelicKit[Ar) is a funKit[Ar). Inbreedv. 2.9.3. Signifiv. 2.9.3. Indirecf Wright. The soff Wright was usedf Wright by testi program was used) method,38.K) was estimated according to the method of Evanno et al.[K is based on the rate of change in the log probability of data between successive K values. Ten runs were carried out for each K, from 1 to 12, to quantify the amount of variation of the likelihood value. Initially, we obtained the highest ΔK value when K was set to 3 (see Results). Three main clusters, therefore, were further analyzed according to the hierarchical island model to probe for possible hidden substructure for each predefined cluster[To assess population structure, STRUCTURE 2.3.3 software was usedo et al., where a cluster.The authors declare that they have no competing interests.SKC carried out the molecular genetic studies, participated in the experiments, data analyses, and drafted the manuscript. KSK and HL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. JEL participated in the experiment. YJK, MSM, IV, AM, JGO, THK, IS, NI, LY, YPZ and NM provided genetic materials and helped to draft the manuscript. All authors read and approved the final manuscript.K), based on 10 iterations per K, generated by the STRUCTURE program [K analysis of LnP(D), according to Evanno et al.[Figure S2: Regression of genetic distance on geographic distance between pairs of East Asian wild boar populations. A. Analysis using all populations included (P = 0.140); B. Analysis after excluding wild boars from Jeju Island (P = 0.002). Mantel’s test for correlations was carried out with 999 permutations. Table S1: Genetic characteristics of 16 microsatellite DNA loci for ten sampling locations in East Asia. See Table Plot of mean posterior probability (LnP(D)) values per clusters ( program , and delno et al.. Figure Click here for file


Environmental tobacco smoke exposure (ETS) was assessed by cotinine level in saliva using a stable isotope dilution LC-ESI-MS/MS method. The mean PAH metabolite concentrations were in the range of 0.15 µg/g creatinine for 9-OH-PHE to 5.9 µg/g creatinine for PHE-9,10-diol. It was shown that none of the individual PAH exposure markers demonstrate a statistically significant influence on birth outcomes. Interestingly a statistically significant association was found between the sum of OH-PHE along with cotinine level and the cephalization index after adjusting for potential confounders (P = 0.04). This study provides evidence that combined exposure of pregnant women to common environmental pollutants such as PAH and ETS might adversely affect fetal development. Thus, reduction of human exposure to these mixtures of hazardous compounds would in particular result in substantial health benefits for newborns.The aim of this study was to assess the impact of PAH exposure on various anthropometric measures of birth outcomes. The study population consisted of 210 nonsmoking pregnant women. Urine samples collected between 20th and 24th week of pregnancy were used for analysis of the following PAH metabolites: 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene , 1-hydroxypyrene (1-OH-PYR), 1,6 + 1,8-dihydroxypyrene (DI-OH-PYR), phenanthrene  Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants that are generated in the course of incomplete combustion and pyrolysis of organic materials such as fuel, coal, wood, garbage, and tobacco. The general population is unavoidably exposed to PAH through polluted ambient air from a variety of sources such as motor vehicles and trucks, coal-fired power plants, residential heating, and environmental tobacco smoke, as well as through contaminated food including charcoal-grilled and smoked meat –4.a]pyrene has been classified by the International Agency for Research on Cancer (IARC) as human carcinogen, whereas other PAH are categorized as probably or possibly carcinogenic to humans  and cephalization index (CI) [head circumference (cm)/birth weight (g) × 104] were calculated.The following anthropometric measures at birth, filled in by gynecologists/neonatologists, were considered in the analysis: birth weight (in grams), birth length, and head and chest circumference (in centimeters). In addition, ponderal index (PI) [birth weight (g)/birth length , medical history, previous and current pregnancies, and lifestyle variables . The questionnaires were administered 3 times during pregnancy  during personal interviews by trained midwives. Factors that could confound the relationship between PAH exposure and anthropometric parameters at birth were selected a priori from a set of characteristics and were tested in a multivariable regression analysis . The folContinuous data were expressed as means ± standard deviations (±SD), as well as the 50th and 95th (for PAH metabolites) percentiles and ranges. Categorical data were presented as numbers and frequencies (%). The creatinine-corrected urinary concentrations of PAH metabolites were analyzed. PAH metabolites and cotinine levels were log-transformed. The 2 following sets of confounders were considered: Model 1: gestational age at birth and child gender; Model 2: all potential confounders significant at a 0.1 level in a multivariable regression .