Author: ecogrow

Our analyses show that false positive and ‘of-target’ GWAS signals that arise because of erroneous physical addresses can typically be identified and rectified by fitting DNA markers associated with causal loci as fixed effects, which is effectively equivalent to fitting a multilocus genetic model in a QTL mapping or candidate gene analysis study using mixed linear models . A preponderance of the erroneous physical addresses are typically going to be found on homoeologous chromosomes, as was the case in our study, and hence could be misconstrued as signals associated with the effects of homoeologous loci.The sheer numerical abundance of sources of resistance to Fusarium wilt race 1 in strawberry did not shed light on the diversity of R-genes that they might carry, if any,or genetic mechanisms underlying resistance . Was resistance to race 1 conferred by dominant R-genes? How many unique Fusarium wilt R-genes exist in wild and domesticated populations of strawberry? To explore these questions, we developed and undertook genetic analyses of S1 populations developed by self-pollinating highly resistant F. × ananassa heirloom cultivars and individuals and full-sib populations developed by crossing highly resistant ecotypes of F. chiloensis subsp. chiloensis , F. virginiana subsp. virginiana , and F. virginiana subsp. grayana with a highly susceptible F. × ananassa individual .

The phenotypes of of spring in each of the segregating populations spanned the entire range from highly resistant to highly susceptible with bimodal distributions . When individuals within each population were classified as resistant or susceptible using 2.0 as the cut of on the disease symptom rating scale,plastic planters the observed phenotypic ratios perfectly ft the expected phenotypic ratios for the segregation of dominant resistance genes . The Guardian and Wiltguard S1 and 12C089P002 × PI602575 and PI552277 × 12C089P002 full-sib populations each appeared to segregate for a single dominant resistance gene, whereas the Earliglow and 17C327P010 S1 populations appeared to segregate for two dominant genes with duplicate epistasis, where a single dominant allele at either locus was sufficient to confer resistance . The statistical inferences were not affected by shifting the cut of downward to 1.5 or upward to 2.5; hence, we concluded that dominant R-genes segregated in these populations .Genome-wide searches for SNPs associated with loci segregating for resistance to Fusarium wilt uncovered a single tightly linked cluster of statistically significant SNP markers in each population . The SNPs most strongly associated with differences in resistance phenotypes were tightly linked to partially to completely dominant R-genes that segregated in these populations and mapped to three non-homoeologous chromosomes . The putative R-genes are hereafter designated FW2 , FW3 , FW4 , and FW5 . FW2 and FW5 genetically mapped proximal to FW1 on chromosome 2B . The effects and positions of the these loci were identified by GWAS or genetic mapping alone .

The SNP markers most strongly associated with each of these loci were different and spanned a 1.6 Mb haploblock . To explore the structure of this haploblock in greater depth, we imputed and phased the haplotypes for 71 50K Axiom array-genotyped SNPs among 653 individuals in the California population . These included the parents and more distant relatives and common ancestors of the S1 and full-sib progeny that were both genotyped and phenotyped . Although FW1, FW2, and FW5 could be alleles, haplotypes for 71 SNPs within the haploblock predicted to harbor these loci were insufficient to rule out paralogs, and confidence intervals for the estimated positions of these loci spanned the gene-rich haploblock . The SNP haplotypes for Fronteras and Portola were identical except for three consecutive SNPs in a short haploblock slightly downstream of the location predicted to harbor FW1 . The haplotype associated with the dominant FW1 allele for that haploblock was ascertained from the genotypes of Fronteras and Portola . The haplotypes observed for the other resistant parents difered from each other and Fronteras and Portola; hence, from SNP haplotypes and approximate physical positions, we could not unequivocally show that the putative R-genes associated with these phenotypically mapped loci were allelic. Finally, the KASP assays we developed for informative SNPs associated with these loci were not cross predictive . The paralog hypothesis seems plausible for the underlying R-genes; however, our data were in sufficient to rule out the single locus, multiple allele hypothesis. Although additional studies are needed to resolve this question, the classes of R-genes hypothesized to underlie these loci are commonly found in tandemly duplicated clusters in plants .

