Eye tracking has been used during face categorisation and identification tasks to identify perceptually salient facial features and infer underlying cognitive processes. However, viewing patterns are influenced by a variety of gaze biases, drawing fixations to the centre of a screen and horizontally to the left side of face images (left-gaze bias). In order to investigate potential interactions between gaze biases uniquely associated with facial expression processing, and those associated with screen location, face stimuli were presented in three possible screen positions to the left, right and centre. Comparisons of fixations between screen locations highlight a significant impact of the screen centre bias, pulling fixations towards the centre of the screen and modifying gaze biases generally observed during facial categorisation tasks. A left horizontal bias for fixations was found to be independent of screen position but interacting with screen centre bias, drawing fixations to the left hemi-face rather than just to the left of the screen. Implications for eye tracking studies utilising centrally presented faces are discussed. Eye-movements provide a way of measuring attention and can highlight perceptually salient facial features for facial identity and expression recognition , 2009. VThe initial centre-of-face bias in gaze behaviour is commonly observed in studies where face stimuli are presented in the centre of the screen , 2014. HA second gaze bias associated with face viewing is the tendency to preferentially view the left hemi-face, from an observers perspective , 2012 orIn order to accurately assess viewing patterns attributed to facial expression categorisation we aim to dissociate generic or methodological gaze biases associated with the use of a screen from face specific biases, by directly comparing viewing patterns between centrally and laterally presented stimuli. Specific biases to be investigated include the central gravitational bias for fixations , 2007, wTo control for a potential gender bias , 2009 onA Tobii T60XL widescreen eye tracker served as eye tracker and monitor displaying at 1,280 × 1,024 pixels at a refresh rate of 60 Hz, stimuli were presented at a size of 900 × 550 pixels subtending a visual angle of 23.110 and 11.674° respectively. Matlab with Psychtoolbox and the Tobii Matlab Toolbox were used for visual stimulus control and to run the eye tracker. The gaze precision of the eye tracker is reported at 0.5 visual degrees with binocular sampling at a distance of 65 cm. Fixations were computed using a dispersion algorithm , 2000. BStimuli were generated using the Karolinska Directed Emotional Faces database , 1998. Tx axes of the screen, for example left presented faces centered on pixel 320  = 0.359, p = 0.701, ηp2 = 0.018), however the main effect of emotion  = 6.754, p = 0.003, ηp2 = 0.252) was significant due to fear expressions being responded to faster  compared to sad expressions . Intensity was also significant  = 9.582, p < 0.001, ηp2 = 0.324) as lower intensities were responded to slower than higher intensities. 10% intensity  was responded to significantly slower than 100% , 20%  was responded to significantly slower than 60%  or 100% (p = 0.041) and 40% intensity  was responded to significantly slower than 50% , 60% (p = 0.037) and 100% (p = 0.006).Reaction times (RT’s) were analysed by entering average RT’s into a 3 × 3 × 7 Repeated Measures ANOVA (Emotion × Screen position × Intensity). Bonferroni corrected pairwise comparisons were used to compare main effects and Greenhouse Geisser adjustment was used where appropriate. Results showed no significant differences in RT’s between each of the possible screen positions  = 0.596, p = 0.556, ηp2 = 0.029); average correct response across all three screen positions was 74 ± 1%. Emotion did have a significant effect on accuracy  = 40.191, p < 0.001, ηp2 = 0.668) which was due to sad expressions being correctly categorised (mean = 89%) more than happy (mean = 71%) or fear . Intensity was also significant  = 27.615, p < 0.001, ηp2 = 0.580), improvements in categorisation performance were seen from 10% intensity (mean 50% correct) to 20% (mean 61%), and 30% (mean 71%) to 40% (mean 79%). At high intensities there were no significant differences of categorisation performance, though the trend to increase performance continued . Finally, emotion and intensity interacted  = 11.515, p < 0.001, ηp2 = 0.365). Compared to sad , for which accuracy did not change significantly from low intensity to high intensity, fear  and happy  had larger improvements from low intensity to high intensity.Accuracy was analysed by entering percentage correct responses for each screen position into a 3 × 3 × 7 Repeated Measures ANOVA (Emotion × Screen position × Intensity). Bonferroni corrected pairwise comparisons were used to compare main effects and Greenhouse Geisser adjustment was used where appropriate. Results showed no significant differences in accuracy between each of the three Screen positions (Face viewing was measured by defining three regions of interest (ROI); the eyes, the nose and the mouth. The eyes ROI included the brows, upper and lower lids and a surrounding area of approximately 2 visual degrees. The nose ROI included the bridge, nasal root and a surrounding area up to 2 visual degrees where this did not impact on other ROI’s. Finally the mouth ROI included the lips, mentolabial sulcus and philtrum and a surrounding area of 2 visual degrees. Each ROI was designed to encompass the face accurately for any expression at all intensities so that gaze biases introduced by the screen or stimulus position would not impact on analyses between expressions. Fixations that were not within the boundaries of the displayed image (900 × 550 pixels) were removed from analysis, however, fixations within the image but not within any ROI  were included in total fixation calculations. Therefore, the total number of fixations for each stimulus were comprised of fixations to the three ROI’s being analysed, as well as fixations to any other area of the face.Due to the free viewing method each stimulus received a variable total number of fixations, therefore these were converted to percentage of total fixations for comparisons between participants and across stimuli, percentage fixations were then averaged across the four models. To analyse the effect of screen position (3), emotion (3) and intensity level (7) on the linear combination of ’Percentage Fixations’ , these percentages were entered in a 3 (Screen position) × 3 (Emotion) × 7 (Intensity) Repeated Measures MANOVA.λ = 0.107, F = 26.575, p < 0.001, ηp2 = 0.677] and univariate analysis showed that the percentage of fixations to eyes  = 5.64, p = 0.007, ηp2 = 0.220), nose  = 100.98, p < 0.001, ηp2 = 0.835) and mouth  = 35.29, p < 0.001, ηp2 = 0.638) all varied significantly dependent on screen position.Multivariate analysis revealed that screen position was significant , which was accounted for by the univariate interaction between Screen position and Intensity for the nose region  = 3.870, p < 0.001, ηp2 = 0.162) and the mouth region  = 2.411, p = 0.006, ηp2 = 0.108), suggest that the effect of screen position for nose and mouth in p’s < 0.001) or right (p’s < 0.001) presented faces and centrally presented faces had fewer fixations to the mouth at all intensities compared to left (p’s < 0.001) or right presentations (p’s < 0.038). However, when left and right screen positions are directly compared, the nose was fixated more in left compared to right presented faces at 100% intensity (p = 0.049) and the mouth was viewed more in left compared to right presented faces at intensity levels 30% (p = 0.001) and 100% intensities (p = 0.020).Bonferroni corrected pairwise comparisons were used to analyse main and interaction effects. The effect of Screen position was compared separately for each ROI see . Percentλ = 0.527, F = 4.775, p < 0.001, ηp2 = 0.274] was accounted for by a significant univariate effect of emotion for percentage fixations to the mouth only  = 15.96, p < 0.001, ηp2 = 0.444). Pairwise comparisons showed this was due to significant differences between all three emotions, with happy receiving the highest percentage of fixations to the mouth (mean = 22.253), fear receiving fewer (mean = 20.627) and sad receiving the lowest percentage . Furthermore the multivariate effect of Intensity  was accounted for by the univariate effect of Intensity on percentage fixation toward the eyes  = 4.539, p < 0.001, ηp2 = 0.185). Pairwise comparisons showed that fixations towards the eyes were higher at intensity 10% (mean = 21.80) compared to 30%  or 60% .A significant multivariate effect of emotion  was found, which was accounted for by a significant interaction between Screen Position and Emotion for the eye-region only  = 5.264, p = 0.001, ηp2 = 0.208). Pairwise comparison found fewer fixations to the eyes of fear expressions that were centrally presented compared to right presentations (p = 0.043). Similarly, sad expressions received fewer fixations to the eyes when centrally presented, compared to right (p = 0.001) presentations. Comparing emotions within each screen position pairwise comparisons found that fear expressions had more fixations to the eyes (mean = 23%) than happy  or sad expressions (mean = 20%) only when faces were presented to the right of the screen, all other comparisons were not significant.A significant multivariate interaction effect between Screen position and Emotion . Fixations to the left hemi-face were significantly lower for fear expressions presented on the left compared to the right or centre (p’s < 0.001). For both happy and sad expressions, fixations to the left hemi-face were lower on faces presented to the left compared to the centre (p’s < 0.001) and higher on faces presented to the right compared to centre . Within each screen position, fixations to the left hemi-face varied significantly only between happy (mean = 43%) and fearful expressions  in left presentations and happy (mean = 70%) and sad  and happy and fearful  expressions in right presentations. Fixations to the right hemi-face when stimuli were presented in the centre of the screen were higher for sad expressions (mean = 23%) compared to happy  or fearful expressions  but when stimuli were presented to the left fearful expressions received more fixations to the right hemi-face (mean = 46%) than happy expressions . Finally, right hemi-face fixations were lower for sad expressions when stimuli were presented on the right of the screen (mean = 11%) compared to happy  or fearful expressions (mean = 13 p = 0.006). Emotion and Intensity  revealed that at 40% intensity, sad expressions had more fixations to the right hemi-face than fear (p = 0.006) and fewer fixations to the left hemi-face compared to happy (p = 0.031); happy expressions had fewer right hemi-face fixations compared to fear at 10% intensity (p = 0.003).Significant interaction effects were further found between Screen position and Emotion [left hemi-face: F = 3.762, p = 0.003, ηp2 = 0.158). Pairwise comparison showed that when presented centrally, all emotions at all intensities had more left hemi-face fixations than when presented on the left . Faces presented on the right of the screen also had more left hemi-face fixations than those presented to the left  except fear at 30% which did not vary significantly between right and left presentations. Right presented faces typically had more left hemi-face fixations than centrally presented faces, this was significant for fear expressions at 40% intensity (p = 0.002), happy expressions at 20% (p = 0.002), 30% (p = 0.014), 50% (p = 0.006) and 60% intensity (p = 0.029) and finally, for sad expressions at 20% (p = 0.003), 30% (p = 0.002) and 50% (p < 0.001) intensity. Next, when faces were presented in the centre of the screen happy expressions received more left hemi-face fixations than fearful expressions at 10% and 30% intensities (p’s < 0.036) and happy expressions also had more left hemi-face fixations than sad faces at 20% and 30% intensities (p’s < 0.048). Sad expressions also received more left hemi-face fixations than fearful expressions at 20% and 40% intensities (p’s < 0.028). When faces were presented to the left of the screen happy expressions had more left hemi-face fixations than sad at 30% and 50% intensities (p’s < 0.049) and fear at 30% (p = 0.018). Sad expressions also had more left face fixations than fear at 20% and 50% (p’s < 0.045) but fewer fixations than fear at 60% intensity (p = 0.049). Lastly, when faces were presented to the right of the screen happy expressions had fewer left hemi-face fixations than sad at 10%, 20%, 50% and 100% (p’s < 0.030) but more left hemi-face fixations than sad at 40% (p = 0.033). Happy expressions also had fewer left face fixations than fear at 20% and 30% (p’s < 0.009) but more left face fixations than fear at 60% (p = 0.032), sad expressions only had more left face fixations than fear at 60% intensity (p = 0.018).Finally, Screen position, Emotion and Intensity was significant for fixations to the left hemi-face only (The present study was designed to differentiate general screen biases in viewing from those associated specifically to faces during categorisation tasks, in particular a tendency for fixations to focus around the centre of the face , 2014 anDisplayed emotions were chosen specifically to contain little or no informative facial characteristics in the nose region, with fear displaying primarily in the eyes and happiness and sadness displaying primarily in the eyes and mouth , 2013. TOur data shows that a screen centre bias, reflected in preferential attending of the hemi-face closest to screen centre, co-occurs with left hemi-face bias. Faces presented to the left of the screen had a similar percentage of fixations to the left hemi-face and right hemi-face, whereas faces presented to the right of the screen received around six times more fixations to the left hemi-face compared to the right hemi-face. Due to the influence of a screen centre gravitational effect , 2007, fIn addition to centre-of-screen and left hemi-face gaze biases, the results of the present study seem to suggest that a small bias towards the left compared to the right side of screen may have influenced gaze patterns, although this effect was small and only observed for the nose and mouth and was restricted to only a few intensity levels. However, this trend is consistent with the horizontal left bias previously reported in free viewing of natural scenes , 2014 anIn summary, a bias to the left hemi-face for fixations was dissociable from a general left horizontal bias , 2015 sp10.7717/peerj.2241/supp-1Supplemental Information 1Click here for additional data file.


