Participants rated liking, saltiness and fat flavor intensity before and after consuming four labeled cheeses. Even though the cheese products were identical, inclusion of health labels influenced consumer perceptions. Cheese with a “light” label had a lower overall expected and perceived liking compared to regular cheese. Although cheese with a “salt reduced” label had a lower expected liking compared to regular cheese, no lower liking was found when consumers actually consumed the labeled cheese. All labels also influenced the perceived intensities of the attributes related to these labels, e.g., for example salt intensity for reduced salt label. While emotional profiles of the labeled cheeses differed before tasting, little differences were found when actual tasting these cheeses. In conclusion, this study shows that health-related labels might influence the perceived flavor and emotional profiles of cheese products.The global increase of cardiovascular diseases is linked to the shift towards unbalanced diets with increasing salt and fat intake. This has led to a growing consumers’ interest in more balanced food products, which explains the growing number of health-related claims on food products . Based on a within-subjects design, consumers ( The frequency of heart disease and hypertension is increasing throughout the world and one of the reasons is a shift towards a more unbalanced diet, which includes a higher salt and fat intake ,2,3. Sali.e., reduced in salt, “light”, etc.). Companies specifically target health-conscious consumers using such labels, which are potentially related to positive health outcomes  in the thoughts of consumers. However, one drawback is that consumers often associate changes in a particular ingredient, like salt reduction, with negative change of flavor. Liem et al. [As a growing group of consumers are becoming more conscious with the health aspects of their diet ,12,13, nm et al. , for exam et al. . While tm et al. ), have sm et al. ,17, it ipriming theory is initially developed in cognitive psychology [et al. [Expectations research has been widely applied in the field of food sensory and consumer research and examines the influence of information cues and expectations of those cues on the evaluation of food products. When one consumes a food or beverage, there may or may not be a disparity between the expected and actual experience. If such discrepancy occurs, a number of different outcomes could occur, as reported in previous literature [halo effect could take place. The halo effect involves cognitive bias when the assessment of one particular characteristic  of an item  strongly affects the perception of other attributes  of the same item [From a theoretical point of view, three concepts are important about the potential influence of labels on the subsequent perception: (1) priming; (2) expectation theory; and (3) halo effect. The ychology ,19 and cychology . In a seychology ,22. As a [et al. . Expectaterature ,24, and terature  containsame item . An examame item  that fouIt is also essential to measure beyond the overall acceptance of food products and obtain a broader perspective of consumers’ food product experience, given the high product failure rates at market introduction ,29. In t® and flash profiling [More recently, a growing number of studies have been carried out where consumers instead of the trained panelists performed sensory profiling of food products ,37. To erofiling . Most nerofiling ,39.The purpose of the this study was twofold: (1) to examine the influence of potential health messages, like “reduced salt content” and “light”, on the expected and perceived sensory evaluation of cheese; and (2) to investigate which emotional conceptualizations consumers associate with such messages.For this study, cheese was taken as a case. Cheese is an important source of dietary calcium, proteins and also vitamins ,41,42. AParticipants were recruited in Ghent area (Belgium) and no information about the aim of the study was provided at recruitment stage. Testing took place in the sensory facilities of Ghent University and consumers completed all the evaluations in sensory booths.In total, 129 consumers participated in this test of which 53.4% were female. The mean participant age was 24.9 years (SD = 9.5), but participants ranged from 18 to 77 years. More than 80% of the participants ate cheese at least 2 to 3 times a week. Subjects were not compensated for their participation in the study.Each participant received two pieces of one cheese at the same time . All cheeses were exactly the same but different information was provided. A 3-digit random number was assigned to each sample and cheese slices were 1.5 cm × 1.5 cm × 1.5 cm . SamplesTo reduce the potential influence of a package , only a label description was included, in line with previous research on soy and organic labels ,46. The The presentation of the four labels were counterbalanced using a Williams design  to avoidet al. [The tests were conducted in the sensory lab of the university. The respondents were told that they were going to evaluate four pieces of young Gouda cheeses and more detailed information about the cheese would follow when the evaluation started. This is comparable to the research of Liem, et al. , which wBefore taking part in the study, potential participants were first required to complete a screening questionnaire in order to assess their suitability for the study. The screening criteria were based on their diet (consuming cheese products), food allergies  and their cheese consumption (at least once a month) ,49.The questionnaire comprised five parts and was computer based using EyeQuestion v 3.12.0 software  .consumption of cheese with questions based upon a focus group discussion, prior research and reviewing the literature. The frequency of consumption was examined using 6 scale labels ranging from “daily” to “once a month”. Next, they indicated which type of cheese they consume followed by choosing their preferred type. Possible options were “hard cheese”, “soft cheese”, “cream cheese”, “light cheese”, “goat cheese”, “blue cheese” or “other”. If they chose “other”, they could specify their answer.The first part examined the shopping behavior, attitudes and awareness of personalsalt and fatintake were asked in the second part of the study. This makes a more detailed classification of the sample possible. Regarding salt labeling, three yes/no questions were asked to assess shopping behavior, based upon previous research [two items reflecting awareness of personal salt intake were included. Participants were asked to which extend they have a diet with a low or high salt intake using a 5-point scale  [Intention to consume less salt in their diet was asked using three possibilities: “no”, “yes, within 6 months” and “yes, within 1 month”. Finally, one question asked if participants thought that they need to have a diet low in salt on most days of the week using a 5-point Likert scale ranging from “strongly disagree” (1) to “strongly agree” (5) to reflect their attitude towards salt consumption. Similar questions were asked about the fat, e.g., “Do you look for the fat content on food products when shopping”, “Does salt content influence your food purchases” and “Do you often buy low-fat labeled food products?” Participants also reported to which extend their diet contains fat using a 5-point scale  [evaluate their fat intakecompared to a men/women of a similar age on a 5-point scale ranging from “much less” (1) to “much more” (5). Intention to consume less fat was questioned using the options “no”, “yes, within 6 months” and “yes, within 1 month”.Questions regarding the participants’ research ,51. Firsn salt”) . To evalin fat”) ,54. Furtexpectations of the salt intensity, fat flavor intensity and desire of the four labeled cheeses were assessed. The labels were given in a random order to avoid order bias and carry-over effects [et al. (2012) and bipolar 7-point scales were used . Recent work also suggests that fat flavor is a basic taste [emotional conceptualizations and sensoryterms that they associate with each cheese. Product specific emotional and sensory terms were determined during preliminary research following a two-step approach suggested by Ng, et al. [et al. [® Wheel, [In the third part, participants’  effects . Therebyic taste ,56. Furt, et al.  and Ares [et al. . First,  [et al. ,61,62,63 [et al. ,64,65,66 [et al. ,58. Sens [et al. . An over [et al. , a rate- [et al. ,68. Part® Wheel, ) using a® Wheel, ,71.i.e., 1 = extremely dislike–7 = extremely like, 1 = not salty at all–7 = extremely salty, 1 = not fatty at all–7 = extremely fatty). Next participants were asked to rate the intensity of the applicable sensory and emotional terms with the following instruction: “Please try cheese sample XXX. Then, tick on each word that applies to describe cheese XXX and rate the intensity. Also, rate the intensity of applicable words which describe how you feel right now.” This instruction was based upon previous work for the sensory [After the participants expressed their expectations during the label only treatment, they received one cheese at the time to perform the perceived treatment. Participants were instructed to consume a first piece of cheese and rate (1) the overall liking; (2) salt flavor intensity and (3) fat flavor intensity of the cheese product using a 7-point bipolar scale .Repeated measures ANOVA and Bonferroni et al [i.e., frequency of selection or weighted frequency of selection (RATA scoring). RATA scorings take the actual points of the scale (ranging from 1 to 5) into account. Next, RATA scores for each emotional and sensory term were calculated by summing up the points. Cochran’s Q test was performed to determine significant differences in the frequency of term selection among the labels in both expected and perceived condition. Friedman’s test was carried out to identify significant differences in RATA scoring between the terms in either the expected or perceived condition. Further, repeated measures ANOVA with Bonferroni correction was performed to examine differences between the quantities of positive/negative emotions between the labels using sums of the frequency of term selection.As suggested by Ares, et al , data obp-value (p ≤ 0.05) have a satisfactory power value above the threshold of 0.80. A 5% significance level (p ≤ 0.05) was considered for all tests, except when stated otherwise.Power analysis was conducted using GPower 3.1   and testMost participants consume cheese several times during a regular week. The participants are fond of hard and soft cheeses while a lot of the participants also consume goat cheeses. Light cheeses are only consumed by around a quarter of the sample. Almost half of the sample prefer hard cheeses, while around 15% of the sample prefer creamy and goat cheeses .Only 3% of the respondents state that they look to the salt content when buying products and a little bit over 2% declares that they often buy food products with a reduced salt content. A high majority (88.4%) of the sample does not see the salt content as a reason not to buy a food product. Given these numbers, unsurprisingly, over 90% of the participants are not planning to consume less salt in their diet. When asking for the consumers’ awareness of their salt intake, ranging from very low in salt to very high in salt, over half of the respondents answer that they have an average salt intake in their diet . If the Roughly one-third of the respondents (36.4%) declare that they have a look at the fat content when buying food products. Just over 51% of the participants declare that fat content can be a reason to not buy a certain food product. In addition, 31.8% of the respondents often buy low-fat products. In total, 30% of the participants are planning to consume more products with a lower fat content in the next six months. Even so, 17% of the respondents state that they are planning to consume fewer products with a lower fat content during the next month. Most participants estimate that their diet is rather average on fat intake and that the total fat