The genotypic means, effects, and PVE estimates for SNP markers tightly linked with FW2 and FW5 were nearly identical to estimates for SNP markers associated with FW1 in the Fronteras and Portola S1 populations . FW2 was nearly completely dominant . The additive and dominance effects of the FW2 locus were 1.5- to 1.9- fold greater than those reported for the FW1 locus, partly because unfavorable allele homozygotes were more strongly susceptible in the Guardian S1 population than in the Fronteras and Portola S1 populations. The estimated marginal means for favorable allele homozygotes ranged from 1.07 to 1.25 in the three populations . The EMM for resistant homozygotes was ̄yAA = 1.1, whereas the EMM for susceptible homozygotes was ̄yaa = 4.5. We could not estimate the degree of dominance for FW5 because the AA homozygote was not observed in the fullsib population; however, the EMM for the heterozygote was 1.06, which implies that the FW5 allele might be completely dominant. Although the statistical evidence for the segregation of a single dominant R-gene on chromosome 1A was strong in the Wiltguard S1 population, the effects of SNPs associated with FW3 were weaker than those associated with FW1, FW2, and FW5 on chromosome 2B . The most significant FW3-associated SNP was AX-123363542 , which only explained 23% of the phenotypic variation for resistance to race 1. Despite this, the EMM for FW3 homozygotes was only slightly greater than the EMMs for FW1 and FW2 homozygotes . Although the PVE estimate was greater for FW4 than FW3 , the EMMs for SNP marker heterozygotes were virtually identical: 1.76 for FW3 and 1.69 for FW4; hence, FW4 appears to be as strong as FW3 .With the genomic locations of FW1, FW2, and FW5 narrowed to a short haploblock on chromosome 2B , we searched annotations in the ‘Royal Royce’ reference genome to identify genes encoding proteins known to play an important role in race-specific disease resistance via pathogen recognition and activation of defense responses, e.g., pathogen-associated molecular pattern -triggered immunity or effector triggered immunity . Eight of 1,208 annotated genes found in the 0.0-5.0 Mb haploblock on chromosome 2B encode proteins with known R-gene domains and functions . These included one coiled-coil domain NLR encoding gene and two tightly linked Tollinterleukin 1 receptor domain type NLR encoding genes . Hence, the most promising candidate genes for FW1 encode NLR proteins. The approximate 95% Bayes confdence interval for the genomic location of FW4 on chromosome 6B was fairly wide and consequently harbored 197 annotated genes in the ‘Royal Royce’ reference genome . Nine of these 197 annotated genes are predicted to encode R-proteins that mediate gene-for-gene resistance in plants . These included multiple NBS-LRR R-proteins . Finally,plastic nursery plant pot the approximate 95% Bayes confdence interval for the genomic location of FW3 on chromosome 1A was slightly wider than that observed for the other mapped loci because the effect of the locus was weaker. There were 535 annotated genes within that interval, of which seven were predicted to encode NLR or other R-proteins . This was the locus with the weakest support for the segregation of a race-specific R-gene; however, as noted earlier, homozygous resistant of spring in the Wiltguard S1 population were highly resistant . Hence, even if FW3 does not encode a race-specific R-protein, this locus merits further study, in part because the favorable allele can be deployed and pyramided to increase the durability of resistance to Fusarium wilt.To accelerate the introduction and selection of Fusarium wilt resistance genes in breeding programs, we developed a collection of high-throughput Kompetitive Allele Specifc PCR markers for SNPs in linkage disequilibrium with FW1–FW5 . Collectively, 25 KASP markers were designed for the five loci using PolyOligo 1.0 .