β) with their standard errors and P values. We considered P values less than 0.05 (P ≤ 0.05) as statistically significant. The statistical analyses were performed using R 3.0.1 statistical package.Robust linear regression was performed for each PAH metabolite and for the sum of selected metabolites. In the multiple regression analysis, data were expressed as regression coefficients . About 70% of the women indicated a big city as their place of residence. About 6% of the women consumed alcohol at least once per month in the pregnancy period. The mean cotinine level in saliva collected in the second trimester of pregnancy was 1.3 ng/mL (±1.3 ng/mL).The mean maternal age was 28.6 (±3.9) years . Most ofAbout 52% of the children were girls . On averP ≤ 0.02). Boys were bigger and weighed more than girls (P ≤ 0.05). Child birth length and head and chest circumference increased along with the increasing maternal height (P ≤ 0.03). In addition, an inverse correlation was observed for cotinine level in saliva and child birth head and chest circumference (P ≤ 0.06). Gestational age was associated with child birth weight and length (P ≤ 0.01).The mothers who had higher prepregnancy BMI and who were taller had infants with higher birth weight (μg/g creatinine for 9-OH-PHE (±0.2 μg/g creatinine) to 5.9 μg/g creatinine for PHE-9,10-diol (±10.6 μg/g creatinine).Characteristics of exposure variables are presented in In the model adjusted for gestational age and newborns' gender, none of the analyzed PAH metabolites was alone associated with any of the anthropometric measures at birth . In analP for interaction = 0.06).The same pattern was observed for the sum of PHE-diol, but the association was of borderline significance (P = 0.04). This association is of potential concern since poor pregnancy outcomes may be linked to later neurodevelopmental and other health problems.In this study we have not found any statistically significant effects of individual urinary biomarker of PAH exposure on birth outcomes. However, a significant interaction effect of the sum of OH-PHE and the cotinine level in saliva as a measure of PAH and ETS exposure, respectively, on the cephalization index was noticed  the association between 1-OH-PYR and biometric parameters at birth was not statistically significant after controlling for the cotinine level in saliva as the biomarker of smoking status . Taking μg/g creatinine and third trimester GM = 0.4 ± 2.4 μg/g creatinine; P = 0.7) [A variety of different PAH metabolites in the urine collected during the second trimester of pregnancy were selected as the biomarkers of exposure. There have been concerns whether the single spot urine tests represent the long-term exposure. The analysis performed by Choi et al. (2008) indicated that measurements during the third trimester were significantly lower compared with the second trimester, whereas those during the first trimester were not . HoweverP = 0.7) .μg/g creatinine) was higher than that noted in the INMA cohort of nonsmoking pregnant women in Valencia (0.1 μg/g creatinine) and postpartum nonsmoking women from Saudi Arabia (0.05 μg/g creatinine) [μg/g creatinine as the reference value for the nonsmoking general population aged between 3 and 69 years [μg/g creatinine). It is important to notice that most of the women included in our analysis lived in a big city (Lodz) which can be the reason for the higher 1-OH-PYR level. This was confirmed by the stationary ambient air monitoring data , which indicated that the annual mean BaP level in various locations within Lodz was substantially higher than the target value of 1 ng/m3 recently set into force by the European Commission [Although the studies evaluating the impact of PAH exposure on pregnancy outcomes exist, it is difficult to compare their results to these obtained in the current study. This results from the fact that researchers