intake is comparable with the mean intake of males/females of the same age . Almost p < 0.001), salt intensity  = 101.478, p < 0.001), fat flavor intensity  = 90.889, p < 0.001) and desire  = 42.265, p < 0.001) was found.A significant main effect of labels on the expected liking  = 81.846, p < 0.001), while it did not differ with the “reduced salt” label .p < 0.001) but lower than the “light label” cheese .The expected salt intensity differed significantly among all labels B. The lop < 0.001). The mean expected fat flavor intensity of the “light + reduced salt” label cheese (4.02 ± 1.42) did not differ significantly between those latter two labels, but was significantly higher compared to the “light label” cheese (p < 0.001).The expected fat flavor intensity varied was significantly lower for the “light label” (2.75 ± 1.04) compared to the other three labels C. Particvs. light label 4.14 ± 1.32, p < 0.001; vs. reduced salt label 4.49 ± 1.24, p < 0.001; vs. light + reduced salt label 4.0 ± 1.42, p < 0.001). The mean expected desire for the “reduced salt label” cheese was significantly higher compared to the “light label” (p = 0.003) and “light + reduced salt label” (p < 0.001). No significant main effect of the label was found on the expected desire between the “light label” and the “light + reduced salt label” (p = 1.0).Consumers mainly show a desire for the “control label” cheese, as its expected desire was 5.16 ± 1.03. This expected desire was significantly higher compared to the other three labels (p < 0.001), salt intensity  = 16.655, p < 0.001) and fat flavor intensity  = 21.671, p < 0.001)  = 8.518, < 0.001) .p < 0.001) and “light + reduced salt label” . Consumers tend to like the “reduced salt label” (4.77 ± 1.35) as much as the “control label”, as no significant differences were found in the overall acceptance between both labels (p = 0.248). Further, the mean consumer liking between the “light label” and “light + reduced salt label” cheeses were very similar (p = 1.0) (The highest perceived liking was for the cheese with the “control label” (5.07 ± 1.20), which was significantly higher compared to the “light label”  D.p < 0.001) and “light label” . There was no main effect of the label on the perceived salt intensity between the “reduced salt label” and “light + reduced salt label” . In addition, no significant effect of the labeling on the saltiness perception was found between the “light label” and “light + reduced salt label” (p = 0.072)  compared with when it had another label. If a “reduced salt label” was given, the perceived salt intensity (3.16 ± 1.41) was significantly lower compared to the “control label” (= 0.072) E.Regarding the perceived fat flavor intensity, the labels could be divided in two groups. When the cheese was provided with a “control label” (4.28 ± 1.13) or “reduced salt label” (4.02 ± 1.24), the perceived fattiness was significantly higher compared with the same cheese labeled as “light label” (3.40 ± 1.22) or “light + reduced salt label” (3.53 ± 1.17) F.Q = 1.892, p = 0.595). Consumers also expect differences on the sensory level between the different labeled cheeses as significant differences for eight out of 11 sensory attributes were found. When taking the actual scores into account for the analysis (RATA scoring), the same significant differences were found as when looking at the frequency of use. However, the RATA scoring approach lead to a higher significance level for three terms: “unpleasant surprise” (p < 0.001 vs.p = 0.001), “dry” (p = 0.001 vs.p = 0.004) and “salty” (p < 0.001 vs.p = 0.003).Results related to frequency of use of both emotional and sensory terms are shown in p < 0.001) and negative emotional conceptualizations  = 38.850, p < 0.001) between the labels. Consumers checked significantly more positive emotional terms with the control label (2.1) compared to the other three labels , light label  and light + reduced salt label ). No significant differences in the association of positive emotional terms were found between the three health-related labels. Consumers tend to associate almost no negative emotions to the regular cheese as a mean term selection of only 0.2 emotional terms was found. This was significantly less compared to the three health labels. The light + reduced salt label (1.2) had the highest association with negative emotions, which was significantly more compared to the “reduced salt label”  but did not differ significantly with the “light label” .If the valence of emotion  is taken into account, the labels could be divided into three groups. The results show that the control label tends to be associated more with positive emotions like glad, happy and enthusiastic compared to the other labels. The other labels are also largely associated with negative emotions in both the frequency of use and rata scoring approach. A distinction between the reduced salt label and the two other labels  could further be made. Consumers have a more positive feeling about the reduced salt label compared to the other two labels, as can be seen in Q = 18.290, p < 0.001), “salty”  and “untasty” . Analyzing the data using the RATA scoring approach revealed additional differences in the sensory perception of the evaluated labeled cheese. Consumers perceived differences in the intensity of the “aftertaste” (χ2 (3) = 7.994, p = 0.046) and “yellow” (χ2 (3) = 15.060, p = 0.002) between the four samples of labeled cheese. Regarding the emotional terms, only a significant difference was reported for the emotion “glad” when taken the intensity into account. Regarding the type of emotions associated with the labeled cheeses, no significant differences were found for either positive  = 0.607, p = 0.611) or negative  = 0.976, p = 0.404) emotional terms. An overview of the differences in the emotional and sensory profiles is listed in When consumers actually consumed the labeled cheese, few significant differences were found in the association with emotional and sensory terms . Signifiet al. [This study illustrated that health labels can influence consumers’ flavor expectations of cheese. Several studies have found that health-related information like fat content ,74,75,76et al. .et al. [et al. [When participants consumed the same cheese, significant differences were not only found for the overall liking but also on the level of salt intensity and fat flavor intensity. These results demonstrate that a health halo effect could occurred when providing health-related information and confirm previous research results that also describe a health halo effect ,27,83. Aet al. . Althoug [et al.  did not  [et al.  who foun [et al. . In the  [et al. , so one  [et al. ,86,87,88 [et al. ,90. Whil [et al. .This study has several limitations. First of all, one should note that the used sample is not representative for the Belgian population. However, the use of a convenience sample at the university has been applied in several studies and provides interesting insights. A future study could use a greater sample size in order to obtain more power for the statistical tests. The tests took place in a lab which could bias the results as it does not mimics the reality but it has the major advantage that the experiment could take place in a better controlled environment. It has been previously reported that the context could influence the sensory ,93 and eThe present study holds practical implications for the role of front-of-package labeling. These labels could influence the sensory expectations and perception of related sensory attributes like fat content and salt content. Further, these labels could impact the emotional conceptualization of a food product. However, these effects are label-specific and the use of a reduced salt label did not lower the overall liking of cheese. Therefore, specific health-related labels might be used as a marketing tool in order to target specific health oriented consumers and even yield potential for priming healthy food products.


We thus propose that the first physiological function of the ancestral gill was acid-base regulation, and that the gill was later co-opted for its central role in gas exchange in more derived aquatic vertebrates.The gill is widely accepted to have played a key role in the adaptive radiation of early vertebrates by supplanting the skin as the dominant site of gas exchange. However, in the most basal extant craniates, the hagfishes, gills play only a minor role in gas exchange. In contrast, we found hagfish gills to be associated with a tremendous capacity for acid-base regulation. Indeed, Pacific hagfish exposed acutely to severe sustained hypercarbia tolerated among the most severe blood acidoses ever reported (1.2 pH unit reduction) and subsequently exhibited the greatest degree of acid-base compensation ever observed in an aquatic chordate. This was accomplished through an unprecedented increase in plasma [HCO Instead, 80–90% of oxygen uptake in hagfishes occurs across the skin at rest10MO2) of any craniate at rest or following severe stress1214151718via the gills through use of a divided chamber systemAs osmo-ionoconformers, hagfish gills are not involved in ionoregulation for the purposes of ion  and hypercarbia , both of which can severely disrupt acid-base status. Indeed, Pacific hagfish, Eptatretus stoutii, are supremely tolerant of hypoxia and anoxia2 is an environmentally relevant challenge, hypercarbia exposure can also be used as a tool to quantify acid-base regulatory capacity. Here we exposed hagfish to sustained hypercarbia to induce a rapid acidosis. Subsequent recovery was used to quantify the rate and degree of blood acid-base compensation for direct comparison with other aquatic craniates, which has not been previously investigated.Acid-base regulation is one of the most tightly regulated physiological processes in animals2 of 10, 30 or 50 mm Hg  for up to 96 h, to simulate the most extreme conditions that hagfish could possibly experience in a benthic burrow, within a decaying carcass, in proximity to deep sea hydrothermal vents2 injection sites2, respectively  values from the 3 h value at the respective CO2 tension].Pacific hagfish were acutely exposed to a water pCOectively . Tissue ectively , but to ectively  relativeectively . Despite3−]  and 75.4 (±8.2) mM during exposure to 30 and 50 mm Hg pCO2 , indicating that the exceptional hypercarbia tolerance of hagfish is not necessarily an agnathan trait.We believe that the hagfish’s tremendous upper limit for blood acid-base compensation may be associated with its’ osmo-ionoconforming strategy and consequent high plasma  and instead, preferentially and completely regulate tissue pHi29−] within the actinopterygiians  were exposed to seawater equilibrated with approximately 10, 30 and 50 mm Hg pCO2 and then sampled either a) immediately after transfer, time 0, or b) after 3, 6, 12, 24, 48 (only 30 and 50 mm Hg pCO2) or 96 (only 50 mm Hg pCO2) h of exposure to elevated pCO2. Blood was obtained from anaesthetized animals for pH, hematocrit, haemoglobin, and mean cell haemoglobin concentration (MCHC) as previously described2 (TCO2), plasma ion composition, and RBC pHi were also measured3−] ΔpH−1, and then expressed in mmol HCO3− pH−1 l−1 of blood or kg−1 of intracellular tissue water, over an in vivo relevant pH range. Data are presented as mean ± SEM . All data was analyzed for normality and equal variance before statistical analysis. Statistical differences were detected using a one-way ANOVA and, when necessary, a post-hoc Dunnett’s test. All statistical analyses were conducted using SigmaStat for Windows 3.5.0.54 , and all analyses were 2-tailed and interpreted using α = 0.05 to determine statistical significance.Pacific hagfish  values as has been done previously for other aquatic species2938394041424344454647For comparison with other species, an estimate of the increase in whole animal net acid excretion rates in hagfish exposed to hypercarbia was calculated  as the iHow to cite this article: Baker, D. W. et al. Hagfish: Champions of CO2 tolerance question the origins of vertebrate gill function. Sci. Rep. 5, 11182; doi: 10.1038/srep11182 (2015).