HNF1a) gene polymorphism on coronary artery disease (CAD) traits in 4631 Saudi angiographed individuals (2419 CAD versus 2212 controls) using TaqMan assay on ABI Prism 7900HT sequence detection system. Following adjustment for confounders, the rs2259820_CC (1.19 (1.01–1.42); P = 0.041), rs2464196_TT (1.19 (1.00–1.40); P = 0.045), and rs2259816_T (1.13 (1.01–1.26); P = 0.031) were associated with MI. The rs2259820_T (1.14 (1.03–1.26); P = 0.011) and rs2464196_C (1.12 (1.02–1.24); P = 0.024) were associated with type 2 diabetes mellitus (T2DM), while the rs2393791_T (1.14 (1.01–1.28); P = 0.032), rs7310409_G (1.16 (1.03–1.30); P = 0.013), and rs2464196_AG+GG (1.25 (1.05–1.49); P = 0.012) were implicated in hypertension. Hypertriglyceridemia was linked to the rs2393791_T (1.14 (1.02–1.27); P = 0.018), rs7310409_G (1.12 (1.01–1.25); P = 0.031), rs1169310_G (1.15 (1.04–1.28); P = 0.010), and rs1169313_CT+TT (1.24 (1.06–1.45); P = 0.008) and high low density lipoprotein-cholesterol levels were associated with rs2259820_T (1.23 (1.07–1.41); P = 0.004), rs2464196_T (1.22 (1.06–1.39); P = 0.004), and rs2259816_T (1.18 (1.02–1.36); P = 0.023). A 7-mer haplotype CATATAC , constructed from the studied SNPs, was associated with MI, and CATATA implicated in T2DM . Hypertriglyceridemia was linked to TGCGGG , and obesity to ACGGGT . Our results suggest that the HNF1a is a common susceptibility gene for MI, T2DM, hypertension, and dyslipidemia.We examined the role of hepatic nuclear factor-1 alpha ( HNF1a, also known as transcription factor-1) on chromosome 12q24 encodes a transcription factor (TF) that binds to promoters of a variety of genes expressed predominantly in the liver [ HNF1a gene are transcribed to specific HNF1a isoforms, which might serve different yet unidentified functions [ HNF1a gene are known to be the most common cause of maturity onset diabetes of the young (MODY), a severe dominantly inherited form of nonketotic diabetes mellitus that is characterized by pancreatic beta-cell dysfunction [ HNF1a mutations have also been implicated in diabetic nephropathy related to type 1 diabetes [The hepatocyte nuclear factor 1a gene  [ HNF1a mutations [ HNF1A in the onset and development of CAD is yet to be clearly defined. Besides, the fact that HNF1A polymorphism has been linked with susceptibility to both CAD and its important risk traits points to some pleiotropic actions of the gene on disease pathways leading to atherosclerosis. In a preliminary linkage study in a heterozygous familial hypercholesterolemia setting, we established a link for early onset CAD to the locus of the HNF1a gene on chromosome 12q24 and suggested a role for this gene in this disease process. In the present study, therefore, we elected to test this notion further and to evaluate the potential role of the gene variants at this locus in CAD risk traits and its manifestation in a large cohort of angiographed Saudi individuals.Apart from diabetic disorders, single nucleotide polymorphisms (SNPs) in thein (CRP) –35, arguin (CRP) , 36 as win (CRP) , 37, wheutations . Howeverutations –41. Henc HNF1a Supplementary Data 1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2014/291419), in which the primary proband underwent triple bypass surgery at the age of 14. Sequencing the HNF1a gene for variants of interest was accomplished in the family members as well as 200 other individuals from the general population. This was followed by a case-control study in a total of 4631 candidates consisting of 2419 CAD patients  with angiographically determined narrowing of the coronary vessels by at least 50% and 2212 angiographed controls  with no evidence of the CAD disease (2 and is classified as the obesity (OBS) subset. Among these subsets of patients, some individuals harboured a combination of two or possibly three of the cardiovascular risk traits. The overall exclusion criteria for the disease cases were major cardiac rhythm disturbances, incapacitating or life-threatening illness, major psychiatric illness or substance abuse, history of cerebral vascular disease, neurological disorder, and administration of psychotropic medication. The controls (CON) for CAD were a group of individuals undergoing surgery for heart valvular diseases and those who may have reported with chest pain but were established to have no significant coronary stenosis by angiography. Exclusion criteria for this group were among others diseases such as cancer, autoimmune disease, or any other disorders likely to interfere with variables under investigation. This study was performed in accordance with the regulations laid down by the King Faisal Specialist Hospital and Research Centre Ethics Committee in compliance with the Helsinki Declaration (http://www.wma.net/en/30publications/10policies/b3/index.html) and all participants signed an informed consent.The initial linkage study was performed in a family of eleven with heterozygous familial hypercholesterolemia . For the whole genome-wide scanning with the Affymetrix Gene Chip 250 Sty1 mapping array , 250 ng of genomic DNA was digested with the restriction endonuclease StyI, mixed with Sty1 adaptors, and ligated with T4 DNA ligase. The mixture was added to four separate PCR reactions, amplified, pooled, and purified to remove the unincorporated dNTPs. The PCR product was then fragmented, biotinylated, hybridized to the 250 Sty1 array for 18 h, washed, stained, and scanned as recommended by the manufacturer. SNP genotypes, linear chromosomal locations, and marker ordering were accomplished using the Affymetrix GeneChip Genotyping Analysis Software (GTYPE) version 4.0. Multipoint parametric linkage analysis was performed using the GeneHunter Easy Linkage analysis software 4.0  for estimating the LOD scores. Genotype calling rates for the samples ranged from 98.23% to 99.83%, which was high and accurate enough for their subsequent evaluation. The disease was assumed to be an autosomal dominant trait with 100% penetrance. Copy Number Analyzer for GeneChip Ver. 3.0 (CNAG)  was employed to establish the shared chromosomal regions of homozygosity. HFN1A gene in the family members and 200 individuals from the general population using the MegaBACE DNA analysis system . Briefly, the DNA was subjected to PCR by standard methods described elsewhere. Five μL of PCR product was then treated with 2 μL of ExoSAP-IT  at 37°C for 30 min to allow the hydrolytic removal of excess primers and dNTPs by Exonuclease 1 and shrimp alkaline phosphatase. The enzymes were inactivated at 80°C for 15 min, and the sequencing reaction was initiated by mixing 2 μL DNA, 1 μL of 5 μmol primer, 8 μL of DYEnamic ET Dye Terminator , and 9 μL of distilled water. The mixture was thermally cycled 40x at 95°C for 20 sec, 50°C for 15 sec, and 60°C for 1 min. Unincorporated dye-labelled terminators were removed by gel-filtration through the DyeEx 96 plate . The eluent was vacuum-dried and dissolved in 10 μL of loading solution  for sequencing. Data were analyzed for SNPs by Lasergene software .The linkage study was followed by sequencing exons and intron-exon junctions and flanking regions of theμL reaction was prepared by mixing 5 μL containing 50 ng DNA, 12.5 μL of 2x Universal mix , 1.25 μL of 20x probe assay mix, and 6.25 μL DNase-free distilled water. Three no-template controls were included in each plate for normalization of emission signal. The thermal amplification profile for the first cycle occurred at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 94°C for 15 sec and 60°C for 30 sec. The plates were then scanned for FRET signal using the 7900HT sequence detection system and data analyzed using SDS 2.0 software .Once the SNPs of interest were identified, genotyping was achieved by TaqMan chemistry using the Applied Biosystems real-time Prism 7900HT Sequence Detection System . Primers and the TaqMan fluorogenic probes bearing a suitable reporter dye on the 5′-end and a quencher dye on the 3′-end were designed using the Primer Express software V2.0  and procured from Applied Biosystems . One probe  was labeled with VIC dye and the other  with FAM dye at the 5′-end, and serial dilutions were run to determine the optimal working concentration. For each reaction, a 25  U (MWU) test, Bonferroni adjustment, and binary logistic regression analyses were employed to compute odds ratios and their 95% confidence intervals as well as estimate the confounding effects of the different cardiovascular risk traits on the respective relationships. For the haplotype-based association analysis, we used the haplo.stats package (http://mayoresearch.mayo.edu/mayo/research/schaid_lab/software.cfm) in the R Statistical Computing Software (http://www.r-project.org/). Odd ratios for haplotypes were calculated using the baseline haplotype CATATAC (frequency = 0.505) as reference, and the haplotype score statistic for the association of a haplotype with the binary trait was calculated as in Schaid et al. (2002) [ P value <0.05 were considered statistically significant.Mann-Whitney. (2002)  and Lake. (2002) . Signifi HNF1a gene on chromosome 12q24 . We then subjected the data to binary logistical regression analysis for the respective conditions, whereby the coexisting disease traits and other covariates were treated as confounders, and Bonferroni adjustment for age and sex was performed on the data (HNF1a Supplementary Data 3). To begin with, the initial univariate analysis suggested that six of these variants rs7310409 A>G (P = 0.041), rs2259820_C>T (P = 0.011), rs2464196_C>T (P = 0.016), rs2259816_G>T (0.007), rs1169310_A>G (P = 0.016), and rs1169313_C>T (P = 0.023) conferred risk for MI (3044 cases versus 1587 controls). However, following adjustment for possible confounding effects of all other risk covariates, only the rs2259820_CC  = 1.19 (1.01–1.41); P = 0.013), rs2464196_TT (1.19 (1.00–1.40); P = 0.042), and rs2259816_T (1.13 (1.01–1.26); P = 0.031) retained their significant association with the disease, while the relationships for the other three variants turned weaker or diminished (P = 0.011) and rs2464196_C (1.12 (1.02–1.24); P = 0.024) further in T2DM (2550 versus 2081), as well as the rs2393791_T (1.14 (1.01–1.28); P = 0.032), rs7310409_G (1.16 (1.03–1.30); P = 0.013), and rs2464196_AG+GG (1.25 (1.05–1.49); P = 0.012) in HTN (3528 versus 