select different PAH metabolites for their analysis so direct comparison of the magnitude of exposure is not possible. However, based on the analysis by Choi et al. (2006), personal airborne PAH exposure was 10-fold higher in Krakow than in NYC . In addiatinine) . The Hum69 years , 34. In mmission , 36.P < 0.01) and in NYC African Americans (P < 0.05), but not in NYC Dominicans [The association between urinary PAH metabolite levels or PAH-DNA damage and reduced biometric measures at birth has been reported in some, but not all, studies. For example, the study performed in the Czech Republic indicated that ambient PAH exposure in the early stage of pregnancy significantly increased the risk of intrauterine growth restriction  . The anaminicans . A recenminicans . This kiminicans .P = 0.04) and head circumference (P = 0.01) [The impact of PAH exposure on the cephalization index was reported in an earlier study by Choi et al. (2008) . Increas = 0.01) . In the  = 0.01) .Fetal toxicity caused by PAH and ETS may result from antiestrogenic effects , bindingMajority of the studies published in this field have focused on the known possible confounding variables such as child gender, gestational age, marital and educational status of pregnant women, prepregnancy BMI, and season of the last menstrual period, which were also addressed in our analysis. By restricting our study population to healthy women without serious pregnancy complications and to single pregnancies, we were able to eliminate several confounding factors.The current analysis is based on the Polish Mother and Child Cohort Study (REPRO_PL cohort). The prospective study design with well-assessed exposure levels based on biomarker measurements  is an important advantage of the current analysis. Thanks to it we have addressed some of the traditional limitations such as exposure misclassification and retrospective or cross-sectional exposure assessment. Additionally, a series of detailed questionnaires (collected once in each trimester of pregnancy and one week after delivery) allow for reliable assessment of coexposure and confounding variables.The current study results may be limited by a modest sample size; however it has sufficient power to perform the presented analysis. As it was mentioned above, the biomarkers were measured only at a single time point which may constitute an additional limitation of our study. Finally, although creatinine correction is commonly used for urinary biomarkers, in some studies the specific gravity was used instead of creatinine in order to adjust PAH metabolites for urine dilution. However, specific gravity is highly correlated with creatinine . TherefoThis study provides evidence that combined exposure of pregnant women to common environmental pollutants such as PAH and ETS might adversely affect fetal development. Thus, reduction of human exposure to these mixtures of hazardous compounds would in particular result in substantial health benefits for newborns and improve prevention of restrictions in their subsequent development.


Our findings outline the characteristics of IAH-induced barrier changes, indicating that intestinal barriers might be treated to alleviate IAH, and the microflora may be an especially relevant target.Intra-abdominal hypertension (IAH) is a common and serious complication in critically ill patients for which there is no well-defined treatment strategy. Here, we explored the effect of IAH on multiple intestinal barriers and discussed whether the alteration in microflora provides clues to guide the rational therapeutic treatment of intestinal barriers during IAH. Using a rat model, we analysed the expression of tight junction proteins (TJs), mucins, chemotactic factors, and Toll-like receptor 4 (TLR4) by immunohistochemistry. We also analysed the microflora populations using 16S rRNA sequencing. We found that, in addition to enhanced permeability, acute IAH (20 mmHg for 90 min) resulted in significant disturbances to mucosal barriers. Dysbiosis of the intestinal microbiota was also induced, as represented by decreased Firmicutes (relative abundance), increased Proteobacteria and migration of Bacteroidetes from the colon to the jejunum. At the genus level, Lactobacillus species and Peptostreptococcaceae  Intra-abdominal hypertension , is a serious complication in critically ill patients, for which there are no well-defined treatment strategies1et al. revealed the associations between mucosal defence mechanisms and the development of gut-derived sepsis4589101112The intestinal mucosa serves