Several studies have assessed the associations between adult migraine and underweight, pre-obesity or obesity. Prevalence, frequency, and severity of migraine appear to increase in relation to the body mass index, although this evidence is not supported by all the studies examined. The link between body weight and headache has hardly been examined in children. Data on the possible association between the body mass index (BMI) and the psychological profile in migraine children are sparse.Aims of the present study were: 1) to study the prevalence of pre-obesity and obesity in migraineur children/adolescents; 2) to analyze the possible relationship between frequency and severity of migraine and overweight; 3) to explore the role of anxiety and somatization on BMI in migraine patients.We studied 92 migraineurs . Patients were divided into 2 groups according to headache attack frequency: 1) high frequency (HF) patients, having from weekly to daily episodes, and 2) low frequency (LF) patients, showing ≤ 3 episodes per month. Pain intensity was rated on a 3-level graduated scale . Given the low frequencies, the “moderate” and the “mild” intensity were collapsed into the same category. The psychological profile was assessed by SAFA Anxiety and Somatization scales. BMI was calculated as the weight in kilograms divided by the height in meters squared.2) = 0.6853, p = 0.41; χ2 = 0.0058; p = 0.94). Compared to normal weight children, overweight patients showed a significant higher score in “Separation anxiety” subscale. In the “overweight” patients, the BMI showed a positive and significant correlation with Anxiety  and with Somatization .Among our patients, fifty-seven (62.0%) were classified as “normal weight”, 15.2% were obese and 17.4% pre-obese (both collapsed into the “overweight” group). Due to their low frequencies, “underweight” children/adolescents (5.4%) were eliminated from our subsequent analysis. The weight  did not correlate with migraine frequency and intensity .


CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3.0 at http://www.bioconductor.org.CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed  The clustered, regularly interspaced, short palindromic repeats (CRISPR) found in many prokaryotes encode RNAs that act together with CRISPR-associated proteins (Cas) and a tracRNA to function as an adaptive immune system to detect and cleave foreign DNA sequences S. pyogeneshttp://crispr.mit.edugRNA-Cas9 complexes recognize specific target sequences composed of two components . The guiCRISPRseek, to facilitate design of target-specific gRNAs for any CRISPR-Cas9 system with a characterized PAM sequence and off-target analysis with any available genome sequence using experimentally-derived off-target scoring schemes To facilitate the use of different classes of CRISPR-Cas9 systems by a wide range of investigators, there is a need for platform-independent and genome-independent tools to identify gRNAs that cleave single or paired target sites for various CRISPR-Cas9 systems and to perform off-target analysis. Two previously described tools can be run locally with different genomes CRISPRseek implements a common gRNA design and off-target analysis workflow for CRISPR-Cas9-derived systems in R, a system for statistical computation and graphics CRISPRseek utilizes the Biostrings package to represent sequences and find matches. To efficiently determine whether off-target sequences overlap with any exon, GenomicRanges is used to represent genomic coordinates. BSgenome is employed to retrieve flanking sequences of off-targets. CRISPRseek leverages various sources of genomes as BSgenome object and annotations such as TransciptDb object in Bioconductor. An experimentally-determined position specific mismatch penalty matrix and scoring scheme were used for predicting cleavage score for potential off-targets CRISPRseek emphasizes flexibility and ease of use. gRNA identification and filtering, and off-target searching, scoring, and annotation are integrated into one workflow function offtargetAnalysis. Once the package is loaded and parameters are set, one line of code  can perform all the analysis by calling various helper functions. A second workflow function compare2sequences is for identifying gRNAs that target one of two related sequences or both sequences.CRISPRseek are described in the Supplemental method file and in the Vignettes and Manual provided with the R package (available at Bioconductor.org).An installation guide and additional generic use cases for CRISPRseek identifies and evaluates candidate gRNAs for CRISPR-Cas9-derived nucleases to target a given input sequence using various constraints and also reports and ranks potential off-target sequences for each gRNAs. The offTargetAnalysis function can be used to perform customized searches for gRNAs to target a given sequence and for off-target sites in a specified genome , findgRNAsWithREcutOnly = TRUE, REpatternFile, minREpatternSize = 6, overlap.gRNA.positions = c, findPairedgRNAOnly = TRUE, min.gap = 0, max.gap = 20, gRNA.name.prefix = “gRNA”, PAM.size = 3, gRNA.size = 20, PAM = “NGG”, BSgenomeName, chromToSearch = “all”, max.mismatch = 4, PAM.pattern = “N[A|G]G$”, gRNA.pattern = “”,min.score = 0.5, topN = 100, topN.OfftargetTotalScore = 10, annotateExon = TRUE,txdb, outputDir, fetchSequence = TRUE, upstream = 200, downstream = 200,weights = c, overwrite = FALSE).findgRNAs command set to FALSE. The default constraints for a target site search is set for sites recognized by the widely used Cas9 nuclease from S. pyogenes, composed of a 20 base guide sequence and a 3 base preferred PAM sequence (NGG). The PAM length and sequence can be altered to use the preferred sequences for Cas9 systems from other species PAM and PAM.size commands. The gRNA.size command can be altered to adjust the identified guide sequence length for Cas9 systems from other species that utilize different gRNA lengths or for truncated gRNAs gRNA.pattern to require or exclude specific features within the target site. For example, synthesis of gRNAs in vivo from host U6 promoters is more efficient if the first base is guanine and gRNA synthesis in vitro using T7 promoters is most efficient when the first two bases are GG gRNA.pattern = “∧G” and “∧GG” respectively .)+$”. In addition, some studies have identified sequence features that broadly correlate with lower nuclease cleavage activity, such as uracil in the last 4 positions of the guide sequence gRNA.pattern = “[ACG]{4,}.{3}$”.Target sites are composed of two components, the guide sequence, which is determined by the variable region of the gRNA, and the PAM sequence, which is recognized by the Cas9 protein . If a spectively . AnotherCRISPRseek can also filter candidate target sites based on other features. Appropriately orientated and spaced pairs of target sites can be used with nickase-derivatives of CRISPR-Cas9 systems to increase cleavage specificity S. pyogenes, it is possible that other spacing constraints is required for CRISPR-Cas9 systems from other species or for novel dimeric nucleases such as CRISPR-RNA guided FokI nucleases findPairedgRNAOnly = TRUE restricts the output to paired sites while the min.gap and max.gap commands allows alternative spacings to be set for paired target sites. An additional feature that can be useful for monitoring nuclease cleavage events is the presence of RE cut sites that overlap the gRNA-Cas9 cleavage site; The findgRNAsWithREcutOnly, REpatternFile, minREpatternSize, overlap.gRNA.positions can be adjusted to require that the Cas9 cleavage site overlaps with RE sites with a particular length recognition site. A list of enzymes and their recognition sites from a commercial supplier is provided with the package.CRISPRseek takes advantage of the Bioconductor BSgenome package, which provides a large number of pre-formated genomes for well-studied organisms and tools to format additional genome sequences. offTargetAnalysis will search all chromosomes of the specified genome in the default setting of chromToSearch . It can be adjusted to skip the off-target search step (“”) or to search individual chromosomes if, for example, a specific gene cluster is of particular interest (“chr1”). The default search of genomic sequences for potential off-target sites allows maximum of 4 mismatches, which can be modified by users with the max.mismatch parameter. If desired, the PAM sequence for off-target searches set by the PAM.pattern parameter can be different from the PAM sequence used for target identification to allow variants of PAM sequences to be scanned as potential off-target sites. For example, Cas9 from S. pyogenes recognizes NGG PAM sequences and, to a lesser extent, NAG PAM = “NGG” for candidate gRNA identification while setting PAM.pattern = “N[A|G]G” for potential off-target finding.An additional criteria for selecting gRNAs is to minimize the likelihood that other sites in the host genome are cleaved by the nuclease . CRISPRsweights parameter can be readily adjusted to incorporate alternative matrices for S. pyogenes Cas9 or different penalty matrices determined for either Cas9 from other species or for gRNAs with truncated guide sequences Prediction of relative off-target cleavage rates is based on previously described approach in which an experimentally-determined penalty matrix is used to score the relative effect of single and multiple mismatches in the off-target sequence Summary.xls (target sequence summaries), REcutDetails.xls (RE cut sites overlapping the nuclease cut site), and pairedSpacers.xls , and Off-targetAnalysis.xls (off-target sequences and scores). Recovered gRNAs are also reported in genbank and fasta formats to readily allow further analysis or visualization with other software.Off-target analysis results are reported as four tab-delimited files: compare2Sequences function facilitates the identification of gRNAs of CRISPR-Cas9-derived systems that target either one or both input sequences , findPairedgRNAOnly = FALSE,min.gap = 0, max.gap = 20, gRNA.name.prefix = “gRNA”, PAM.size = 3,gRNA.size = 20, PAM = “NGG”, PAM.pattern = “N[A|G]G$”, outputDir,weights = c, overwrite = FALSE).offTargetAnalysis function, except that two input sequences are provided and no genome-wide off-target analysis is performed. Instead, the predicted cleavage scores are calculated for target sites in both sequences. Sites with the desired properties (either present in both or specific to one) can then be further characterized via the offTargetAnalysis command.These settings are largely similar to those for the CRISPRseek to select gRNAs that target allele-specific sites in the human Huntington’s disease gene (HTT). Expansion of CAG repeats in one copy of HTT can result in adult onset neurodegeneration. Because HTT is an essential gene, nucleases cannot be used that inactivate both alleles HTT. Two sequences that differ only at the polymorphism site were used as inputs for compare2sequences (http://en.wikipedia.org/wiki/Ape) and gRNA positions were visualized using the Graphic Display function. For each target site, a cleavage score was predicted for each input sequence where the maximum cleavage score is 100 Click here for additional data file.File S2Fasta file with sequence for T allele of human single nucleotide polymorphism RS362331.(FA)Click here for additional data file.File S3compare2sequences function of CRISPRseek using Tab delimited file outputs from (XLSX)Click here for additional data file.File S4compare2sequences function of CRISPRseek using Genbank formated files with gRNA identified by (GBK)Click here for additional data file.File S5compare2sequences function of CRISPRseek using Genbank formated files with gRNA identified by (GBK)Click here for additional data file.File S6compare2sequences function of CRISPRseek using Fasta formatted file of gRNAs identified for the C allele by (FA)Click here for additional data file.File S7R commands used to identify gRNAs that target the C allele of a SNP from the Huntington’s Disease locus.(DOCX)Click here for additional data file.File S8A basic introduction to installing Bioconductor, CRISPRseek, and different data packages required for gRNA identification by CRISPRseek.(DOCX)Click here for additional data file.