The genotypic clusters for 17 of these were codominant , co-segregated with the predicted resistance loci, and were robust and reliable when tested on diverse germplasm accessions . For each target locus, at least one KASP-SNP marker had a prediction accuracy in the 98-100% range when tested in the original populations where they were discovered . To further gauge their accuracy when applied in diverse germplasm, they were genotyped on 78 California and 66 non-California individuals, mostly cultivars . Because the causal genes and mutations underlying FW1–FW5 are not known, the SNPs we targeted are highly population-specific . They are strongly predictive when applied in populations where specific genes are known to be segregating and moderately predictive when assayed among random samples of individuals because of recombination between the SNP markers and unknown causal mutations.The deployment of Fusarium wilt resistant cultivars has become critical in California since the early 2000s when outbreaks of the disease were first reported . This disease has rapidly spread and become one of the most common biotic causes of plant death and yield losses in California, the source of 88-91% of the strawberries produced in the US . The scope of the problem was initially unclear, as were the solutions, because the resistance phenotypes of commercially important cultivars, genetic mechanisms underlying resistance, and distribution and race structure of the pathogen were either unknown or uncertain when the disease unexpectedly surfaced in California . A breeding solution instantly emerged with the discovery of FW1 , and was further strengthened with the discovery of additional homologous and non-homoeologous resistance genes in the present study . Genetic and physical mapping of these race-specific R-genes has enabled the rapid development and deployment of Fusarium wilt resistant cultivars through marker-assisted selection. The transfer of R-genes from race 1 resistant donors to susceptible recipients via MAS has been rapid because the resistant alleles are dominant, found in both heirloom and modern cultivars, and identifiable without phenotyping using SNP markers tightly linked to the causal loci . Once FW1 was discovered, we knew that we had a robust solution to the race 1 resistance problem; however, we had virtually no knowledge of the diversity of Fusarium wilt R-genes in populations of the wild octoploid progenitors and heirloom cultivars of cultivated strawberry that might be needed to cope with pathogen race evolution . We did not purposefully set out to identify redundant R-genes but rather to scour global diversity for ancestrally diverse R-genes, both to facilitate R-gene pyramiding and inform future searches for sources of resistance to as yet unknown races of the pathogen, in addition to assessing the frequency, diversity, and distribution of R-genes in the wild and domesticated reservoirs of genetic diversity . Our results paint a promising picture for the identification of genes for resistance to race 2 and other as yet unknown races of the pathogen. As our phenotypic screening studies showed, the frequency of resistance to race 2 was comparable to that observed for race 1 . Similar to our findings for race 1, the sources we identified for resistance to race 2 were symptomless, which suggests that gene-for-gene resistance might underlie their phenotypes. The genetic basis of resistance to race 2 and other races of the pathogen, however, has not yet been elucidated. There is empirical evidence that resistance to Australian isolates of the pathogen might be quantitative . Henry et al. showed that the non-chlorotic symptom syndrome caused by Australian Fof isolates differs from the chlorotic symptom syndrome caused by California and Japanese Fof isolates . Hence, the genetic basis of resistance to the wilt- and yellowsfragariae diseases could be markedly different. We identified several strong sources of resistance to Australian and other non-California isolates of the pathogen that should accelerate the discovery of novel race-specific R-genes, elucidation of genetic mechanisms, and development of resistant cultivars . Growing resistant cultivars is indisputably a highly effective and cost-free method for preventing losses to Fusarium wilt race 1 in strawberry . We estimate that approximately two-thirds of the cultivars grown in California since the earliest outbreaks in 2005 were highly susceptible, whereas the other one-third were highly resistant .

These physical activities have been used in the exercise literature as measures of physical fitness and are both safe and simple to learn in an in-lab setting . For all activities, duration of time spent in each activity and number of attempts were assessed. For study 2, participants were asked to complete a shortened Trier Social Stress Test , during which participants delivered a 3-minute speech presenting their qualifications for a dream job to a panel of two evaluators while physiological stress responses were assessed .Across both studies, we used the same measures obtained at the same intervals to assess subjective well being. We replaced missing values with the mean of available items if ≤half of the items were missing, otherwise we treated the scale as missing . State optimism, considered a manipulation check for the optimism intervention, was measured at baseline, mid-study, and post-intervention with seven items from the validated State Optimism Measure . Participants were asked how they felt ‘right now’ on a scale of 0 to 10 regarding expectations for their present and the future. A total score was created by averaging scores across all seven items. In Study 1, α’s ranged from 0.91–0.92 and, in Study 2, from 0.89–0.90. Confidence about the future was assessed by asking participants to rate, on a ten-point scale ranging from 0 to 10 ,black flower buckets how they believe the challenges in their lives right now will turn out. It was measured at baseline and post intervention using the validated Life Orientation Test Revised .