1103), following adjustment for possible confounding effects of coexisting disease traits and other covariates (HNF1a Supplementary Data 4). Interestingly, dyslipidemia was linked to all the variants, with four of them the rs2393791_T (1.14 (1.02–1.27); P = 0.018), rs7310409 (1.12 (1.01–1.25); P = 0.031), rs1169310_G (1.15 (1.04–1.28); P = 0.010), and rs1169313_CT+TT (1.24 (1.06–1.45); P = 0.008) being implicated in hypertriglyceridemia  as well as rs2259820_T (1.23 (1.07–1.41); P = 0.004), rs2464196_T (1.22 (1.06–1.39); P = 0.004), and rs2259816_T (1.18 (1.02–1.36); P = 0.023) conferring a risk for harbouring high low-density lipoprotein-cholesterol (hLDLC) levels. None of the studied SNPs was associated with gender or history of CAD.We first performed the MWU test on the data, which demonstrated significant associations of the various SNPs with different disease traits  conferred a risk for MI (HNF1a Supplementary Data 5). Notably, its 6-mer (1–6) CATATA  and 5-mer (1–5) CATAT  derivatives were equally implicated in the disease. Further analysis pointed to the G>T change at this locus as explaining the difference between being causative and protective, as demonstrated by the fact that the 7-mer CATAGAC  was equally protective. Interestingly, the haplotype CATATAC  and its 4-mer derivative CATA was also implicated, albeit less significantly so in T2DM, pointing to MI and T2DM sharing common causative genomic sequences at this locus. No haplotype was positively associated with either hypercholesterolemia (hChol) or the harbouring low levels of high density lipoprotein-cholesterol (lHDLC). However, hypertriglyceridemia (hTG) was linked to TGCGGG  and its 5-mer TGCGG  derivative, while obesity was associated with ACGGGT  and its 5-mer derivative ACGGG .We further evaluated the possible impact of the haplotypes at this locus on the various disease traits. The linkage disequilibrium plot for the studied SNPs is given in  HNF1a gene, possibly pointing directly to this gene as the potential culprit. Currently, there appears to be stealth of information on the role of the HNF1a gene or its genomic locus in CAD/MI, in general. The available literature is somewhat conflicting, with some investigators implicating this locus in the disease [The present study evaluated the relationships of gene variants on chromosomal 12q24 with CAD/MI and its various risk traits. We described the association for at least three of the studied SNPs, rs2259820, rs2464196, and rs2259816, and a weak link for two others, with the risk for MI. These variants reside in the chromosomal region that harbours the disease , 36 and  disease –41. For  disease , 32–34,  disease , rs22598 disease , others  disease . The obs HNF1a gene, suggesting that this genic region is important for the manifestation of this disease. However, as in the case of CAD, a number of investigations addressing the role of the HNF1a gene polymorphism with respect to diabetic disease conditions have yielded somewhat inconsistent results. Thus, for example, among the variants included in the present study, the rs2259816_G>T has been associated with diabetic nephropathy in type 1 diabetes patients but not with T2DM [ HNF1a polymorphism on disease manifestation, in general.Our present data also implicated both the rs2259820 and rs2464196 in T2DM, reaffirming a role for this genomic region in diabetic disease pathways. Interestingly, the rs2464196 and two other variants linked to MI were also implicated in HTN, pointing to a possible common link for the three cardiovascular diseases. To our knowledge, this is the first report linking the rs2259820 to T2DM. This variant resides on exon 7 of theith T2DM . On the  HNF1a gene variants in dyslipidemia, T2DM [ HNF1a have also shown altered plasma cholesterol levels [ HNF1a gene is a susceptibility locus for MI, HTN, T2DM, and dyslipidemia, the important causes for atherosclerosis. This scenario also points to possible complex interactions between diabetes and dyslipidemia in atherosclerosis disease pathways. Similar interaction has been suggested recently between HNFIa gene, hyperglycemia, cardiovascular risk, and T2DM [As demonstrated in ia, T2DM , hTG, ania, T2DM . Specifiia, T2DM , and a sia, T2DM . Besidesl levels . Put togand T2DM . HNF1a variants associated with disease to date are coding, in the present study, it was primarily noncoding SNPs that were implicated. These include the rs2259816 located on intron 7 of the gene, which was associated with MI but was protective against T2DM. These observations seem to point to the likelihood that other entities related to the function of this locus might offer an explanation for these observations. Hence, we thought it worthwhile to explore further the probability of haplotyping this region as being more informative than individual SNPs in establishing the impact of the changes in the HNF1a gene locus on disease manifestation. Indeed, our study revealed several stretches of sequences that were associated with MI, T2DM, and changes in triglyceride and LDL-cholesterol levels. Notably, the difference between the causative 7-mer CATATAC and protective CATAGAC for MI was the genomic position of the rs2259816, which displayed the most significant association with the disease. Even more interesting was the observation of an association of the former haplotype with T2DM, demonstrating a link for the two diseases at haplotype level. Thus, MI and T2DM share not only common causative variants but also a common genomic region at the locus of the HNF1a gene. However, hTG was also associated with a number of other haplotypes that differed, however, from those implicated in MI and T2DM, suggestive of other independent entities as the possible underlying causes of these disorders. This could occur in various fashions, including the likelihood of the changes influencing gene expression as cis-acting regulators of nearby genes. For potential mechanisms involving SNPs in the 3-untranslated region (3′-UTR), it can be speculated that such genic changes are likely to interfere with actions of gene regulatory elements, such as the microRNAs, thereby influencing mRNA maturation processes, for example. However, this notion needs to be verified further.Even more important is the fact that, while the large majority of HNF1a locus constitutes a risk factor for MI, T2DM HTN, and dyslipidemia. Furthermore, since the majority of implicated variants were noncoding, we speculate that some other entities at this genomic locus, rather than the HNF1A gene per se, are likely to be the primary contributors to the disease pathways of atherosclerosis.We conclude that theThe HNF1a Suppl data consist of four data sets. The first summarises the important clinical and demographic characteristics and genotyping of the studied heterozygous familial hypercholesterolaemia family. The second set (HNF1a Suppl data 2), is a summary of the Mann-Whitney-Wilcoxon analysis tests, while the third set (HNF1a Suppl data 3) depicts the Bonferroni adjustment for age. In the fourth (HNF1a Suppl data 4) we provide the summary of the Binary logistical regression analysis for the HNF1a gene variants with the disease traits.


Spirulina maxima (SM) were tested. SM cultures grown in Zarrouk medium containing different N2 (in NaNO3 form) concentrations  for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2 concentration. The cultures of SM at large scales  showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2 cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production in S. maxima.L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA from Streptomyces gulbargensis, Enterobacter cloacae, and Serratia marcescens), fungi, and yeast. All the forms of the enzyme have similar functionality and received important attention [Enzymes are proteins that catalyze biochemical reactions. They are more important than vitamins and minerals for general health . Hydrolattention –11.2=CH-CO-NH2), a potential carcinogen, which is formed from the reaction of L-asparagine (L-AsnA) and reducing sugars contained in foods during heating processes [Recently, L-asparaginase is used in food technology as a potent mitigating agent for reducing the acrylamide . It was grown in the modified Zarrouk medium [−1): Na2CO3, 4.03; NaHCO3, 13.61; NaCl, 1.00; K2SO4, 1.00; NaNO3, 2.50; K2HPO4, 0.50; MgSO4, 0.20; and CaCl2·2H2O, 0.04. All nutrients were dissolved in redistilled water containing (per liter) 6 mL of metal solution  and 1 mL of trace-nutrient solution .k medium  containi3 with the concentrations of 1.25, 2.5 g/L, and 5.0 g/L NaNO3 into a different flask containing 1.7-liter Zarrouk medium in a 2 L flask. Aeration was accomplished utilizing air pumps to achieve an air flow rate of 20 L/h. The cultures were gassed with 0.03% volume CO2 in the air and temperature was maintained at 25°C ± 3°C. The pH of all media was adjusted to 9.5 and monitored at 24 h intervals. The cultures were illuminated with perpetual 10 cool white fluorescent lamps (Philips 40 W) that provided an illumination of 2500 lux. In all cultivated flasks, conductivity, salinity, pH, and temperature were daily quantified with Hanna (HI 09812-5) conductivity meter. The purity of cultures was periodically checked by microscopic observation following taxonomy guidelines. All solutions and glassware were autoclaved at 121°C for 15 min prior to utilization.Nitrogen was supplied as NaNO3 Spirulina maxima culture was cultured in 320 L glasses aquarium containing 300 L Zarrouk medium. The aquarium was incubated at the same conditions as described previously.The inoculum from 5 g/L NaNO S. maxima was monitored every three days through the entire cultivation period by determining the dry weight (dw) and optical density (OD) at 560 nm by UV-vis spectrophotometer [The growth rate ofotometer . A good The algal cells were harvested at the stationary phase, by centrifugation at 10,000 ×g (4°C) for 15 min, and the cell masses were stored at −20°C until analysis.Rapid screening of L-asparaginase produced from SM cultures was assessed based on Gulati et al.  method wExtraction of crude enzyme was done by adding 10 mL of sodium phosphate buffer (pH 7) to the MS cultures, kept in rotator shaker for 45 min. One mL of the extract was transferred to the Eppendorf tube and centrifuged at 10,000 ×g for 10 minutes. The obtained supernatant was used as crude extract for L-asparaginase assay.