as the first line of defence in protecting the host from enteric toxins and pathogenic microorganisms. In the 1990 s, Deitch Among the multiple intestinal barriers, a few studies have specifically investigated alterations to the mechanical barrier function of the intestine during IAH111416171819P < 0.01) in the concentration of FD-4 in the plasma of portal blood (178.2 ± 26.0 μg/mL).Intestinal permeability to macromolecules was evaluated by measuring FITC-dextran  leakage from the gut cavity into the portal circulation. As shown in 2) for 90 min affected the staining intensity, as well as the percentage of cells staining for TJ proteins, mucins, chemotactic factors, and TLR4 . Rarefaction curves and estimators are shown in The PCA score plots showed significant differences in the microfloral structure of the intestinal content between rats with IAH and controls . In the vs. Control, jejunal content: 83.4% vs. 95.6%; colonic content: 53.3% vs. 58.1%) and an increase in the RA of Proteobacteria  in both the jejunum and colon  and an increase in the RA of Bacteroidetes species in the jejunum .The patterns seen in microbial composition were quite dissimilar in the IAH experimental group and in controls. At the phylum level, the microflora of the jejunum and colon in the controls were dominated by species of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. IAH did not alter the dominant phyla inhabiting either the colon or jejunum. However, an alteration in the relative abundance (RA) of these three phyla was observed in rats with acute IAH. IAH resulted in a decrease in the RA of Firmicutes species , two belonged to the phylum Bacteroidetes (S24-7 no rank and Bacteroides), and two belonged to the phylum Proteobacteria (Helicobacter and Pseudomonas). Amongst the Firmicutes species, IAH resulted in a downregulation of lactobacilli  without any apparent impact on lactococci (incertae sedis load in the jejunum was reduced by IAH (IAH vs. Control: 0.4 ± 0.6% vs. 3.3 ± 5.2%). An increase in the abundance of Ruminococcaceae incertae sedis and Ruminococcus species was observed in rats with acute IAH, although the differences were not significant. The RA of Helicobacter, Pseudomonas, Bacteroides, and S24-7_no rank species increased.The influence of IAH on the RA of the intestinal microflora at the genus level is shown in ctococci . In addiThe damage to the barrier functions of the gastrointestinal (GI) tract, triggered by IAH associated ischaemia and subsequent oxidative injury, is considered a likely pathophysiological basis for the IAH-associated multi-organ failure (MOF) cascade15162324incertae sedis significantly decreased.In this study, we found that, accompanying enhanced permeability, acute IAH (20 mmHg for 90 min) resulted in significant disturbances to multiple intestinal barriers, together with a marked decrease in the immunoreactivity of TJ proteins and a significant increase in the immunoreactivity of mucin 1, mucin 2, CXCL 1, MIP 1beta, MCP 1, and TLR4 . Dysbioset al. reported that a reduction in intestinal microcirculatory blood flow (MBF) was associated with disruption of the ultrastructure of TJs, resulting in marked dilation between adjacent epithelial cells in animals with IAH1129et al. reported that complete ischaemia in the splanchnic artery of rats for 30 min, resulted in breakdown of intestinal mucin accompanied by increased intestinal permeability to FITC-dextran, as well as the degradation of TLR4Among the markers of distinct intestinal mucosal barriers investigated in this study, only the changes of TJs under IAH had been previously investigated, to the best of our knowledge111427incertae sedis, Ruminococcus, Helicobacter, Pseudomonas, Bacteroides and S24-7_no rank species, were all shown to increase in abundance.Not only does the intestinal microbiota act as biological barrier, but it also participates in the formation of other three barriers. In addition, an imbalance in the microbial communities can provide a higher load of bacterial antigens3233via interferon-mediated TLR gene regulation, and by interacting with specific receptors on dendritic cells. The effectors involved may include lipoproteins, glycolipids, bacterial short chain fatty acids, bacterial proteases, and certain cell surface proteins such as S-layer proteins3940414243444546The specific mechanisms by which IAH disturbs the interaction between host immunity and the microbiota needs further investigation. With reference to other diseases, we speculate that the changes in microbial composition detected in the