Responses were modified to a seven-point continuum, ranging from ‘strongly disagree’ to ‘strongly agree,’ based on prior work suggesting the 7-point scale is more suited for electronically distributed questionnaires . The total optimism score is the sum of the three positively worded items and the three negatively worded items reverse-scored, with higher scores reflecting higher optimism. In Study 1, α’s ranged from 0.79–0.80 and, in Study 2, from 0.74–0.85. Anxiety, depression, and aggression were assessed at baseline, mid-study, and post-intervention with items from the State-Trait Personality Inventory . Using a seven-point scale, respondents rated the degree to which they agreed with each statement. The final anxiety, depression, and aggression scores were calculated by averaging across items, with higher scores reflecting higher levels of each construct.Positive affect and negative affect were assessed at baseline, mid-study, and post-intervention by the Positive and Negative Schedule , which included ten items capturing positive affect and ten items capturing negative affect . Response options ranged from 1 to 7 . After completing the in-lab writing tasks and interview, participants were asked to complete a stepping task and a sit-to-stand task. For the stepping task, participants were asked to step up and down on a 14-inch high step platform at a rate of 22 steps per minute , until they were too fatigued to continue . Participants were asked to make as many attempts as they wanted for up to a total of 15 minutes. The research assistant recorded the duration of time participants spent stepping across all attempts, as well as the total number of attempts made. The final stepping task score was calculated by the duration divided by the number of attempts.Participants were asked to perform a sit-to-stand exercise.

This involved sitting in the middle of the chair and crossing their arms with each hand on the opposite shoulder and then completing sit-to-stand cycles at a rate of 22 cycles per minute. Participants were first asked to attempt this task for up to 60 seconds. If they decided to continue, the duration requested for each subsequent attempt increased by 30 seconds whereby the second attempt lasted up to 90 seconds and the third attempt lasted up to 120 seconds. As with the stepping task, the research assistant recorded the number of attempts and total duration completed. The sit-stand task score was created by dividing total duration by number of attempts.Physiological measures included measures derived from electrocardiography and impedance cardiography using ECG and NICO modules integrated into an MP150 Data Acquisition System with signals sampled at 1000 Hz. Spot sensors placed in a modified Lead II configuration measured ECG, and four mylar bands that completely encircled the neck and torso measured ICG. From these measures, we derived high-frequency heart rate variability and pre-ejection period . PEP is a time based measure of the contractile force measured from the time the ventricle contracts to the opening of the aortic valve. Shorter time indicates greater increases in sympathetic nervous system activity. RSA provides a relatively pure measure of parasympathetic nervous system responses, whereas PEP provides a relatively pure measure of sympathetic nervous system responses. We also collected blood pressure responses using a Colin Prodigy blood pressure monitor at 7 targeted times during the 2-hour visit. Our BP timing included end of the resting baseline, post-writing, post-interview of writing, beginning of speech preparation, beginning of speech delivery, beginning of math task, and end of math task.For both studies, we trained coders to evaluate the in-lab writing tasks to assess the optimism and affect conveyed in the essays. Coders were trained by first coding approximately 10% of the essays, and the Inter-rater reliability was assessed and found to be satisfactory.