μmole of ammonia per mL per min (μmole/mL/min) at 37°C.L-asparaginase activity was determined by measuring the amount of ammonia released by nesslerization according to Wriston Jr. and Yellin  method. The purification was carried out using crude enzyme extract. The enzyme was purified by the following steps: ammonium sulfate precipitation and Sephadex G-200 gel filtration, according to the modified method of Distasio et al. , 22. AftThe powder of ammonium sulfate was added gradually to the crude extract (20–80%) reaching 80% saturation solution. The mixture was left for 12 h at 4°C, followed by centrifugation at 8,000 ×g for 20 min at 4°C. The fractions precipitate was dissolved in a 0.01 M phosphate buffer pH 8.5 and dialyzed overnight against the same buffer at 4°C.The dialyzed ammonium sulfate fraction was applied to a Sephadex G-200 column that was preequilibrated with a 0.01 M phosphate buffer pH 8.5. Then, the protein fraction was eluted with the 0.01 M phosphate buffer at a flow rate of 5 mL/25 min. It was assayed for protein quantity at 280 nm as well as for enzyme activity. The active fractions were pooled, dialyzed against the 0.01 M phosphate buffer pH 8.5, and concentrated.The total protein content in soluble protein content and protein fractions was determined spectrophotometrically at 595 nm, using Coomassie Blue (G 250) as mentioned by Bradford . Bovine  S. maxima L-asparaginase was assessed by incubating the assay mixture under assay conditions as a function of varying ranging of pH  and temperature (20–50°C).To determine the optimum pH and temperature of the AsnA enzyme, the activity of the purifiedThe optimum pH of the enzyme was determined as reported by Singh et al.  using thThe optimal temperature of purified L-asparaginase enzyme activity was determined by preincubating the enzyme at desired temperature (20–50°C) for 15 min and the amount of ammonia liberated was determined. At each temperature, blank assay was done without adding the enzyme.P < 0.05 were considered statistically significant. The results were presented as mean values .All measurements were carried out in triplicate. Statistical analyses were performed using one-way analysis of variance (ANOVA), and the significance of the difference between means was determined by Duncan's multiple range tests. Differences at 2 (as NaNO3) concentrations  in Zarrouk medium on algae growth for 18 days, as monitors by dry weight yields of biomasses (DWYg/L). The results showed that the DWY was significantly different (P > 0.5%) among all algae cultures at the incubation time interval. The biomass yield (g/L DW) was increased significantly among all cultures as the function of incubation times and nitrogen concentrations. The highest biomass yield recorded after 18 days of incubation was 3.456 mg/L, 2.456 mg/L, and 1.948 mg/L in SM cultures grown in Zarrouk's medium containing 5.0 g/L, 2.5 g/L, and 1.25 g/L N2, respectively. Thus, high biomass yield was noticed in S. maxima cultured in either high (5.0 g/L) or optimal (5.0 g/L) N2 concentrations compared with that noted in low N2 (1.25 g/L) culture. It is known well that nitrogen is an essential element of S. maxima growth and it grows well with high biomass yield [ A. platensis biomass grown in rich or optimal N2 was higher than that in low N2 cultures [2 concentration [2 levels had high protein content. In general, environmental factors, nutrient status, and salinity lead to changes in cellular metabolic pathway and cellular composition [Nitrogen is the major structural and functional element of algal cells and plays an important role in the nutrition in states. Nitrogen is required for all biosynthesis leading to reproduction, product formation, and cell maintenance. Production of the primary (proteins and carbohydrates) metabolite by microorganisms is highly influenced by their growth conditions. cultures . Howevercultures , 27. Simcultures  that somntration . In thisposition . These c Spirulina maxima (SM) growth in Zarrouk medium. These results indicate that the increase of nitrogen concentration in the Zarrouk nutrient media led to high changes in TP, SPr, and SPY contents in SP cells. At high N2 5.0 g/L, the values of these constituents were 56.36% of DW, 40.52 mg/g of DW, and 1397.9 mg/L, respectively. At limited N2 1.225 g/L, these values were 33.42% of DW, 20.12 mg/g of DW, and 390 mg/L, respectively. Thus, the protein constituents were increased in algal cells grown at high N2 by 2.51%, 4.11%, and 9.01%, respectively, as great as that in SP grown at low N2 concentration. Thus, the increase of N2 concentration in nutrient medium caused a significant increase of protein constituents in SM cultures. It could be noted that the total protein content increased with the increasing of N2 concentration in Zarrouk media. By this way, increasing the concentration of N2 in the nutrient medium over that optimal level can be manipulated with respect to its total proteins and soluble protein content. One would say that increase in N2 levels in growth medium may lead to increase in the protein synthesis required for increasing the metabolic pathway intracellular nitrogenous compounds in order to balance the high N2 concentration. However, it is well known that the nitrogen concentration in a medium has a great influence on protein content and its constituents in several species of Spirulina species. Piorreck et al. [ Spirulina, Chlorella, and many algae species grown in high nitrogen level showed high protein contents. Zeng and Vonshak [ Spirulina cells grown under stress conditions, including salinity-stress, have a lower protein synthesis capacity. The finding results are in harmony with that of our findings [ Spirulina grown in medium containing high nitrogen with a high biomass production and total protein content as factors for macro- and microelements such as nitrogen source and concentrations. On the other hand, Becker [ Spirulina spp. grown in a nitrogen-rich medium had a high ability to accumulate a considerable quantity of proteins (>60%) and up to 20% of this protein fraction was identified as a phycocyanin blue pigment.Data in k et al.  reported Vonshak  found thfindings  in Spiru, Becker  and Abd , Becker  reported Spirulina maxima grown in Zarrouk medium that contained various nitrogen concentration was colorimetrically assessed based on pH indicator phenol red (PR). Algal L-asparagine was incorporated into 0.009% methanolic phenol red-pH indicator; phenol red at acidic pH is yellow and in alkaline pH turns into pink colors. Thus, appearing of pink color indicates the hydrolysis of L-asparagine into aspartic acid and ammonia was released by L-asparaginase (L-AsnA) presence in Spirulina maxima (SM). Here, again, initial pH was changed from acidic (yellow) to basic (pink) due to the release of ammonia and this could be considered as positive result. After 5 min incubation period, aliquot Spirulina extract with PR was changed to pink coloration and it indicates the presence of algal L-asparaginase with high activity (L-asparaginase (L-AsnA) activity of microalgae was rapid assay based on Gulati et al.  method. activity . However S. maxima grown in various nitrogen cultures was determined by evaluating enzyme activity IU, total protein, specific activity, and yield of enzyme in an aliquot of the SM enzyme extract , these values were found to be 356 IU (594 IU/L), 212 mg/g (332 mg/g), 1.667 IU/mg protein (1.79 IU/mg protein), and 32.39 IU/L (43.86 IU/L), respectively. This result revealed that the highest enzyme activities were recorded in SM cultures grown in high N2 medium when compared with that in optimal and low N2 cultures. Thus, algae grown in rich N2 medium were considered as high substrates and high enzyme activity correlated with good algae biomass yield. This result also showed significant differences among all various nitrogen cultures on the production of asparaginase by S. maxima.The enzyme activity of asparaginase produced by extract . In rich S. maxima cultures purification of crude enzyme was performed by 80% ammonium sulfate precipitation and gel filtration GF purification steps is summarized in  S. maxima purified by GF had a high specific activity of 19.1 IU/g and was found to be of approximately 2-fold purity compared to 80% AS purification method. However, 80% AS and GF purified process showed a high enzyme purification 5.28- and 10.91-fold greater than that in crude enzyme extract ( Pseudomonas aeruginosa reached 106-fold by 3 steps of purification (AS precipitation and GF on Sephadex G-100 column followed by CM-Sephadex C50 column).Among all three SP cultures examined for synthesis of L-asparaginase, SP culture grown in high N concentration medium characterized by high yields enzyme 51.28 U/L and high enzyme activity (898 IU) and protein enzyme (405 mg/g) content was selected for further studies. The characterization parameters of its high synthesis of L-asparaginase in extract . However extract . El-Bess S. maxima AsnA enzyme purified by GF step are . Makky et al. [ Streptobacillus sp. was recorded to be 21.77 U/mg with 39.58-fold purification and 39% of yield. Finally, the maximum enzyme activity of S. maxima was found to be at/or near physiological pH and temperature, making it extremely valuable in the chemotherapeutic treatment of some diseases such as cancer.In general, the optimum activity of purified AsnA enzyme was recorded at the pH 8.5 and 37 ± 0.42°C the optimum temperature in many of microbial species. In this regard, El-Bessoumy et al. [ectively . In conty et al.  reportedSpirulina maxima (SM) cultures grown in Zarrouk medium containing high N2 (5 g/L) as NaNO3 level for 18 days showed high dry biomass yields (1.948 g/L) and L-asparaginase (L-AsnA) enzyme production, total protein 405 mg/g, specific activity 2.21 IU/mg protein, and yield of enzyme 51.28 IU/L compared with those obtained in either optimal N2 (2.5 g/L) or low N2 (1.25 g/L) concentration culture. The partial purification of crude MS L-asparaginase enzyme achieved by 80% ammonium sulfate prepetition and gel filtration had a high specific activity greater by 5.28 and 10.91 than that in crude enzyme extract. The optimum activity of purified enzyme was recorded at 8.5 and 37 ± 0.2.