Staphylococcus aureus (VISA) derived from the same parent MRSA that have changes in cell wall composition and decreased autolysis. A variety of mutations were found in the VISA, with more in strain 13136p−m+V20 (vancomycin MIC = 16 µg/mL) than strain 13136p−m+V5 (MIC = 8 µg/mL). Most of the mutations have not previously been associated with the VISA phenotype; some were associated with cell wall metabolism and many with stress responses, notably relating to DNA damage. The genomes and transcriptomes of the two VISA support the importance of gene expression regulation to the VISA phenotype. Similarities in overall transcriptomic and metabolomic data indicated that the VISA physiologic state includes elements of the stringent response, such as downregulation of protein and nucleotide synthesis, the pentose phosphate pathway and nutrient transport systems. Gene expression for secreted virulence determinants was generally downregulated, but was more variable for surface-associated virulence determinants, although capsule formation was clearly inhibited. The importance of activated stress response elements could be seen across all three analyses, as in the accumulation of osmoprotectant metabolites such as proline and glutamate. Concentrations of potential cell wall precursor amino acids and glucosamine were increased in the VISA strains. Polyamines were decreased in the VISA, which may facilitate the accrual of mutations. Overall, the studies confirm the wide variability in mutations and gene expression patterns that can lead to the VISA phenotype.Complete genome comparisons, transcriptomic and metabolomic studies were performed on two laboratory-selected, well-characterized vancomycin-intermediate  Staphylococcus aureus showing decreased susceptibility to vancomycin, minimum inhibitory concentration (MIC) 8 μg/mL, appeared in 1997 [S. aureus (VISA), and these have been reviewed [S. aureus to cell wall-active antibiotics induces the expression of a set of genes that comprises a cell wall stress stimulon [The first report of a clinical isolate of reviewed ,3,4. Desreviewed ,3,4. Expstimulon . Alteredstimulon .et al. [in vitro step selection from vancomycin-susceptible S. aureus (VSSA) in the presence of increasing concentrations of vancomycin. Among these VSSA was heterogeneously methicillin-resistant S. aureus (MRSA) strain 13136p−m+ . This strain was originally isolated in 1960 as MRSA 13136, one of the handful of MRSA among several thousand clinical S. aureus isolates surveyed [−m+ is a penicillinase-negative derivative selected from 13136 [geh). The result is a truncated, inactive gene product (lipase) [In 2000, Pfeltz et al.  reportedsurveyed . Strain (lipase) . L54a, a(lipase) . Althoug(lipase) ,12.−m+ was the parent strain from which VISA 13136p−m+V5 was derived, and subsequently VISA 13136p−m+V20 was then derived from 13136p−m+V5. The MICs of the VISA strains were 8 and 16 μg/mL for 13136p−m+V5 and 13136p−m+V20, respectively [−m+V5 was isolated after two selection cycles in one week and strain 13136p−m+V20 after a total of five cycles of selection across five weeks. Each selection cycle consisted of growth in liquid medium that was inoculated onto solid medium containing the same concentration of vancomycin. The 64 µg/mL minimum bactericidal concentration (MBC) of strain 13136p−m+V20 was significantly higher than that of 13136p−m+V5 (16 µg/mL) or the parent strain (8 µg/mL). These strains have been extensively studied for their cell wall-related properties [−m+V20 in particular, as judged by cell wall phosphorus content. The VISA strains showed decreased whole cell autolysis, decreased lysostaphin susceptibility and decreased isolated crude cell wall autolytic activity. Differences were noted in the amount and profile of peptidoglycan hydrolase activities in the VISA strains [atl, the gene encoding two major S. aureus autolysins [−m+V20 compared to 13136p−m+.MRSA 13136pectively . Strain operties ,13. The operties . Muropeptolysins , showed We have used these strains to further probe the mechanism(s) of decreased vancomycin susceptibility by a combination of genomic, transcriptomic and metabolomic approaches. Metabolomics typically involves the comparative analysis of small, cellular organic compounds of a molecular weight less than 1000 daltons . A metab−m+V5 and 16 SNPs across nine genes in 13136p−m+V20, relative to VSSA parent strain 13136p−m+. Although the MIC of 13136p−m+V20 was only double that of 13136p−m+V5, strain 13136p−m+V20 was subjected to three more selection cycles than 13136p−m+V5, and this was reflected in the higher number of SNPs. Six of the nine 13136p−m+V5 SNPs were also present in 13136p−m+V20, but three were not.The mutations present in the two VISA strains are summarized in comGB, fusA, infA, ssb1 and dinG [pepF encodes an endopeptidase that hydrolyzes internalized peptides to provide nutrients or endogenous peptides for protein turnover [glpF encodes a glycerol transporter (carbon and energy acquisition) [apt encodes an enzyme in a purine salvage pathway catalyzing the production of AMP from adenine and phosphoribosyl pyrophosphate [apt is associated with the stringent response in other Gram-positive bacteria [fmtB and tarO gene products are cell envelope proteins involved in cell wall biosynthesis, β-lactam resistance and virulence [fmtB is located downstream of glmM and is also known as mrp and sasB [tarO, also known as tagO, llm and farO, catalyzes the initial step of cell wall teichoic acid synthesis. Locus SACOL2451 encodes a putative membrane protein, a homolog of OpuBC, the permease component of an ABC-type osmoprotectant transport system [A total of 12 genes were associated with 19 protein amino acid changes in one or both VISA . Five ofand dinG ,27,28,30isition) ; and apthosphate ,31. apt bacteria ,33,34,35irulence ,29. fmtBand sasB . FmtB haand sasB . The indand sasB . tarO, at system .stp1, encodes a eukaryotic-like serine-threonine phosphatase and is located in tandem with its cognate serine-threonine kinase gene, stk1 [stp1 have previously been associated with laboratory-selected glycopeptide resistance, along with cell wall alterations characteristic of the VISA phenotype [et al. [stp1 mutation. Additionally, Cameron et al. [Mycobacterium tuberculosis that leads to an adaptive response involving modifications to cell wall structure and virulence factor production [pepF, can be linked to stress response either by expression change or gene product function.The final mutated gene, ne, stk1 . These tne, stk1 , in addine, stk1 ,40. Mutahenotype ,42,43. Poduction . Eleven −m+V5 and 13136p−m+V20 relative to VSSA progenitor strain 13136p−m+ are listed in versus VSSA 13136p−m+ were downregulated as were upregulated. Unfortunately, but not unexpectedly, the gene functional group with the largest number of changes was hypothetical functions, and along with the unclassified and unknown function categories accounted for 91 of the 338 genes (27%) with expression changes in VISA strains. Among genes with assigned functions, seven functional groups each had at least 5% of the genes each with expression changes in VISA vs. VSSA 13136p−m+: cell envelope , transport and binding proteins , purines, pyrimidines, nucleosides and nucleotides , protein synthesis , regulatory functions , and biosynthesis of cofactors, prosthetic groups and carriers (18 genes) and central intermediary metabolism (16 genes) with 5% each. Together these eight functional groups accounted for 86% of the genes represented in Gene expression differences in VISA 13136p−m+V5 and 13136p−m+V20. Approximately the same number of genes were upregulated in each VISA strain, 83 in VISA13136p−m+V5 and 80 13136p−m+V20. Of these, 53%–55% of those upregulated in one VISA were also upregulated in the other VISA strain. Approximately the same number of genes were also downregulated in each VISA strain, 187 in VISA13136p−m+V5 and 177 in 13136p−m+V20. Expression concordance was greater for underexpression patterns, as 70%–73% of genes downregulated in one VISA were also downregulated in the other VISA strain. As shown in −m+V5 had noticeably more expression changes than 13136p−m+V20 among the cell envelope (41 vs. 29) and transport and binding protein (11 vs. 20) genes and fewer than 13136p−m+V20 among protein fate (two vs. six) and protein synthesis (11 vs. 20) genes, with downregulated genes outnumbering upregulated genes at least 2:1 for each VISA and these functional groups, except the 1:1 ratio for the two 13136p−m+V5 protein fate expression changes. Total numbers of regulatory function gene expression changes were comparable between the two VISA, at 19 for VISA13136p−m+V5 and 17 for 13136p−m+V20, but the ratio of the number of upregulated to downregulated genes varied between the strains at 4:15 and 6:11, respectively.versus their progenitor VSSA, specific patterns within these data were not consistent between the two comparisons. For example, 13 of the 16 ribosomal proteins listed in −m+V20, with three unchanged, compared to 13136p−m+. In contrast, four were downregulated and 12 unchanged for the 13136p−m+V5 to 13136p−m+ comparison. However, for the expression of 18 pur and pyr genes associated with central intermediary biosynthetic pathways, 12 were downregulated and six unchanged in 13136p−m+V5, and 15 were downregulated and three unchanged in 13136p−m+V20. For either ribosomal protein or pur and pyr genes, when expression was unchanged in one VISA, it was always downregulated in the other VISA versus 13136p−m+. Genes encoding secreted proteases were generally downregulated in both VISA , as were cap genes encoding capsular polysaccharide biosynthesis, but the pattern of expression changes was inconsistent between the two VISA. The expression of genes encoding cell surface protein, regulatory, biofilm-related and cell wall metabolism gene products was more variable, with more frequent upregulation and greater ranges of expression fold-change values. A large number of genes previously identified as associated with the VISA strain, including members of the cell wall stress stimulon, showed expression changes in one or both VISA versus 13136p−m+. These include vraR and prsA, which are overexpressed in both VISA and are part of the cell wall stress stimulon [et al. [Although overall gene expression trends were similar between the two VISA cmk, agrA and ddh were shown to be in good agreement with microarray results (data not shown).Three genes, which were altered in expression, as determined by microarray analysis, were validated using qRT-PCR. The