Coders then met with other coders to clarify conceptual categories and calibrate their evaluations. Subsequently, each essay was then coded by at least two different judges. We averaged coders ratings at the item level and took the mean of the items to obtain final scores for optimism, positive affect, negative affect, and explanatory style. Optimism was judged by evaluating goal-oriented thinking and perceived resources . Judges provided ratings ranged from 1 to 7 . Study 1: alpha for the intervention group: α = 0.86 and control group: α = 0.82; Study 2: intervention group: α = 0.90 and control group: α = 0.74. Affect was evaluated by judges by completing a PANAS 20-item scale, which we modified to include feelings of optimism, gratitude, resentment, and pessimism in addition to the standard items. All items were rated on a 5-point scale ranging from 1 to 5 . Items that describe positive feelings were averaged to obtain a score for positive affect, and items that described negative feelings were averaged to obtain a score for negative affect. , α = 0.85 ; negative affect α = 0.71 , α = 0.77 ; Study 2: positive affect α = 0.95 , α = 0.93 ; negative affect α = 0.81 , α = 0.70.For both studies, we first evaluated the effectiveness of the randomization procedure by comparing the distribution of the demographic characteristics across the intervention and control groups, using chi-square tests or t-tests as appropriate. We then examined the pooled data from the two studies and conducted multilevel mixed-effect models to evaluate the combined effect of the manipulation on changes in optimism and the common self-report measures. We treated time as a fixed effect and study sites as a random effect. Next, we examined the effect of the intervention on self-reported outcomes and study specific outcomes. In Study 1, we conducted an ANOVA with condition as the between-subjects factor and self reported outcomes and in-lab exercise outcomes as separate dependent variables. For in-lab exercise outcomes,french flower bucket we conducted sensitivity analyses to additionally adjust for baseline self-report physical activity which was assessed by asking participants the number of days they had engaged in moderate physical activities, such as bicycling at a regular pace or doubles tennis. In Study 2, we used an ANOVA to evaluate reactivity values across the four physiologic responses: RSA, PEP, and systolic and diastolic blood pressure . Reactivity was derived by subtracting the last minute of baseline from each minute of the stress task for each physiological response. In order to understand whether the intervention effects were due primarily to increases in positive affect rather than optimism per se, we conducted sensitivity analyses on all physical activity and stress reactivity outcomes to also control for increases in positive affect. For essay coding, we conducted moderated regression analyses to predict psychological, physical activity, and physiological outcomes with condition, essay-coded optimism, and their interaction. The goal of these analyses was to determine if there was an association among more optimistic terms in the essay and better health-related responses in the lab. All analyses were performed using SAS 9.4 and figures were created using R. This study examined effects of induced optimism on in lab physical activity and stress reactivity through two experimental studies with community-dwelling individuals. Across both studies, the intervention led to greater increases in short-term optimism and positive affect in the intervention group compared with the control group. In general, coder-rated optimism and positive affect were associated with better performance on the in-lab physical activity tasks and healthier forms of stress reactivity.

However, we found little to no evidence that the intervention led to reliable changes in self-reported anxiety, depression, and aggression nor did we observe differences in in-lab physical activity and stress reactivity. Our findings that the writing tasks led to greater improvement in self-reports of optimism and positive affect in the intervention versus the control group are consistent with previous studies conducted in other populations. For example, in a study of 82 students, individuals who did versus did not receive a positive psychological intervention consisting of writing tasks exhibited greater improvement in optimism and positive affect . In another study of 54 Dutch-speaking participants, most of whom were students, a best possible selves writing intervention led to larger increases in optimism compared with writing about a daily activity task . Our research extends these findings to two larger samples of diverse, community-dwelling populations and provides further evidence that this type of intervention can be effective in boosting short-term optimism and positive affect.These findings notwithstanding, we did not find evidence that experimentally induced optimism influenced in-lab physical activity. In contrast, observational studies have repeatedly demonstrated that higher self-reported optimism levels are associated with greater engagement in physical activity or buffer against the harmful effects of stress on health . However, it is notable that our intervention was able to change participants’ exercise beliefs and improve perceived benefits of exercise. Perhaps the small increases in optimism that we obtained from the intervention were insufficient to change subsequent health-related behavior. This may be especially true for health behaviors like physical activity, which are largely habitual. Once established, health behavior is deeply embedded in individuals’ daily routines, and thus changes in behavior may require longer interventions with more substantial changes in optimism. We observed some differences in stress reactivity by condition with induced optimism associated with less vagal withdrawal and lower sympathetic nervous system activation – that is, both less PNS withdrawal and less SNS activation during the stress task. Together these findings suggest participants in the experimental condition had less arousal than participants in the control condition. We might speculate that the optimism condition increased a sense of calm resulting in less arousal during the stress task. However, it is important to note that the intervention did not result in differences in resting physiology nor in blood pressure reactivity, which are more closely associated with better physical health. Despite the null effects of our optimism intervention on in-lab physical activity, we found effects of coder-rated optimism and affect from essays on outcomes in both studies. Overall, greater coded optimism was associated with greater persistence in both physical activity tasks and less vagal withdrawal during the stress tasks. These findings raise several interesting possibilities for understanding our findings and the relationship of optimism with our outcomes of interest. One possibility is that optimism as reflected in writing rather than based on direct self-report more accurately captures participants’ true levels of their positive expectations. A second possibility is that optimism as reflected in writing provides a type of dose-level response such that individuals with more optimistic orientation in their writing benefitted most from the intervention. Our study has several limitations. Although we excluded individuals who were highly physically active from Study 1, we did not establish a pre-intervention exercise assessment. Doing so might have increased our precision in determining if the intervention increased exercise in the lab.