Background and Objectives. A prospective, randomized, controlled, double-blind clinical trial to assess the effect of tramadol and ketamine, 50 mg, added to ropivacaine in brachial plexus anesthesia. Methods. Thirty-six ASA physical statuses I and II patients, between 18 and 60 years of age, scheduled for forearm and hand surgery under axillary brachial plexus block, were allocated to 3 groups. Group R received 0.375% ropivacaine in 40 mL, group RT received 0.375% ropivacaine in 40 mL with 50 mg tramadol, and group RK received 0.375% ropivacaine in 40 mL with 50 mg ketamine for axillary brachial plexus block. The onset times and the duration of sensory and motor blocks, duration of analgesia, hemodynamic parameters, and adverse events  were recorded. Results. The onset time of sensorial block was the fastest in ropivacaine + tramadol group. Duration of sensorial and motor block was the shortest in the ropivacaine + tramadol group. Duration of analgesia was significantly longer in ropivacaine + tramadol group. Conclusion. We conclude that when added to brachial plexus analgesia at a dose of 50 mg, tramadol extends the onset and duration time of the block and improves the quality of postoperative analgesia without any side effects. Regional anesthesia provides a safe anesthesia for upper extremity surgery. Brachial plexus block with axillary approach for hand and forearm surgery is commonly used.The use of adjuvants in combination with local anesthetics for peripheral nerve blocks enhances the quality and duration of anesthesia and postoperative analgesia. Numerous studies have been published on the effects of different adjuvants on local anesthetics for axillary brachial plexus block –5. Tramaα2-adrenoceptor antagonists as well as opioid antagonists [Tramadol is a synthetic analgesic drug that is antagonized by agonists . Ketaminagonists .The effects of different doses of tramadol, ranging between 40 and 200 mg, and ketamine, ranging between 1 and 1.5 mg/kg, with different local anesthetics, have been reported in several studies –13. HoweWe designed a prospective, randomized, controlled, double-blind clinical trial to assess the effect of lower doses of tramadol and ketamine, 50 mg, added to ropivacaine in brachial plexus anesthesia to determine effectivity of tramadol and ketamine.After institutional ethics committee approval and written informed consent were obtained, 36 ASA physical statuses I and II patients, between 18 and 60 years of age, scheduled for forearm and hand surgery under axillary brachial plexus block, were included in this randomized (envelope method), controlled study. Pregnant women and patients with a history of cardiac, respiratory, hepatic, or renal failure were excluded from the study. A 20-gauge intravenous cannula was inserted into the contralateral dorsal hand. Routine monitoring of electrocardiogram, noninvasive measurement of arterial blood pressure, peripheral oxygen saturation, and respiratory rate monitoring were conducted.Patients were allocated to 3 groups in a controlled, randomized, double-blinded fashion. Group R received 0.375% ropivacaine in 40 mL, group RT received 0.375% ropivacaine in 40 mL with tramadol 50 mg, and group RK received 0.375% ropivacaine in 40 mL with ketamine 50 mg for axillary brachial plexus block.μg/kg and midazolam 0.03 mg/kg intravenously 10 minutes before the axillary block. Axillary block was performed in the supine position with the upper arm abducted at 90° and the elbow flexed at 90°. The area was shaved the day before and disinfected. The axillary artery was palpated in the proximal part of the axilla, and a skin wheal was injected using 1 mL of lidocaine 2%. A nerve stimulator  was used to identify the plexus. The position of the needle was judged adequate when an output current of less than 0.5 mA still elicited a slight distal motor response. With intermittent aspiration, the total volume was injected into the perivascular area. All the blocks were performed by the same anesthetist.Patients were premedicated with fentanyl 0.75 The onset times and the duration of sensory and motor blocks, duration of analgesia, hemodynamic parameters, and adverse events  were recorded. Patients were considered sedated according to Ramsay Sedation Scale > 2 .All local anesthetic solutions and adjuvant drugs were prepared by an anesthesiologist not involved in performing brachial plexus block or data collection. All blocks were performed by one of the authors, who was unaware of the contents of the injected solution. Sensory block and motor block of musculocutaneous, radial, median, and ulnar nerves were assessed and recorded at 5-minute intervals. Sensory block of each nerve was assessed by pinprick and compared with the same stimulation of the contralateral arm as reference. Motor block was evaluated by modified Bromage scale . Duration of sensory block was considered as the time interval between the local-anesthetic administration and the complete offset of anesthesia. Motor-block duration was defined as the time interval between the local-anesthetic administration and the recovery of motor function. Heart rate, peripheral oxygen saturation, respiratory rate, and blood pressure were measured before the axillary block, 5, 10, 15, 30, 45, and 60 minutes after the axillary block, and every 60 minutes thereafter for 2 hours postoperatively. Additional adverse events were recorded .t-tests and analyses of variance for parametric data to compare the differences between groups. We used Mann-Whitney U tests for nonparametric comparison of the data. A value of P < 0.05 was considered statistically significant. The results are expressed as means ± standard errors.Statistical analyses of the data were performed using P ≥ 0.05). Also, heart rates and oxygen saturations of all of the groups were not significantly different (P ≥ 0.05).There was no statistically significant difference between the groups in age, weight, gender, and duration of surgery . No diffThe distribution of the surgery types among groups is shown in P = 0.015), and ropivacaine+ketamine groups, 9.85 ± 0.41 (P = 0.007). The onset time of motor block was 10.8 ± 0.38 min, 9.3 ± 0.28 min, and 11.1 ± 0.43 min, respectively, and P values were P = 0.005 and P = 0.002.The onset time of sensorial block was the fastest in ropivacaine + tramadol group, 8.17 ± 0.33 min, compared to the ropivacaine, 9.55 ± 0.34 min (P = 0.023), and ropivacaine + ketamine groups, 13.6 ± 0.30 h (P = 0.009). Duration of motor block was similar, in groups 15.8 ± 041 h, 13.60 ± 0.30 h, and 13.74 ± 0.33 h, respectively, and P values were P = 0.001 and P = 0.001.Duration of sensorial block was also the shortest in the ropivacaine + tramadol group, 13.16 ± 0.40 h, compared to the ropivacaine, 14.90 ± 0.33 h (P = 0.001), and 22.6 ± 0.30 h in group RK compared with group R (P = 0.08).Duration of analgesia was 21.6 ± 0.40 h in group R, 24.9 ± 0.33 h in group RT  antagonist, was evaluated when the drug is delivered via caudal , 8, epidIn our study, addition of 50 mg ketamine to 0.375% ropivacaine did not provide any additional effect on axillary brachial plexus. The possible cause of this may be the low concentration of 50 mg ketamine in a volume of 40 mL. In our opinion, a higher concentration of ketamine is necessary for ketamine to be used as an adjuvant to local anesthetics in brachial plexus block. However, using a higher concentration means more ketamine-induced adverse effects.Few studies report adverse effects when various doses of tramadol and ketamine are added to local anesthetics as adjuvants. These studies found no difference in adverse effects when comparing the ropivacaine alone group to a group in which 100 mg tramadol was added for brachial plexus. Our study confirmed these results –25.We found no statistically significant differences with regard to the side effects between the groups R and RT, but all side effects in the RK group, except dizziness and sedation, were significantly different.Nausea and vomiting were observed in the RK group, possibly due to the emetic effect of ketamine that is caused by release of endogenous catecholamine. Feelings of discomfort are also known effects of ketamine.We conclude that when added to brachial plexus analgesia at a dose of 50 mg, tramadol extends the onset and duration time of the block and improves the quality of postoperative analgesia without any side effects. Further studies of the effects of lower doses of tramadol with various combinations of tramadol and local anesthetics are needed, before final recommendations can be made.Our findings do not encourage the use of ketamine for brachial plexus block. Ketamine does not enhance the local anesthetic effect at the level of the axillary brachial plexus nerve roots and does not prolong postoperative analgesia with the dose of 50 mg when combined with ropivacaine. In addition, the high incidence of ketamine-induced adverse effects at this dose is disturbing.These findings make us ask the question of “do we need to use ketamine as an adjuvant to local anesthetics in brachial plexus blockage?”The answer is that we have more than one alternative.


Listeria monocytogenes in cold smoked salmon (CSS) is of concern as it is a refrigerated processed food of extended durability (REPFED). The objectives of this study were to compare and optimize the antimicrobial effectiveness of films and coatings incorporating nisin (Nis) and sodium lactate (SL), sodium diacetate (SD), potassium sorbate (PS), and/or sodium benzoate (SB) in binary or ternary combinations on CSS. Surface treatments incorporating Nis (25000 IU/mL) in combination with PS (0.3%) and SB (0.1%) had the highest inhibitory activity, reducing the population of L. monocytogenes by a maximum of 3.3 log CFU/cm2 (films) and 2.9 log CFU/cm2 (coatings) relative to control samples after 10 days of storage at 21°C. During refrigerated storage, coatings were more effective in inhibiting growth of L. monocytogenes than their film counterparts. Cellulose-based coatings incorporating Nis, PS, and SB reduced the population of L. monocytogenes, and anaerobic and aerobic spoilage flora by a maximum of 4.2, 4.8, and 4.9 log CFU/cm2, respectively, after 4 weeks of refrigerated storage. This study highlights the effectiveness of cellulose-based edible coatings incorporating generally regarded as safe (GRAS) natural and chemical antimicrobials to inhibit the development of L. monocytogenes and spoilage microflora thus enhancing the safety and quality of CSS.The relatively high incidence of  Listeria monocytogenes has long been established as an important food-borne pathogen with a fatality rate of 25–30% [ L. monocytogenes has been listed in the top five highest-ranking pathogens with respect to the total cost of foodborne illness in the United States in terms of loss of income by the affected individual, cost of health care, loss of productivity due to absenteeism, costs of investigations of an outbreak, loss of income due to closure of businesses, consumer litigations, or losses of product sales when consumers avoid particular products [f 25–30% . HoweverL. monocytogenes infection has been associated with consuming a variety of meat, poultry, fish, and dairy products [ L. monocytogenes if the raw salmon harbored the pathogen or acquired it from the processing environment [ L. monocytogenes and therefore this product cannot be completely free of this pathogen [products . The preproducts , 5. Thisironment . Hence, pathogen . For thipathogen . L. monocytogenes is highly problematic, antimicrobial additives are sought after to prevent growth of this bacterium in food products and to ensure safety. Several studies have reported the effectiveness of nisin  in delaying and reducing growth of Listeria spp. in model systems [Since postprocess contamination of smoked fish with systems , 9 and i systems  includin systems , 12. Nis systems . In the  systems , 15. The systems . These i systems , as well systems . L. monocytogenes in media, meat [Thus, a multiple-hurdle approach relying on the combination of nisin with other antimicrobials, such as chemical preservatives, is desirable. Sodium lactate (SL) is a GRAS additive that is widely used to enhance flavor, control microbial growth, and increase shelf life of meat, poultry, and fish products –22. The ia, meat –30, and ia, meat , 32. L. monocytogenes on CSS. Neetoo et al. [ L. monocytogenes in CSS during a 30-day storage at 4°C. Moreover, Ye et