expression ratios of −m+V20 and 13136p−m+V5. A total of 60 changes ≥8-fold were present between the three strains across 52 different genes. The hypothetical/ unclassified/unknown function gene functional group accounted for 21 (40%) of the 52 genes with ≥8-fold changes, followed by 11 cell envelope genes (21%), four genes each (8%) in regulatory functions and biosynthesis of cofactors, prosthetic groups and carriers and three genes each (6%) for purines, pyrimidines, nucleosides, nucleotides and transport and binding proteins; together, these groups accounted for 89% of the ≥8-fold change gene subset. This pattern differed from that in agrA, sarS, rot, mecR1 and locus SACOL2147) all had very different expression patterns between the two VISA versus parent VSSA3136p−m+. The only two genes with ≥8-fold expression changes between both VISA and the parent VSSA and between the two VISA themselves were locus SAR2275, which encodes a hypothetical protein, and asp23, whose gene product is the σB-regulated alkaline shock protein 23. This gene is downregulated by cell wall-active antibiotics and often upregulated during the general stress response and many specific stress responses, but its function is not known [Five genes in the ≥8-fold change subset that encode regulatory proteins (Genes with ≥8-fold expression changes were grouped into four VISA-related categories in −m+V5 versus VSSA 13136p−m+ comparison. These figures were six and 20 (23% and 77%) and 10 and 13 (43% and 57%) for the 13136p−m+V20 versus VSSA 13136p−m+ and 13136p−m+V20 versus 13136p−m+V5 comparisons, respectively.Ten of the ≥8-fold expression changes were upregulations (29%), and 24 were downregulations (71%) for the 13136p−m+ and its two VISA derivatives, 13136p−m+V5 and 13136p−m+V20. Twenty-four were in the amines and polyamines metabolite class, 25 were amino acids, 26 were polar organic acids and the remaining 23 were sugars. The concentrations of 43 metabolites were increased and 42 were decreased in 13136p−m+V5 cells compared to the parent strain 13136p−m+ (12 were unchanged). In 13136p−m+V20 cells, the concentrations of 47 metabolites were increased and 48 were decreased compared to their concentrations in the parent strain 13136p−m+ (only two were unchanged). Thirteen metabolites were increased in both VISA and twelve others were decreased in both VISA compared to the parent VSSA. The relative concentrations of the identified metabolites in the three strains are shown in versus parent VSSA relative concentrations, as well as fold-changes for 13136p−m+V20 compared to its immediate passage-selection predecessor, 13136p−m+V5. In 13136p−m+V20 cells, the concentrations of 46 metabolites were increased, and 48 were decreased compared to their concentrations in predecessor 13136p−m+V5 13136p−m+ (four relative concentrations were unchanged).Ninety-eight total metabolites were identified in VSSA 13136p−m+V5 in comparison to the parent VSSA, 13136p−m+V20 in comparison to the parent VSSA, and 13136p−m+V20 in comparison to 13136p−m+V5. In general, a decrease in sugars was evident in strain 13136p−m+V20 versus its VISA predecessor 13136p−m+V5. Relative concentrations of 10 sugars increased and 12 decreased (one unchanged) in 13136p−m+V5 versus the parent VSSA, but the tallies were only five increased and 18 decreased for the 13136p−m+V20 versus parent VSSA and five increased and 16 decreased in 13136p−m+V20 in comparison to 13136p−m+V5. Most of the increased relative concentrations of sugars in 13136p−m+V20 in comparison to 13136p−m+V5 were still below those in VSSA 13136p−m+. In contrast, the relative concentrations of 15 amino acids increased and eight decreased in 13136p−m+V5 in comparison to 13136p−m+, 11 increased and 13 decreased in 13136p−m+V20 in comparison to 13136p−m+, and 12 increased and 11 decreased in 13136p−m+V20 in comparison to 13136p−m+V5.versus the parent VSSA and VISA 13136p−m+V20 vs. VSSA 3136p−m+V5). For clarity, only metabolite relative concentration changes of five-fold or greater are presented. Forty five of the 98 metabolites identified had at least one relative concentration change of five-fold or greater among the three strain comparisons. By metabolite class, these were: 13 amines and polyamines, eight amino acids, nine polar organic acids and 15 sugars. These 45 metabolites, their relative concentrations per strain, and relative concentration fold-changes for all three strain comparisons are listed in −m+V5 and the accumulation of AMP, N-acetylglutamic acid, pantothenate and phenylpyruvic acid in 13136p−m+V20. Polyamine relative concentrations were sequentially diminished as greater vancomycin tolerance accrued over the course of VISA selection. Initially, the spermidine relative concentration was reduced 9.4-fold by the selection of 13136p−m+V5 from VSSA 13136p−m+, while putrescine’s was unchanged; then the selection of 13136p−m+V20 from 13136p−m+V5 led to a 5.4-fold reduction in putrescine that was accompanied by a further, small reduction in spermidine. Several sugars were sequentially diminished in a similar fashion, notably glucose-1-phosphate, glucose-6-phosphate, and fructose.A number of metabolites were only detected in one or two of the three strains employed in this study. Particularly striking were the absence of guanosine from 13136p−m+V5 and 13136p−m+V20 compared to the parent strain. Aspartic acid was increased 2.3- and 2.4-fold, respectively, in the two VISA strains, glutamic acid 14- and 15-fold, leucine 2.8- and 3.8-fold and proline 14- and 22-fold.The amino acids consistently present in the highest concentrations were alanine, aspartic acid, glutamic acid, leucine, lysine, proline and pyroglutamic acid. In general, the concentrations of these amino acids were increased in strains 13136pS. aureus.There were high concentrations at a similar level in all three strains of pyroglutamate, which is the cyclic lactam of glutamic acid . AlthougS. aureus. Lactic acid was present at high levels in all three strains.Glycerol was present at similar high levels in all three strains. Alpha glycerophosphate was present in all three strains. Mannitol relative concentration was significantly decreased in the VISA strains. Acid production from mannitol is a species characteristic of S. aureus cell to serve as substrates for central intermediary metabolic pathways. Metabolic pathways are modulated to minimize damage from host immune response oxidative and nitrosative assaults [S. aureus is density-dependent and regulated by a complicated network of global regulatory loci, but also by stress-related adjustments of central metabolic pathways, including the tricarboxylic acid (TCA) cycle. Some TCA cycle enzymes are upregulated in biofilm-associated cells versus planktonic culture [B and members of the staphylococcal accessory regulator (Sar) family, exert regulatory influence over not only virulence factors, but the S. aureus general stress response and expression of antimicrobial resistances, including vancomycin intermediate susceptibility [It has become apparent that the stress response network and virulence are intrinsically interwoven with central intermediary metabolism. assaults . Cell su culture ,63. Manytibility .S. aureus starvation response involves expression changes to genes associated with DNA repair and oxidative stress resistance [rpoB mutations associated with conversion from VSSA to VISA also impart characteristics of the stringent response, reduced expression of genes encoding ribosomal proteins and upregulation of genes encoding amino acid biosynthesis enzymes, in addition to a slowed growth rate [rpoB and rpoC were downregulated 3.1- and 2.1-fold in 13136p−m+V20, respectively, but expression was unchanged in 13136p−m+V5. The stringent response is activated in MRSA in response to β-lactam antibiotics and leads to intermediary metabolic pathways optimized for energy production [S. aureus and other bacteria [mecA gene in VISA 13136p−m+V5 (48.5-fold vs. parent VSSA or 13136p−m+ V20), the oxacillin MIC of 13136p−m+V5 was comparable to those of the other two strains. Expression of two other genes encoding penicillin-binding proteins changed only slightly, pbp2 down 2.3-fold in 13136p−m+V20 and pbp3 down 2.5-fold in13136p−m+V5, despite the reported importance of PBP2 to laboratory-selected VISA [The alarmone-mediated nutritional stress response, known as the stringent response, has been linked to activated stress responses and methicillin resistance . The S. sistance . rpoB muwth rate . RNA poloduction ,68. Strioduction . Additiobacteria . Despiteted VISA .et al. [walKR, vraRS and graRS, and the gene encoding RNA polymerase subunit (rpoB) have been most frequently associated with the VISA phenotype. WalKR is an essential two-component system involved in the control of cell wall metabolism, particularly in controlling the expression of peptidoglycan hydrolases [stp1, fmtB and tarO, which are all involved in cell wall metabolism. Matsuo et al. [Multiple studies have identified genes associated with decreased susceptibility to vancomycin. These studies have been summarized by Howden et al. ,4, who hdrolases . VraSR cdrolases ,73. GraSdrolases  and moredrolases . We did o et al.  have ide−m+ VISA mutations are in genes encoding products that interact with nucleic acids in protein synthesis or are associated with DNA damage/SOS responses: comGB, fusA, infA, ssb1 and dinG. Often, a single stress will activate components from multiple stress responses. Oxidative stress and heat shock are known to elicit very broad stress responses [Many of the mutations we encountered in our VISA strains have either not been or have only been rarely associated with the VISA phenotype. Five of the 12 13136pesponses . Howeverssb1 is intriguing. Single-stranded DNA binding proteins of L54a and similar prophages are thought to function as positive transcriptional regulators of integrase and perhaps other recombination-related phage genes [agr system [ssb1), interpreted as a reinforcement of the lysogenic state, because stress is known to activate the SOS system and thereby the entry of the prophage into the lytic cycle [−m+V5 and additional mutations accumulated during the selection of increased resistance in VISA 13136p−m+V20, and that there were no mutations in other L54a genes such as those encoding the excisionase, suggests that ssb1 may have a regulatory function that was the target of VISA selection. It seems unlikely that so many mutations in one gene would be necessary to simply disable lytic cycle capabilities. Likewise, mutations in fusA, infA and stp1 may serve