present study might be initiated by oxidative damage, and may be the consequence of subsequent disturbance to the local immune functioning153738In conclusion, we characterised the dysfunctions of intestinal barriers during acute IAH. Upon production of acute IAH, we observed a striking alteration in the microbial composition of the GI tract. The disturbed host-microbiota interactions produced conditions that could, in principle, result in gut-derived sepsis. Our results in this animal model strengthen the clinical case for restoring intestinal barrier function, perhaps using probiotics (like lactobacilli), to treat critically ill patients that develop IAH.To study the effects of acute IAH on intestinal barrier functions, 21 male SPF Sprague-Dawley rats  were randomly assigned to the IAH group , or control group (n = 9). The rats in each group were further randomly assigned to three subgroups, for the detection of intestinal permeability to macromolecules, assessing microflora community diversity, and characterising the immune and chemical barrier functions . All experimental procedures, including the care and handling of animals, were performed following international guidelines. All surgeries were conducted under sodium pentobarbital anaesthesia  and all efforts were made to minimise pain. After the procedure was complete, the animals were euthanized with an overdose of sodium pentobarbital  to minimise pain49The acute IAH animal model was established using the 90 min-nitrogen pneumoperitoneum procedure, which was previously describedThe intestinal permeability to FD-4  was assessed as previously describedAfter 90 min, the nitrogen pneumoperitoneum was decompressed gradually and laparotomy was performed by median abdominal incision. The samples of jejunal and colonic tissues (1 cm × 1 cm) were collected and gently flushed using cold saline. The tissue samples were then fixed in 10% neutral buffered formalin (pH 7.4) for immunohistochemical analyses. For detection of microfloral diversity, 10-cm long jejunal and colonic segments were isolated, and their contents were collected and stored in sterile freezing tubes. The samples were frozen by immersion in liquid nitrogen and stored at −80 °C until needed for experiments.Tissue preparation and immunohistochemical staining for TJ proteins, Toll like receptor 4, chemotactic factors, and mucins was performed as previously describedMicrobial DNA was extracted using the E.Z.N.A. stool DNA Kit  according to the manufacturer’s protocols. The V4-V5 region of the bacterial 16S ribosomal RNA gene were amplified by PCR . The primers used were 338F 5′-barcode- GTGCCAGCMGCCGCGG)-3′ and 806R 5′-CCGTCAATTCMTTTRAGTTT-3′: barcode is an eight-base sequence unique to each sample. All PCR reactions were performed in triplicate with 20 μL final reaction mixture containing 4 μL of 5× Fast Pfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μΜ), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions, and quantified using QuantiFluor-ST . The purified amplicons were pooled in equimolar quantities and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to standard protocols.QIIME (version 1.17) with the following criteria: (i) Reads with 250 bp, truncated at any site, received an average quality score <20 over a 10-bp sliding window. Reads shorter than 50 bp were discarded. (ii) For exact barcode matching, reads with two nucleotide mismatches in primer matching. Reads containing ambiguous characters, were discarded. (iii) Sequences that had overlaps longer than 10 bp were assembled according to their overlap sequence. The reads that could not be assembled were discarded. Sets of sequences with ≥97% identity were defined as an Operational Taxonomic Unit (OTU) using UPARSE (version 7.1 http://drive5.com/uparse/). Any chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was analysed by RDP Classifier (http://rdp.cme.msu.edu/) against the SILVA 16S rRNA database  using a confidence threshold of 70%53Raw ‘fastq’ files were demultiplexed and quality-filtered using QIIME. Unweighted UniFrac distance-metrics analysis was performed using OTUs for each sample55Rarefaction curves, alpha diversity , and beta diversity calculations were performed using SPSS 16.0 software . P-values < 0.05 were considered statistically significant.The Mann-Whitney test and independent t-test were performed using How to cite this article: Leng, Y. et al. Effects of acute intra-abdominal hypertension on multiple intestinal barrier functions in rats. Sci. Rep. 6, 22814; doi: 10.1038/srep22814 (2016).