al. [ L. monocytogenes on CSS during subsequent refrigerated storage. Reductions ranging from 0.5 to 4.5 log CFU/cm2 compared to uncoated samples were reported. Growth of L. monocytogenes on the surface of CSS was inhibited by whey-protein films incorporating a lactoperoxidase system [ L. monocytogenes. Similarly, Tammineni et al. [ L. monocytogenes on CSS. The films reduced the inoculum by greater than 2 log cfu/g during storage at 4°C for 28 days. However, all aforementioned studies have tested the antilisterial efficacy of films or coatings in isolation and lack a systematic comparison of the relative efficacy of each approach.Antimicrobial coatings and films allow the controlled diffusion and gradual release of embedded antimicrobials onto the food surface . Signifio et al.  found the et al.  showed te system . Seydim e system  showed ti et al.  develope L. monocytogenes and spoilage microflora on vacuum-packaged CSS during refrigerated storage (4°C).The objective of this study was to compare cellulose-coated plastic films and edible cellulose-based coatings incorporating binary or ternary combinations of food-approved antimicrobials to inhibit growth of L. monocytogenes strains, PSU1 (serotype 1/2a), PSU21 (serotype 4b), PSU9 (serotype 1/2b), F5069 (serotype 4b), and Scott A (serotype 4b) , were used. The strains were maintained on tryptic soy agar plus 0.6% yeast extract (TSAYE) plates and stored at 4°C. Each strain was grown independently in tryptic soy broth plus 0.6% yeast extract (TSBYE) for 24 h at 37°C and a loopful of each overnight culture was transferred to 10 mL of fresh TSBYE and incubated at 37°C for 24 h. On the day of the experiment, 1 mL volume of each culture was combined to provide a five-strain mixture and then readjusted with 0.1% peptone water to a final cell density of ca. 108 CFU/mL, which served as the inoculum. Serial dilutions were plated onto TSAYE plates and incubated at 37°C for 24 h to determine cell numbers.FiveSalmo salar) was obtained from a producer. It was kept frozen at −20°C and thawed at 2 ± 2°C (<4°C) for 1 day immediately before use. Slices of CSS were punched aseptically into 5.7 cm diameter round pieces weighing 10 ± 1 g. The samples were surface-inoculated with a 108 CFU/mL dilution of the five-strain cocktail of L. monocytogenes to achieve final concentrations of 105 CFU/cm2 (or 5 × 105 CFU/g) of salmon surface. After inoculation, salmon samples were kept at room temperature for 30 min to allow bacterial attachment. L. monocytogenes populations in CSS are generally low (1–103 CFU/g) with 90–99% of cases below 102 CFU/g and less than 1% between 103 and 104 CFU/g as reported by Jørgensen and Huss [5–107 CFU/g have also been reported previously [Freshly processed CSS  and hydroxypropylmethylcellulose  were mixed with 200 mL of 95% ethanol and 200 mL of sterile distilled water and stirred to which 6 mL of polyethylene glycol 400 was subsequently added. This coating stock solution was then supplemented with the various antimicrobial preparations. Briefly, 1.3 g of nisin was dissolved in 60 mL of 0.02 M acetic acid, and 12 mL of the prepared nisin solution was then supplemented with either 1.29 g SD, 1.54 g PS, 0.51 g SB, or 2.57 g of a-60% SL syrup. The antimicrobial solution was then made up to 50 mL with the MC/HPMC carrier solution. The solution was sufficient to coat 10 salmon discs.2 each, using a thin layer chromatography (TLC) plate coater.The MC and HPMC were mixed with ethanol and water to prepare a coating solution as described above. The coating solution was then supplemented with the various antimicrobials. Briefly, 3.0 g nisin was dissolved in 60 mL 0.02 M acetic acid, and 12 mL of the prepared nisin solution was then supplemented with 3.0 g SD, 3.6 g PS, 1.2 g SB, or 6 g of 60% of SL syrup. The antimicrobial solution was then made up to 42 mL with the MC/HPMC carrier solution. The solution was then used to coat 3 glass plates, lined with LDPE films covering a surface area of 400 cmμL aliquot of a HPMC/MC coating solution containing nisin  alone or in combination with SL (0.3%), SD (0.25%), PS (0.3%), or SB (0.1%) as summarized in The inoculated CSS discs were coated with a 500 Inoculated samples without films or coatings were also prepared as untreated controls. Controls and treated samples (film-wrapped or coated) were then inserted into 3 mm thick high barrier pouches and subsequently sealed using a vacuum-packaging machine. The samples were stored at room temperature (21°C) for 10 days and analyzed microbiologically every other day. L. monocytogenes were determined by an overlay method [Listeria monocytogenes PCR assay. The numbers of L. monocytogenes per cm2 of salmon were calculated by dividing the total count of L. monocytogenes per salmon disc by the total surface area (51.4 cm2). The absence of the pathogen in the CSS samples was confirmed by a primary enrichment in UVM broth (Difco Laboratories) and a secondary enrichment in Fraser broth (Difco Laboratories) according to the USDA Microbiology Laboratory Guidebook [For microbial analysis, the package was aseptically cut and the sample transferred to a sterile stomacher bag that contained 40 mL of 0.1% sterile peptone water and stomached for 2 min. Serial dilutions were made in 0.1% peptone water and counts ofy method . Brieflyuidebook .5 CFU/cm2 of salmon surface. Antimicrobials with the highest antilisterial activity (PS and SB) were selected for further testing. Inoculated salmon discs were coated with a 500 μL aliquot of HPMC/MC coating solution on each side containing Nis (25000 IU/mL) with PS  or Nis (25000 IU/mL) with PS  and SB . The samples were air-dried and packaged. In addition, inoculated CSS samples were wrapped with LDPE films coated with a solution containing Nis (25000 IU/mL) with PS  or Nis (25000 IU/mL) with PS  and SB . Samples were then packaged and stored at 21°C for 10 days. The formulations for the various binary or ternary combinations of antimicrobials incorporated in films or coatings are summarized in  L. monocytogenes as described earlier.The CSS samples were inoculated to a final concentration of 103 CFU/cm2 (or 5 × 103 CFU/g). Uninoculated samples were also prepared. Inoculated and uninoculated samples were wrapped with film or coated with an antimicrobial coating solution, packaged, and stored at 4°C for 4 weeks. The combinations of antimicrobials incorporated in films and coatings for the refrigerated storage study are summarized in The CSS samples were surface-inoculated to a final concentration of approximately 10 L. monocytogenes as described previously. Uninoculated samples were also analyzed weekly for spoilage aerobic and anaerobic bacteria. Spoilage anaerobic bacterial counts were determined by plating on Liver Veal Agar and plates incubated in anaerobic jars with anaerobic GasPak (BBL) for two days at 35°C. Aerobic bacterial counts were determined by plating onto TSAYE and plates incubated aerobically for two days at 35°C.Inoculated samples were analyzed weekly for10 CFU/cm2 and means and standard deviations were calculated using Microsoft Excel. A Tukey-Kramer test was used to determine differences in the populations of L. monocytogenes, aerobes, and anaerobes on CSS samples. Significant differences were considered at the 95% confidence level (P < 0.05).Three independent trials were conducted for all experiments. Colony counts were converted to log L. monocytogenes on CSS slices treated with different binary combinations of antimicrobials in films and coatings is represented in Figures 2. After 2 days, the control (untreated) samples had higher counts than all other treatments with the Nis + SB (5.2 log CFU/cm2) and Nis + PS (4.8 log CFU/cm2) coatings showing significantly (P < 0.05) lower counts than the control groups. After 10 days, L. monocytogenes population on samples treated with Nis + SB (4.9–6.3 log CFU/cm2) and Nis + PS (4.3–4.7 log CFU/cm2) was appreciably lower compared to the control untreated samples (7.7 log CFU/cm2) although the results were not statistically significant (P > 0.05).The fate of L. monocytogenes on CSS. The initial counts of L. monocytogenes on CSS were about 5.6 log CFU/cm2 and increased steadily over the 10-day period reaching a maximum count of 7.6 log CFU/cm2. Over the storage period, the counts for all treatments were consistently lower than the control for either method of antimicrobial application. Ternary combinations of Nis (25000 IU/mL), PS (0.15 or 0.3%), and SB (0.05 or 0.1%) in films and coatings significantly (P < 0.05) inhibited the growth of L. monocytogenes after 10 days, lowering the population by 2.0–3.3 log CFU/cm2 and 2.2–2.9 log CFU/cm2, respectively.Figures  L. monocytogenes counts on inoculated CSS treated with antimicrobial films and coatings incorporating Nis + PS or Nis + PS + SB are shown in Figures  L. monocytogenes on CSS recovered just after inoculation was 2.7 log CFU/cm2. The pathogen grew unhindered in untreated samples stored at 4°C, reaching approximately 4.8 log CFU/cm2 after 4 weeks. All the antimicrobial treatments (films or coatings) brought about a significant reduction (P < 0.05) in the population achieving reductions of 2.0–2.7 log CFU/cm2 by the end of the storage period. Ternary combinations of Nis (25000 IU/mL) with PS (0.3%) and SB (0.1%) were more effective than binary combinations of Nis (25000 IU/mL) with PS (0.3%) for either method of application although the difference was not statistically significant (P > 0.05).The two combined treatments chosen for this study were Nis + PS (0.3%) and Nis + PS (0.3%) + SB (0.1%).2 by the end of the storage period. Although both films and coatings were effective in delaying the development of background flora, coatings incorporating Nis + PS and Nis + PS + SB resulted in greater (P > 0.05) population reduction than their film counterparts.The total anaerobic and aerobic counts of the uninoculated samples are shown in Figures  L. monocytogenes. In addition, CSS is a type of minimally processed food, also called Refrigerated Processed Foods of Extended Durability (REPFED) [ L. monocytogenes contamination [Cold smoked salmon (CSS) is considered a high-risk food because the temperature used during the cold smoking operation is not lethal to(REPFED) , and conmination  and it hmination . Moreovemination , 46 have L. monocytogenes in these products: (1) elimination or reduction of L. monocytogenes on the outside surface of frozen or fresh fish before filleting, (2) prevention of recontamination and growth of L. monocytogenes during all stages of processing, and (3) the inhibition of any possible survivors or recontamination during processing and distribution [ L. monocytogenes in cold smoked fish using physical interventions including gamma irradiation [Several forms of interventions have thus been recommended to reduce the risks fromribution . Numerouadiation , X-ray iadiation , E-beam adiation , high-pradiation , chemicaadiation –53, natuadiation , 55, andadiation , 57. Extadiation , which cadiation . Moreoveadiation . L. monocytogenes, following chitosan application on CSS, have been reported previously [μL/mL), EDTA (150 μg/mL), and nisin (2000 IU/mL) alone and with their mixes against Pseudomonas spp., TVC, and psychrotrophic bacteria during storage at 4°C. The inhibitory effect of those antimicrobials on TVC followed the order: cinnamon + EDTA + nisin or cinnamon (5.5 log CFU/g) > nisin + EDTA (1.5 log CFU/g) compared to controls. Song et al. [ E. coli O157:H7 and L. monocytogenes on salmon after 15 days of storage at 4°C. The antilisterial effect of a calcium alginate coating incorporating oyster lysozyme in the presence or absence of nisin on the surface of smoked salmon was also investigated previously [ L. monocytogenes, the treatment was not highly inhibitory. Taken together, these findings point to the highly variable antimicrobial efficacy of edible films and coatings (reductions of 0.5–5.5 log CFU/g), which is dependent on the type, concentration, and combination of antimicrobials as well as the test product of interest.Because of the aforementioned reasons, films and coatings have garnered more interest by virtue of their ability to not only provide a barrier against gases and moisture , but alseviously , 62. Moreviously  coated ceviously  tested, g et al.  reportedeviously . Althoug L. monocytogenes and anaerobic and aerobic spoilage flora by a maximum of 4.2, 4.8, and 4.9 log CFU/cm2, respectively, after 4 weeks