to modulate regulatory functions. Competence damage-inducible protein CinA, with functional similarities to both the comGB and dinG gene products, was identified as a member of the cell wall stress stimulon by Utaida et al. [comGB, L54a itself cannot induce competence as a prophage [That seven of the nineteen mutant loci identified in this study were in L54a prophage gene ge genes ,77. Propr system , and casr system ,80. Cellic cycle . The facprophage .et al. [−m+V20 (6:11) than 13136p−m+V5 (4:15) supports this observation. This is also true for the stp1 SNP reversion when VISA 13136p−m+V20 was selected from VISA 13136p−m+V5. Several regulatory genes showed modest downregulation in 13136p−m+V5: arlSR, glnR, pyrR, rsbU, agrA, agrB, agrC2 (but not agrD), icaR, sarA, sarV, rpoF (encodes stress response RNA polymerase sigma factor σB) and rsbV (encodes an anti-anti-sigma factor). However, saeS, sarR and vraR showed modest upregulation in 13136p−m+V5, and the downregulation of both rpoF and rsbV together was unusual in that this would have opposite effects relative to σB-mediated regulation. In 13136p−m+V20, sarA homologs sarS and rot (repressor of toxins) were highly upregulated at 8.9- and 12-fold versus parent VSSA, respectively. agrA, agrB, agrC2 and agrD genes, as well as saeS, sarA and rsbW were downregulated in 13136p−m+V20, while arlSR, glnR, pyrR, rpoF, rsbV, sarR, sarV and vraR had no expression change versus the progenitor 13136p−m+. saeS is the sensor histidine kinase gene of a two-component regulation system that is upregulated by β-lactams and vancomycin in some other strains [sarA is important for intrinsic antimicrobial resistance [cap genes have been found to be overexpressed in VISA strains [Matsuo et al.  describe strains , and sarsistance . The com36p−m+V5 :15 suppo strains .aur, encoding the zinc metalloproteinase aureolysin, was overexpressed 8.2-fold in 13136p−m+V5. Aureolysin has been shown to cleave complement component C3 and is proposed to mediate immune evasion [sspB encodes a cysteine protease precursor and was upregulated 3.7-fold in 13136p−m+V5, but unchanged in 13136p−m+V20. This gene was reported as downregulated in another VISA strain, JH9 [sspC and sspA, encoding a cysteine protease and V8 serine protease, respectively, were downregulated in both 13136p−m+V5 and 13136p−m+V20. Ingavale et al. [spa gene, encoding protein A, had no expression change in 13136p−m+V5 but surprisingly was upregulated 11.5-fold in 13136p−m+V20. Downregulation of this gene is the typical observation in VISA strains [Secreted and cell surface-associated virulence factor gene expression patterns were similarly complicated.  evasion . Gene sse et al.  suggestee et al. . The spa strains .−m+V20 were in genes with known associations to stress responses, primarily to cell wall antibiotics and DNA damage. The tarO gene product is a membrane-associated protein that catalyzes the initial step in the biosynthesis of cell wall teichoic acid, a polymer of ribitol phosphate subunits [dlt gene products, promotes biofilm formation and cell adhesion and is associated with the regulation of autolysis and virulence [dlt genes were downregulated three-fold, but only in VISA 13136p−m+V5. Only VISA 13136p−m+V5 had the mutation in tarO, and it had the largest decrease in the metabolite ribitol relative concentration . VISA 13136p−m+V20 has only 70% of the cell wall phosphorus of the parent VSSA, whereas VISA 13136p−m+V5 has 92% of the parent level [All 16 SNPs in VISA 13136pirulence . Both dl−m+V5 and 13136p−m+V20, respectively, versus the relative concentration in the parent VSSA. Fructose phosphate levels were slightly lower in the two VISA than the parent. Expression of locus SAV0698, encoding a putative fructose utilization (fru) operon repressor, was upregulated 2.3- and 4.3-fold in 13136p−m+V5 and 13136p−m+V20, respectively. Factors contributing to the diminished fructose pool could be the repression of genes encoding enzymes required for uptake and utilization or the diversion of available fructose toward upregulated peptidoglycan synthesis. There are problems with both of these hypotheses, however. The uptake mechanism encoded by the fru operon is a phosphotransferase system, but it was unphosphorylated fructose whose relative concentrations changed dramatically. Furthermore, the thickened cell walls characteristic of the VISA phenotype are generally only produced in the presence of glycopeptides. Analyses in this study were performed on cells grown in antibiotic-free medium. Additionally, in 13136p−m+V5, the glmS gene is downregulated, but not in 13136p−m+V20. GlmS catalyzes the initial step in UDP-N-acetylglucosamine biosynthesis, in which fructose as fructose-6-phosphate is incorporated into the cell wall biosynthetic pathway.Fructose levels were 10- and 23-fold lower in 13136pS. aureus general stress response network were selectively deployed in the two VISA, as three of the eight genes whose expression was highly downregulated (≥8-fold versus parent VSSA) were associated with stress responses. These were asp23, encoding alkaline shock protein 23, locus SAV2185, encoding a putative transporter of osmoprotectant glycine betaine, and SACOL1114, encoding a putative Mn2+/Fe2+ transporter associated with oxidative stress management. The remaining five genes encoded two enterotoxins , a pseudogene with homology to integrase/recombinase proteins and two hypothetical proteins. No genes were upregulated ≥8-fold in both VISA.Components of the ica operon was strongly upregulated in VISA 13136p−m+V5, but not VISA 13136p−m+V20. Production of a number of virulence factors, including the intercellular adhesin proteins encoded by the ica operon, are regulated by TCA cycle changes in response to stress. An increase in ribose concentration has been linked to TCA cycle stress and an accompanying pentose phosphate pathway regulation modulation [−m+V5 than the parent VSSA, although only up 2.9-fold in VISA 13136p−m+V20, while concentrations of ribose itself were comparable in VISA 13136p−m+V5 and the parent VSSA but two-fold lower in VISA 13136p−m+V20 than the parent strain.There was evidence of stress response activation or modification in metabolomic data. The dulation . Ribose-adh1 gene, whose expression was decreased 7.8-fold in VISA 13136p−m+V5 versus the parent VSSA, representing a reduced potential for fermentation, while expression was increased 4.7-fold in VISA 13136p−m+V5 versus the parent VSSA, representing an increased potential for fermentation. The pdhC gene encodes a component of the pyruvate dehydrogenase complex, which converts pyruvate to acetate. Expression of pdhC versus the parent 13136p−m+ was unchanged in 13136p−m+V5 and downregulated 3.1-fold in 13136p−m+V20, indicating that fermentation to acetate was not occurring. A shift away from anaerobic growth is consistent with the fact that acetate was not detected by the metabolomic analysis, pyruvic acid levels declined only slightly in the two VISA in comparison to the VSSA parent, and nitrate extrusion protein gene narK was downregulated in 13136p−m+V5.Alcohol dehydrogenase is a fermentation pathway enzyme encoded by the −m+ or 13136p−m+V5, but was at detectable levels in 13136p−m+V20. In S. aureus, the PurA protein is a substrate for phosphorylation by Stk1, the cognate serine threonine phosphatase to the Stp1 serine threonine kinase encoded by the stp1 gene [−m+V5 that reverted during selection of 13136p−m+V20. The purA gene encodes adenylosuccinate synthase, which catalyzes the final step in AMP biosynthesis. However, purA was downregulated 2.4-fold in 13136p−m+V20, but unchanged in 13136p−m+V5, so the mutant Stp1 protein was not responsible for the increased AMP level in 13136p−m+V20. Therefore, the accumulation of AMP and downregulation of a gene encoding its biosynthesis may be due to a reduced demand for ATP in 13136p−m+V20. Energy conservation is a stringent response characteristic, as is the general downregulation of purine and pyrimidine metabolism genes observed in both VISA.Adenosine monophosphate (AMP) was not detected in 13136pS. aureus is through arginine and ornithine via the urea cycle, not from glutamate [S. aureus. Trehalose was present in 13136p−m+V5 and 13136p−m+V20 at relative concentrations seven- and 1.4-fold higher than in the parent strain. This is consistent with an activated stress response network in VISA, but not with the gene expression data for the trehalose-specific phosphotransferase system encoded by treP . The TreP system is phosphoenolpyruvate (PEP) dependent, however, and PEP was not among the identified metabolites, suggesting that other uptake systems may have been utilized for trehalose and indicating that no significant gluconeogenesis was occurring in any of the three strains.The major proline biosynthetic pathway in lutamate . Accumulversus the parent VSSA in both 13136p−m+V5 (14-fold) and 13136p−m+V20 (22-fold). Citrate, cis-aconitate and glutamate, metabolites prior to the urea cycle branch point in the TCA cycle, all accumulated in 13136p−m+V5, while fumarate and malate concentrations, which are at or after the urea cycle branch point, were reduced. TCA cycle intermediate cis-aconitate was not detected in parent VSSA or 13136p−m+V20, only in 13136p−m+V5. In 13136p−m+V20, the identified TCA cycle intermediates, except cis-aconitate, were all within five-fold of parent strain relative concentrations, but many intermediates in glycolysis were reduced at least five-fold versus the parent. This suggests elevated flux through glycolysis to allow the TCA cycle to maintain such high proline levels. However, S. aureus also imports proline via the ProP low-affinity transport system in response to osmotic stress or a high-affinity PutP transport system in response to nutrient limitation [−m+V5 versus the parent strain, consistent with elevated proline concentrations and an activated stress response network. Data were not available for 13136p−m+V20 proP expression. Overall, the results suggest that 13136p−m+V5 was more reliant on transport into the cell via ProP to accumulate proline than 13136p−m+V20, which, in turn, relied more on proline biosynthesis from arginine via the urea cycle. Osmoprotectant uptake from the environment is a more rapid response to osmotic stress than de novo biosynthesis. However, that becomes irrelevant if proline biosynthesis is an aspect of the stress response network in VISA 13136p−m+V20 that is permanently activated to maintain high proline concentrations. Glutamate accumulation may have been due to reduced demand from nucleotide