of refrigerated storage. The listeriostatic activity of nisin on cold smoked fish, at levels ranging from 100 IU/mL to 2000 IU/mL, alone or in combination with LAB protective cultures [ L. monocytogenes as well as psychrotrophic bacteria on vacuum-packaged shrimps by 1.3 and 4.0 log CFU/g, respectively. Neetoo et al. [ L. monocytogenes previously [ in vitro as well as on packaged beef kept at refrigeration temperature for up to 4 weeks.In our current study, the antimicrobial efficacy of cellulose-coated films and cellulose-based coatings incorporating Nis, SL, SD, PS, and SB in different concentrations and combinations was compared. Findings revealed that coatings incorporating nisin 25000 IU/mL), PS (0.3%), and SB (0.1%) were most effective and reduced the population ofcultures , or natucultures , or chemcultures  has beencultures  reportedcultures  showed to et al.  also ind000 IU/mLeviously , 71 and eviously . Other aeviously , 73 have L. monocytogenes and spoilage bacteria to a greater extent than their film counterparts. Recent studies have also highlighted the application of organic acids or their salts alone [2 [Moreover, our data indicate that edible coatings containing nisin (25000 IU/mL) + PS (0.3%) or nisin (25000 IU/mL) + PS (0.3%) + SB (0.1%) reduced the population ofts alone  or in coalone [2  to delayalone [2 , 77 whenalone [2  for the alone [2 , 79. L. monocytogenes, although the maximum allowable level of this additive in smoked fish has not been stipulated [Antimicrobials tested in this study are regarded as direct food additives and their application is thus limited by governmental legislation . Sorbateipulated . The US ipulated , 81. In  L. monocytogenes, (ii) a refrigerated processed food of extended durability, and (iii) consumed without any heat-killing step, it is regarded as a high-risk product. This study reiterates the usefulness of antimicrobial packaging for cold smoked salmon, and it also underscores the effectiveness of cellulose-based coatings incorporating GRAS antimicrobials to control the development of pathogens and spoilage microbiota, thereby enhancing the microbiological safety and quality of this product.In recognition of the fact that CSS is (i) susceptible to postprocess surface contamination by


NDSB cosolubilization allowed us to perform successful structural and biochemical experiments with substantially fewer artifacts, which represents a strategy to directly resolve the problematic oligomerization and aggregation of compounds.Numerous small organic compounds exist in equilibrium among monomers, soluble oligomers, and insoluble aggregates in aqueous solution. Compound aggregation is a major reason for false positives in drug screening, and even soluble oligomers can interfere with structural and biochemical analyses. However, an efficient way to manage the equilibrium of aggregation-prone compounds, especially those involved with soluble oligomers, has not been established. In this study, solution NMR spectroscopy was used as a suitable technique to detect compound oligomers in equilibrium, and it was demonstrated that cosolubilization of nondetergent sulfobetaines (NDSBs) can largely suppress compound oligomerization and aggregation by shifting the equilibrium toward the monomers. The rotational correlation time was obtained from the ratio of the selective and nonselective longitudinal NMR relaxation times, which directly and quantitatively reflected the apparent sizes of the compounds in the equilibrium. The rotational correlation time of the aggregation-prone compound SKF86002 (1 m A number of small organic compounds, including those relevant for drug development, have the tendency to form aggregates in aqueous solution.–4 Such aBecause of their small Stork radius, oligomers are soluble, invisible to the naked eye, and absorb UV light without extensive scattering. Therefore, distinguishing oligomers from monomers on the basis of visibility to the naked eye or by UV absorption is difficult. However, aggregates of compounds are rather obvious, because they are insoluble and appear turbid as a result of the scattering of visible light. Extensive aggregation often leads to spontaneous precipitation of the compounds. Otherwise, the aggregates can easily be sedimented by low-speed centrifugation (≈3000 g). After sedimentation, these aggregates no longer contribute to UV absorbance. The proportion of compound in each state within the above-mentioned equilibrium depends strongly on the experimental conditions. At low concentrations, the compounds are more in equilibrium between the two soluble states, that is, the monomers and oligomers, than between the soluble states and the insoluble aggregates. In contrast, higher concentrations favor aggregation. Often, the equilibrium is transient and is shifted toward aggregates in a time-dependent manner. This occurs on various timescales from seconds to days, and in tImportantly, oligomers and/or aggregates may not exert the same activity as the monomers, because the active moiety within the compounds can be masked by molecular contacts within the oligomers and aggregates. Recent work has suggested that compound aggregates are a major reason for artificial inhibitions  in drug screening, because they segregate macromolecules from the experimental system.,9,10 Aggτc>microseconds) make the relaxation of resonances from the aggregates too fast to be detected. More importantly, the populations of the two soluble states are reflected in the relaxation properties of the compounds, as the apparent τc of the compound oligomers is substantially larger (sub-nanoseconds to nanoseconds) than that of the monomers , tIn this study, we showed that the ratio of selective and nonselective longitudinal relaxation times and the apparent rotational correlation time deduced from solution NMR spectroscopy measurements were sensitive in detecting soluble oligomers of aggregation-prone compounds. In addition, we found that cosolubilization of nondetergent sulfobetaines (NDSBs) reduced oligomerization and subsequent aggregation of compounds by shifting the equilibrium toward the monomers. We successfully reduced the artifacts in solution NMR spectroscopy and biochemical assays by NDSB cosolubilization. Therefore, NDSBs represent a class of cosolubilization reagents that directly resolve the oligomerization and aggregation of compounds for successful structural and biological analyses.N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, and Tween-80 were purchased from Sigma–Aldrich. According to the provider’s information, all chemicals were confirmed to have more than 95 % purity in HPLC or TLC.SK86002, SB203580, NDSB195, and NDSB256 were purchased from Calbiochem . COS was purchased from Cambridge Isotope Laboratories. I4PTH and benzyl benzoate were purchased from TCI . Clotrimazole was purchased from Wako . Arginine hydrochloride, chymotrypsin, the photometric substrate I84A was expressed in E. coli cells, BL21(DE3) strain. The E. coli cells that harbor the p38αI84A expression plasmids were grown in H2O or D2O M9 media supplemented with appropriate stable isotopes. The recombinant protein was purified by Ni-NTA resin affinity chromatography and size- exclusion chromatography, as published elsewhere.[The cDNA of human p38α (2–360) was cloned into the pET15b vector , and the I84A mutation was introduced by QuikChange mutagenesis (Agilent technologies) by using the vendor-provided protocol. p38αlsewhere.m p38αI84A in 25 mm deuterated Tris-buffer (pH 7.2) in D2O containing 150 mm NaCl and 1 mm deuterated dithiothreitol (DTT) at 25 °C with a Bruker Avance 600 MHz spectrometer equipped with a cryocooled triple resonance (TXI) probe. The p38α inhibitors (SKF and SB) were prepared in deuterated dimethyl sulfoxide (DMSO) and were added directly to the protein solution. The final concentrations of SKF and SB were 1.0 and 0.4 mm, respectively. The final concentration of deuterated DMSO was 10 % (v/v). NDSBs were prepared as 1 m stock solutions and were added to the protein solution prior to the addition of the ligands. Tween-80 was prepared as 10 % (v/v) and was diluted to a final concentration of 1.3 or 0.01 mm. 2048 and 256 points were recorded for the direct and indirect dimensions, respectively. The number of scans was set to 64, and the repetition delay was 1.2 s. Mixing time was set to 0.2 s. The typical experimental time was 7.5 h.INPHARMA experiments were performed with 25 μT1s and T1ns experiments for SKF were performed in 25 mm deuterated Tris-buffer (pH 7.2) in D2O containing 150 mm NaCl and 10 % (v/v) deuterated DMSO at 25 °C, and T1s and T1ns experiments for benzyl benzoate were performed in 50 mm potassium phosphate buffer (pH 7.0) in D2O containing 5 % (v/v) deuterated DMSO at 25 °C with a Bruker Avance 600 MHz spectrometer equipped with a cryocooled TXI probe. Selective inversion was performed with a Gaussian-selective pulse (12.5 Hz), whereas nonselective inversion was performed with a rectangular hard pulse (16.7 kHz). 8192 points were recorded for the direct dimension. The number of scans was set to 8, but 64 and 16 scans were used under the 0.1 mm SKF and 0.2 mm SKF conditions, respectively. The repetition delay was 15 s. The inversion recovery delays were set to 0.01, 0.25, 0.5, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0, 5.0, and 6.0 s. The typical experimental time was 1 h. The τc values were calculated from selective (Ts1i) and nonselective (Tns1i) proton spin-lattice relaxation times by assuming a pure dipolar mechanism according to Equations (1)–(4):[ 1)–4):1, 2, 3, –(4):1, 2:1, 2, 3,ijρ and ijσ are the autorelaxation and cross-relaxation terms for a proton pair, respectively; rij,ω, ħ, and γ are the distance between ith and jth protons, the proton Larmor frequency, the reduced Planck constant, and the gyromagnetic ratio, respectively.in which All NMR spectra were processed with the Topspin 2.1 program (Bruker) and were analyzed with Sparky (http://www.cgl.ucsf.edu/home/sparky/).λ=324 and 314 nm, respectively, in 25 mm deuterated Tris-buffer (pH 7.2) in D2O containing 150 mm NaCl and 10 % (v/v) deuterated DMSO at 25 °C. The UV absorbance of I4PTH was measured at λ=316 nm with 50 mm potassium phosphate buffer (pH 7.0) in H2O at 25 °C. Prior to the UV measurements, the samples were centrifuged at 3000 g for 5 min to sediment the compound aggregates.The UV absorbance of the SKF and SB compounds was measured at m potassium phosphate buffer (pH 7.0) at 25 °C. Stocks of inhibitors were prepared in DMSO, and the final concentration of the inhibitors was 0.2, 0.4, and 1.0 mm for I4PTH, clotrimazole, and benzyl benzoate, respectively. No more than 5 % DMSO was present in the assay. The NDSB was directly added to the assay buffer. Chymotrypsin (28 nm) was incubated with the inhibitor for 5 min before the reaction, and the reaction was initiated by the addition of 200 μm photometric substrate, N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide. The progress of the reaction was monitored by the absorbance at λ=405 nm.[Assays were performed in 50 m