biosynthesis pathways.Proline relative concentrations were extremely elevated mitation . Gene exet al. [cit operon. Of seven pyrimidine ribonucleotide biosynthetic pathway genes with expression changes in this study, two were downregulated and five unchanged in 13136p−m+V5 and six downregulated and one unchanged in 13136p−m+V20. The relative concentrations of pyrimidines, as well as purines, did not correlate well with biosynthetic gene expression patterns. McAleese et al. [pyr gene downregulation in VISA, and Reiss et al. [pur gene downregulation in MRSA COL during a mupirocin-induced stringent response. Hypoxanthine and inosine were detected in 13136p−m+V20, but not in 13136p−m+V5 or the parent 13136p−m+. These are purine salvage pathway substrates either taken up from the growth medium, perhaps due to activated elements of the starvation response, or are degradation products from excess adenine and adenosine after downregulated purine biosynthesis [versus the parent VSSA in 13136p−m+V5 and were substantially elevated again in 13136p−m+V20. Orotate levels were comparable in parent and 13136p−m+V5, with substantial accumulation only in 13136p−m+V20.Nelson et al.  have sugs et al.  reportedynthesis . Likewis−m+V20) and spermidine (both strains). Polyamines play important roles in many cellular processes [et al. [S. aureus. Lower levels of polyamines in VISA strains may facilitate the development of the VISA phenotype by increasing the frequency of mutations. Within the cell polyamines are often complexed with nucleic acids, where they are thought to serve a protective function against stress and possibly participate in the regulation of protein synthesis and DNA-binding protein interactions. Polyamines protect E. coli against oxidative stress and are a component of the acid stress response. On the cell surface polyamines have been shown to influence biofilm formation in Bacillus subtilis and a number of Gram-negative pathogens. Spermidine biosynthesis exerts a regulatory influence on Streptococcus pneumoniae autolytic activity, possibly through cell wall structural interactions [There were significant decreases in the polyamines putrescine  and 13136p−m+V20 . In the parent 13136p−m+, there was almost five-times the relative concentration of the unphosphorylated mannitol as in its VISA derivatives. The lower concentrations in the VISA can be explained by downregulated mtlA expression, which encodes a mannitol phosphotransferase system component, by 2.9- and 4.5-fold in 13136p−m+V5 and 13136p−m+V20, respectively. One reason for the dramatic proline accumulation in the VISA might have been to compensate for the unavailability of mannitol for service in osmoprotection.Mannitol is suspected of playing a role in osmoregulation in . aureus . Relativssb1, dinG and stp1). A large proportion of gene expression changes identified by transcriptome analysis encode products in the hypothetical protein, unclassified, and unknown function gene functional groups . This is not a novel observation [relA up- or down-regulation give no guidance on which activities are active at the time of sample processing. Furthermore, the single gene expression change in the mobile and extrachromosomal element functions gene functional group identified by the transcriptional analysis does not reflect the dominant position of the L54a prophage mutations within the genomic analysis.Ideally, it would be possible to reveal elements of the VISA resistance mechanism by working backwards from phenotypic characteristics through metabolomic, then transcriptomic, and finally genomic data. However, interpretation of the omics data from this study presents several challenges. In-depth knowledge of the function of the gene products of many of the genes with SNPs is lacking, profoundly so for some of them  were used for transcriptional profiling and metabolomics analysis. studied . They we−m+ to which the genomes of 13136p−m+V5 and 13136p−m+V20 were compared.These were performed with an array-based service provided by Nimblegen Systems Inc.  ,101. TheS. aureus genome microarrays version 6.0, provided by the Pathogen Functional Genomics Resource Center of the National Institutes of Allergy and Infectious Diseases, were used.This was carried out as described in previous publications from this laboratory ,103. S. et al. [This was carried out as described by Song et al.  on selecet al. [This was carried out as described by Singh et al. . Brieflyet al. [et al. [−m+ in this study had been laboratory-propagated for decades, although for much of that time it was in frozen storage, and its VISA derivatives were selected in vitro and have never been passaged through animals. The extensive modulation of virulence determinants in these and other in vitro-selected and -propagated VISA demonstrates that virulence and central intermediary metabolism are inextricably intertwined. Extended in vitro passage has been shown to change the expression pattern of virulence determinants, but it does not eliminate them [This study added new members to the lists of mutations and transcriptomes that are associated with the VISA phenotype. Overall trends in VISA characteristics were consistent with previous studies, including a great variability in the mutations and gene expression patterns among VISA of different lineages. This variability has been noted previously by Howden et al. ,4,105. T [et al.  also conate them ,108.−m+V20). The pattern of downregulation of central metabolic processes related to protein synthesis and purine and pyrimidine biosynthetic pathways indicates a stringent response-like state for the two VISA, as has been reported for both MRSA and VISA [versus their VSSA parent, for while central metabolism in VISA 13136p−m+V20 was clearly less active than that in 13136p−m+V5, their doubling times are very similar [The importance of activated elements of the stress response network was reinforced by the results from this study. Stress response modulation in the VISA phenotype was evident across all three omics analyses, from mutations and expression changes with genes known to respond to stresses or encode stress-related products, to modulation of stress-protection metabolites, such as proline and polyamines. It was surprising how many mutations were identified in the two VISA strains that were associated with stress related to DNA damage and the SOS response, especially so for the strain with the higher vancomycin MIC (13136pand VISA ,66. Down similar .−m+ and 13136p−m+V5, and then between 13136p−m+V5 and 13136p−m+V20. Numbers of stress response-related mutations consistently changed in this fashion, as did broad expression patterns for genes associated with aspects of metabolism related to the stress response network and central intermediary metabolism. This pattern was also evident in metabolite levels of osmoprotectants, polyamines, and intermediates in nucleotide biosynthesis and central carbon and energy utilization pathways.The greatest value derived from the metabolomics analysis was in providing physiologic evidence supporting the association of stress response activation and stringent response characteristics with the VISA phenotype. This was evident in progressive changes in metabolite relative concentrations that paralleled the selection of greater vancomycin resistance between VSSA 13136p


Tm, has been detected. For this kind of series, for the first time, the calorimetric melting enthalpy  and transition entropy (ΔSm) parameters have been determined simultaneously throughout. The first analysis indicated a two-phase impact implying (a) the initial, dramatic drop in both ΔHcal and ΔSm, obviously connected to specific, direct interaction between the [ch][dhp] components and α-CT's charged groups (within 0 to 1 mol/L [ch][dhp]), leading to the essential rearrangement of the interfacial hydrogen-bonded (HB) network; and (b) the follow-up (within 1 to 3.0 mol/L [ch][dhp]), modest changes in ΔHcal and lack of changes in ΔSm, seemingly connected with a subsequent steady strengthening of already reformed HB network, respectively. These changes, presumably, are primarily facilitated by Coulombic interactions between the [dhp] anions and solvent-exposed positively charged amino groups of α-CT.An impact of 0.5 to 3 M choline dihydrogen phosphate, [ch][dhp], the biotechnologically relevant ionic substance, on the thermal stability of a model globular protein, α-chymotrypsin (α-CT), has been studied exploiting the highly sensitive differential scanning calorimetry (DSC) technique. The notable overall stabilizing effect of 11 ± 2 K regarding the thermal transition (melting) temperature,  Currently, there is a great interest in the multipurpose application of recently revived substances, organic salts possessing relatively low melting points . These substances, when mixed with water, are able to form a huge amount of unique liquid or semisolid compositions apt for the novel nanotechnological and bionanotechnological developments –4. BecauTm, the unfolding (melting) calorimetric enthalpy, ΔHcal , and calorimetric entropy, ΔScal [dhp], on the thermal stability of model protein, α-CT. Determination of both enthalpic and entropic components contributing to the protein melting process allowed for a conjecturing of fine interfacial effects behind the notable stabilization impact.Numerous papers on DSC studies for the stabilizing/destabilizing impact of various organic additives –26, 30, e, e.g., , 8, 9; fe, e.g., , 26, 30)nterpart , 31–33. entioned , 26, 30.−2 M buffer solutions (pH 4.2).Highly purified and lyophilized α-CT was purchased from Fluka and [ch][dhp] was from Iolitech, GmbH, and used without further purification. Other chemicals were of highest purity available. The α-CT samples for the DSC experiments were prepared at concentrations of 1.5 mg/mL, by dissolving in phosphate 10Microcalorimetric measurements for temperature-induced melting (denaturation) of α-CT in buffered [ch][dhp] aqueous solutions were performed with a DASM-4A adiabatic scanning calorimeter  integrated with a PCI-DAS1001 (Measurement Computing Corporation) interface unit, providing direct PC access . The heaHcal, were determined as areas under the calorimetric curves of T is the absolute temperature and T1 and T2 are the temperatures that correspond to the start and completion of heat absorption due to the protein's thermal melting (T1 < Tm < T2). The values of the counterpart parameter, melting entropy, ΔScal, can be determined as follows [dhp]  and ΔSm, as determined through (Tm versus the [ch][dhp]) content (from 0 to 1 mol/l [ch][dhp]) was a little bit larger than one for the rest of the concentration range  a a3) and ch][dhp] . The val through  and (3),on range . This fa Figures , Table 1steadily   is not reported in [Tm ≈ 74°C at 2.5 mol/L [ch][dhp] for our system); however, the overall dependence of Tm on the [ch][dhp] content for lysozyme, in contrast to our case  at any concentration of the additive. The lacking information would allow one to see the variable balance between ΔHcal and ΔSm behind the resultant behavior of Tm in these studies, hence, to compare the complete data for all three model proteins and gain better insight into atomistic mechanisms of stabilization.Interestingly, Constatinescu et al.  recentlyure 2 of ). Howeveure 2 of , the incorted in . In anotorted in  reportedour case , exhibitHcal and ΔSm and further slight increase with and virtual independence of Tm, respectively, are obviously different. When going from zero to 0.5 ÷ 1 mol/L additive concentration, seemingly, strong association between the [ch][dhp] components and proteins' charged groups takes place that should be rather specific to the protein entity and its ionization state (pH); note different patterns of Tm for α-CT and ribonuclease A on the one hand and lysozyme on the other hand . Around this point of the additive concentration, the specific interactions, probably, arrive at the saturation level, after which more gradual/monotonic changes of Tm  and very small/no variation of ΔHcal and ΔSm (as detected in our case) can be observed. The small changes in ΔHcal, seemingly, are connected with the formation of additional contacts and some redistribution of existing ones between the protein and the [ch][dhp] components. Development of the latter process seems to favor proteins' gradual stabilization via the ΔHcal rather than the ΔSm parameter. According to the analysis of [dhp] on the thermal stability of a model globular protein α-CT and found the notable stabilizing effect of 11 ± 2 K regarding the thermal unfolding (melting) temperature, Hcal) and transition entropy (ΔSm) parameters were determined simultaneously throughout the series, and the trend analysis indicated a two-phase pattern including the low-concentration (0 to 1 mol/L [ch][dhp]) and high-concentration (1 to 3 mol/L [ch][dhp])) regions, connected to the initial dramatic and the follow-up minor or no changes of ΔHcal and ΔSm, respectively.(b) The calorimetric melting enthalpy (Δ(c) The atomistic mechanism behind the observed overall stabilizing effect, presumably, is guided by the ion pair formation between the protein's charged groups and the [ch][dhp] components, with a prevailing role of restructured hydrogen-bonded networks facilitated mostly by Coulombic interaction between the [dhp] anions and solvent-exposed positively charged amino groups of α-CT.Hcal and ΔScal (ΔSm) throughout this kind of series, for the proper understanding of atomistic mechanisms behind the stabilizing/destabilizing impact of various additives on proteins, is clearly demonstrated.(d) A need for the determination of parameters Δ


The results showed that seed protein, sucrose, fructose, and glucose were higher in W + B treatment than in W − B, WS + B, and WS − B. The increase in protein in W + B resulted in lower seed oil, and the increase of oleic in WS − B or WS + B resulted in lower linolenic acid. Foliar B resulted in higher nitrogen fixation and water stress resulted in seed δ15N and δ13C alteration. Increased stachyose indicated possible physiological and metabolic changes in carbon and nitrogen pathways and their sources under water stress. This research is beneficial to growers for fertilizer management and seed quality and to breeders to use 15N/14N and 13C/12C ratios and stachyose to select for drought tolerance soybean.Therefore, the objective of the current research was to investigate the effects of foliar B nutrition on seed protein, oil, fatty acids, and sugars under water stress conditions. A repeated greenhouse experiment was conducted using different maturity group (MG) cultivars. Plants were well-watered with no foliar B (W − B), well-watered with foliar B (W + B), water-stressed with no foliar B (WS − B), and water-stressed with foliar B (WS + B). Foliar B was applied at rate of 0.45 kg Soybean is a major crop in the world, and its nutritional benefits reside in its seed protein 37–42%), oil 19–23%), fatty acids , and minerals contents [9–23%, fa2%, oil 1It is well-known that seed composition  is genetically controlled, but it is also reported that these constituents have been found to vary depending on biotic and abiotic stress factors, including water stress/drought, temperature, agriculture practices, fertilizer application, genotype, and maturity. For example, the influence of environment and maturity group on seed protein, oil, fatty acids, sugars, and minerals has been reported previously , 6–11. IBoron nutrient is essential for crop growth, development, production, and seed quality –15. Boroδ15N and δ13C isotopes were also investigated.Based on the above introduction it is clear that in spite of the well-established literature on the structural role , 13, 16 ·kg−1). Water stress conditions were achieved by weighing soil in pots then saturating them with deionized water and were left to drain and weighed again to obtain the water field capacity [·ha−1 and was applied twice at flowering and at seed-fill stages. Leaf samples for B measurements were taken after the second application at seed-fill stage. Seeds at harvest maturity were collected for seed nutrition assessments. Four replicates were used for each treatment and each pot with four individual plants was considered one replicate. The greenhouse conditions were as follows: temperature ranged from about 32°C ± 11°C during the day and about 29°C ± 9°C at night with a photosynthetic photon flux density (PPFD) of about 1800–2500 μmol·m−2·s−1, as measured by Quantum Meter . The two experiments were conducted simultaneously at the same time and during the normal growing season (April-September) for the Early Soybean Production System in the midsouth USA.The experiment was conducted under greenhouse conditions twice. Soybean seeds were germinated in flat trays in vermiculite, and uniform size seedlings at about V1 stage were transplanted into 9.45 L size pots filled with field soil. Physical and chemical analysis of soil showed that the soil was a Dundee silt loam  with pH 6.3 and 1.1% organic matter. The soil contained enough B concentration  , 34, 35 −1. A volume of 100 μL sample was added to 1 mL of the Glucose (HK) Assay Reagent and incubated at room temperature for 15 min. Glucose concentration in samples was determined at absorbance of 340 nm using the Beckman Coulter DU 800 spectrophotometer. The concentration of glucose was expressed as mg g dwt−1.Glucose concentration in seed was conducted by an enzymatic reaction using a Glucose (HK) Assay Kit, Product Code GAHK-20  . In this−1.Fructose concentration was determined by an enzymatic reaction using a Fructose Assay Kit, Product Code FA-20  . In thisδ15N and 13C natural abundance was conducted from ratios of nitrogen isotope (15N/14N ratio) and carbon isotope (13C/12C ratio) using about 0.9 mg of ground seeds. Isotopic analysis was conducted using a Thermo FinniGlyn Delta Plus Advantage Mass Spectrometer with a FinniGan ConFlo III, and Isomass Elemental Analyzer . Isodat software version 2.38 was used to obtain Delta values [Analysis of a values –41. The The experiment was a split plot design with irrigation as a main plot, cultivar, and subplot, and B treatments were sub-subplot. The data were subjected to analysis of variance using Proc ANOVA in SAS . Means wAnalysis of variance (ANOVA) showed that cultivar (Cv), water treatment , and boron treatment (Treat) had significant effects on protein, oil, oleic, linoleic, and linolenic acids . GeneralUnder well-watered conditions , foliar 15N and 13C isotopes, below, indicated lower nitrogen fixation rates and alteration in 15N and 13C under water stress conditions, indicating changes in nitrogen and carbon fixation pathways. On the other hand, nitrogen fixation rates were higher and no alteration in 15N and 13C isotopes occurred in well-watered conditions.In well-watered plants , FB applFurther, it was found that the activity of sucrose synthase, the main enzyme involved in sucrose hydrolysis in nodules, was significantly inhibited under drought conditions , 46, whiThe higher accumulation of stachyose concentration under water stress may indicate possible role of this oligosaccharide in abiotic stress conditions. Effects of water stress on sugars were previously reported , 45, 47.15N/14N and 13C/12C ratios  and decreasing 14N . Also, the alteration of 15N/14N indicated that water stress inhibited nitrogen fixation due to nitrogenase sensitivity to water stress  in seed of plants grown under water stress conditions also indicated change in carbon fixation source. Previous research reported that δ13C value in plant tissues can be affected by water supply and temperature [δ13C in plant tissues is affected by environmental conditions , and this occurs by affecting plant gas exchange through stomatal conductance and CO2 fixation [13C/12C ratio indicates that drought stress led to stomatal closure and 13C fixation increase, leading to less discrimination against δ13C and a shift in carbon fixation metabolism from ribulose bisphosphate (RuBP) carboxylase pathway to phosphoenolpyruvate carboxylase (PEP), resulting in δ13C enrichment [δ15N and δ13C values changed and water stress resulted in enrichment of δ15N and δ13C. This indicated that nitrogen and carbon metabolism pathways altered, explaining the possible association between nitrogen and carbon fixation pathways and seed protein, oil, and sugars accumulation in seed, impacting seed quality.The possible mechanisms of how plants shift tabolism , 40, 53.perature , plant pperature , and mycperature . This infixation , 58. Therichment . Our curFoliar B application Figures  and 4 inδ15N (15N/14N ratio) and δ13C (13C/12C ratio) isotopes, but water stress resulted in shifting nitrogen metabolism towards nitrogen assimilation due to higher sensitivity of nitrogen fixation to water deficit. The lower accumulation of B in seeds in water-stressed seeds may be due to limited translocation of B from leaves to seed.The current research demonstrated that foliar B application and water stress altered seed composition, especially protein, oleic acid, and sugars. Foliar B application did not alter the dynamics of