Fusarium oxysporum, an important replant disease pathogen in Pseudostellaria heterophylla rhizospheric soil. Moreover, HPLC was used to identify phenolic acids in root exudates then it was further to explore the effects of the phenolic acid allelochemicals on the growth of F. oxysporum f.sp. heterophylla. The amount of F. oxysporum increased significantly in P. heterophylla rhizosphere soil under a consecutive replant system as monitored through qPCR analysis. Furthermore, the growth of F. oxysporum f.sp. heterophylla mycelium was enhanced by root exudates with a maximum increase of 23.8%. In addition, the number of spores increased to a maximum of 12.5-fold. Some phenolic acids promoted the growth of F. oxysporum f.sp. heterophylla mycelium and spore production. Our study revealed that phenolic acids in the root secretion of P. heterophylla increased long with its development, which was closely related to changes in rhizospheric microorganisms. The population of pathogenic microorganisms such as F. oxysporum in the rhizosphere soil of P. heterophylla also sharply increased. Our results on plant-microbe communication will help to better clarify the cause of problems associated with P. heterophylla under consecutive monoculture treatment.In this study, quantitative real-time PCR (qPCR) was used to determine the amount of  P. heterophylla root tubers are commonly used to treat chronic diseases associated with the lung and as a spleen tonicP. heterophylla under consecutive monoculture.PP. heterophylla, but also in many other greenhouse crops, fruits, forages, horticultural and medicinal plants. Several groups of chemicals have been implicated for autotoxicity, including terpenoids, phenolics, steroids, alkaloids and cyanogenic glycosides789Three major causes have been described for consecutive monoculture problems in previous studies: imbalance of soil nutrients, autotoxicity of root exudates and shifts in the microbial community345p-coumaric acid in cucumber root exudates significantly affects soil microbial communities in the rhizosphere and promoted the growth of soil-borne pathogensHowever, many other studies suggested that the autotoxicity of root exudates could shape rhizosphere microbiology by deterring or attracting specific microbial species1113P. heterophylla monoculture system. We hypothesize that some allelopathic compounds may have an indirect detrimental impact on plants, and predispose roots to infection by pathogenic fungi through certain biochemical and physiological interactions. Additionally, root allelochemicals in the rhizosphere have an ecological role in plant-microbe-soil interactionsBased on our field observations, severe fungal diseases, especially root rot and Fusarium wilt, are commonly found in the Fusarium oxysporium has numerous specialized forms, and is considered one of the most important fungal pathogens of crops that causes root rot and wilt diseaseP. heterophylla. Previous studiesP. heterophylla growth and development. Nevertheless, how the specific pathogenic microorganism such as F. oxysporum varies in consecutive monoculture system and how the root exudates affect the soil-borne fungi under in vitro conditions still remain unknown. Therefore, the aim of the study is to quantify the population of the specific pathogen in rhizosphere soils of P. heterophylla with different years of monoculture using qPCR, and consequently to further assess the stimulatory effect of allelochemicals produced by host on the growth of soil-borne fungi and its relationships with consecutive monoculture problems. This paper provides a new insight into understanding the chemoecological process of host–pathogen interactions in rhizosphere, and attempts to explain the increased incidence of root rot and Fusarium wilt disease in the monocultured P. heterophylla.2 = 0.993) (Amplification efficiency E = 10−1/slope = 1.898) for our qPCR analysis. The result of qPCR analysis showed a significant increase in the amount of pathogenic F. oxysporum in the rhizosphere of P. heterophylla as the monoculture years increased , the growth rate reached a plateau at 0.67 μg/mL concentration. Therefore, a single phenolic acid may promote the mycelium growth of F. oxysporum f.sp. heterophylla within a specific concentration range. This result is consistent with the effect of total root exudates on the mycelium growth of F. oxysporum f.sp. heterophylla as observed above. However, for phenolic acids mixture, the promoting rate on mycelium growth increased as the concentration increased, implying that the synergistic effects of phenolic acids might be involved in the mixture.The mycelial growth patterns of m growth . Among tF. oxysporum f.sp. heterophylla spore production at concentrations from 0 to 54 μg/mL (F. oxysporum f.sp. heterophylla at a range of concentrations. Vanillin showed the greatest enhancement (357%), followed by p-hydroxybenzoic acid (314%) and mix-7 (300%).Phenolic acids showed a significant dose-response effect on the capacity of 54 μg/mL . TherefoF. oxysporum f.sp. heterophylla increased dramatically as compared with control ; vanillin, y = 13050x – 14191 (R2 = 0.9991); ferulic acid, y = 12194x + 38266 (R2 = 0.9998).Based on the previous results, standard curves for ferulic acid, F. oxysporum f.sp. heterophylla. We observed different patterns in the usage of the three compounds by F. oxysporum f.sp. heterophylla as compared to the control. After 22 h, p-hydroxybenzoic acid and vanillin were completely consumed and after 26 h ferulic acid was completely consumed in the fermentation media  method to study the rhizospheric microbial composition under consecutive monoculture system of P. heterophylla and we found that the poor growth of P. heterophylla under consecutive monoculture system resulted from an altered microbial community F. oxysporum in the rhizosphere of P. heterophylla significantly increased under a monoculture system, to a level fivefold higher than that of the control  in a consecutive monoculture system.The population of Pseudostellaria heterophylla Demonstration District of Agroecological Institute, Fujian Agriculture and Forestry University, Zherong , Fujian, is known as a geo-authentic production zone, i.e., an optimal production area for P. heterophylla. The rhizosphere soil samples of P. heterophylla were collected from both newly planted and replanted fields at the harvesting time, and the adjacent uncultivated field soil sample was collected as a control. A total of five random plots (15 m2) for each rhizosphere soil sample were used in our trial tests.In this study, the trial field located at the Fusarium oxysporum f.sp. heterophylla was isolated from infected P. heterophylla plants grown in the Pseudostellaria heterophylla Demonstration District, the Agroecological Institute, Fujian Agriculture and Forestry University. The separation frequency was 88.5%, and it only infected P. heterophylla plants but was not able to infect the seedlings of other crops . We isolated the pathogenic fungus from the infected parts of P. heterophylla plants again and verified it to be exactly the same as the original strain (Fusarium oxysporum) based on morphological observation, infection characteristic and ITS sequence analysis. It has been preserved in the Agroecological Institute, Fujian Agriculture and Forestry University, P.R. China.The sequencing information of the pathogenic strains was searched using BLAST and homologous comparison. Specific primers YD1/YD2  were desF. oxysporum, F. graminearum, F. solani, Aspergillus flavus, Bacillus amyloliquefaciens, and the different-year monoculture soils was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and used to test the specificity of the primer sets YD1/YD2 and for subsequent PCR analysis2O. Cycle conditions were 5 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 62.5°C and 20 s at 72°C, followed by 10 min of extension at 72°C and a final hold at 4°C. PCR products were analyzed on an agarose gel.DNA from 2O). The parameters were as follows: 95°C for 5 min (denaturation and hot-start activation), followed by 40 cycles of 95°C for 10 s and 62.5°C for 20 s. After the qPCR ran, melting curve analysis was performed to verify the specificity of the amplified product under the following conditions: 95°C for 15 s, 60°C for 15 s, followed by an increase to 95°C over 10 min and a hold at 95°C for 15 s.The Foh106 fragment was gel-purified, cloned into the PeasyTM-T4 Zero Cloning Kit , and sequenced. The sequenced DNA was reamplified using YD1/YD2 from the plasmid and purified using a TIAN pure Mini Plasmid Kit (TIANGEN BIOTECH (BEIJING) CO., LTD). The concentration of the target DNA (Foh106) was determined using a spectrophotometer , and was then diluted to 5, 4, 3, 2, 1, 0.1, 0.01, and 0.001 ng/μL. qPCR was monitored on a Mastercycler ep realplex (Germany). Standard curve plotting and melting-curve analysis was performed following the qPCR amplification protocol3 lux and the relative humidity was 50% in the growth chamber. All mediums were then collected after the tissue culture plantlets were incubated for one month and two months, respectively. Blank culture medium was used as a control. A total of 200 mL of methyl alcohol (chromatography) was added to each group mediums. After ultrasonic processing and incubating for several minutes, the extracting solution was centrifuged and concentrated using rotary evaporation at 40°C until the final volume was 5 mL, and then dried with N2, weighed and stored at −80°C. Thereafter, half of the exudates were dissolved with sterile distilled water to 100 μg/mL26F. oxysporum f.sp. heterophylla was inoculated onto a 9 cm diameter petri dish and cultivated at 28°C for 5–7 days. F. oxysporum f.sp. heterophylla growth diameter was measured when the control hyphal diameter covered two thirds of the culture dish.Batches of uniform seedlings with healthy roots at the 4-leaf-stage were transferred into sterile robust seedling medium . Each group included three replicates in 30 mL of MS medium and three tissue culture plantlets. The tissue culture plantlets were grown at 25°C under bacteria-free condition. The illumination time was from 6:00 to 18:00, the light intensity was 3.97 ± 0.15 × 10F. oxysporum f.sp. heterophylla spores were flooded with sterilized water and the conidia suspensions were filtered through three layers of gauze to separate conidia from myceliumUnder sterile conditions, P. heterophylla were filtered through a 0.22 µm filter prior to the analysis of the root exudation profiles. The analysis was conducted using an HPLC system  with an ODS - C18 column . The root exudation profiles were determined under the following HPLC conditions, by which eight standard phenolic compounds  could be completely separated. Under these conditions, the mobile phase consisted of methanol (A) and 1% phosphoric acid (B) with a gradient elution of 100–50% A plus 0–50% B at a rate of 0.8 mL/min for 12 min, 50% A plus 50% B at a rate of 0.8 mL/min for 20 min, 50–30% A plus 50–70% B at a rate of 0.8 mL/min for 30 min, 30% A plus 70% B at a rate of 0.8 mL/min for 32 min, and 30–50% A plus 70–50% B at a rate of 0.8 mL/min. The UV detector wavelength was set at 280 nm and the column temperature was maintained at 40°C. The standard compounds were chromatographed alone and in mixtures. Retention times for the standard compounds and the major peaks in the extracts were recorded. The phenolic compounds from each fraction were identified based on their retention times and the addition of standards to the samplesRoot exudates (100 μg/mL) of the monocultured p-hydroxybenzoic acid, vanillin, vanillic acid, syringic acid, salicylic acid and cinnamic acid were made in the same ratio as the results by HPLC analysis and diluted to the same concentrations as single phenolic acid dissolved in the basal medium . After 7 h, 0.1% HgCl2 was added to all the experimental groups to stop spore germination. Then the spore germination rate was calculated. Each treatment had three replicates.Based on our preliminary study, seven standard compounds identified in root exudates were selected and made as mother solution, then diluted to 54, 18, 6, 2 and 0.67 μg/mL in each medium alone, at the same time, the mixtures with ferulic acid, F. oxysporum f.sp. heterophylla (3 × 106 microconidia/mL) and 64 μg/mL of each standard phenolic acid  were added to modified Czapek's medium (with sucrose removed). Basal medium was used as the control. F. oxysporum f.sp. heterophylla was then cultivated at 28°C with 150 rpm rotation. Each group comprised three replicates. The concentration of each phenolic acid was quantified in the liquid fermentation media using HPLC at 4–7 h intervals. Standard compounds of the three concerned phenolic acids  were calibrated with 5, 1, 0.5, and 0.1 μg/mL standard solutions for HPLC chromatography. The standard compounds were injected alone and in mixtures. Retention times for the standard compounds and the major peaks in the medium were recorded. The phenolic compounds from each fraction were identified based on their retention times and the addition of standards to the samples. The HPLC conditions were as following: The column was ODS - C18 , with a mobile phase of methanol and 1% phosphoric acid aqueous solution (30: 70). The UV detector wavelength was set at 280 nm at a rate of 1 mL/min, and the column temperature was maintained at 40°C.A total of 100 μL of Analysis of variance was performed using DPS7.05. The differences in the means were determined by calculation of the least significant difference (LSD) at the 5% level.W.X.L. designed the experiments, and Y.P.Z., Z.F.L. and L.X.C. executed the experiments and wrote the manuscript. W.X.L. improved the manuscript. Y.Q.Y., L.K.W., S.A., Z.X.Z. and C.X.F. helped to polish the language. All authors reviewed the manuscript.Dataset 1