The Yael variety ael shows moderate levels of anthocyanins, hydroxycinnamates, and proanthocyanidins, as well as a high mean degree of polymerization, but with lower levels of the other measured factors. These compounds can contribute bitter and astringent characteristics to grapes and wines, impacting overall flavor and mouthfeel properties. Wild varieties are an incredibly valuable genetic resource for grape breeding, particularly for enhancing metabolites such as resveratrol, as well as for disease resistance. Considering the aroma profiling, most indigenous Israeli varieties showed low levels of terpenoids and esters. Such volatile aroma profiles are often relatively neutral in sensory properties, making wines made from these varieties useful for blending purposes. However, some interesting differences in the volatile profiles were observed that may provide valuable germplasms for breeding unique aroma and flavor traits. For example, a unique high terpene variety, Dumiat, was observed, which may provide an aromatic quality similar to Muscat and Riesling varieties. In addition, the Madvar variety has a unique profile among this set of varieties that is high in benzenoid/phenylpropanoid compounds such as cresol, eugenol, methyl salicylate, benzyl alcohol, and 2-phenylethanol. Depending on their concentrations, these compounds can contribute spicy, smoky, and honey-like aromas. Further sensory analyses will be necessary to fully relate the chemical composition of these accessions with specific aroma and flavor attributes. However,procona buckets this initial analysis demonstrates the potential for these field-grown indigenous varieties to serve as sources for breeding new varieties with unique flavor profiles.

Such varieties not only provide resources for breeding against a wide range of biotic and abiotic stresses, but also have oenological potential with health benefits. This high throughput analysis of berries from many different varieties, grown under the same geographic and agronomic conditions, can only be achieved by working in the germplasm collections. This comprehensive analysis can help determine possible uses of these varieties, especially when considering newly found ones that have never been studied and categorized.Viruses are obligate intracellular pathogens that require living host cells to replicate and spread in the infected plant. During compatible interactions, viruses overcome the plant immune system and hijack host cellular processes to establish active infections . Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological deffects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species . Although these viruses are generally transmitted by plant-feeding insects or soil borne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices .

In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, they manifest as irregular chlorotic regions on the leaf blades. GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening. Most metabolic pathways that promote desired quality traits in grape berries are induced during ripening. The onset of ripening is accompanied by significant changes in berry physiology and metabolism, including softening, sugar accumulation, decrease in organic acids, and synthesis of anthocyanins and other secondary metabolites that define the sensory properties of the fruit . Berry ripening is controlled by multiple regulatory pathways, and occurs in an organized and developmentally timed manner. Interactions between transcriptional regulators and plant hormones regulate the initiation and progression of ripening processes . Like other non-climacteric fruit, grape berries do not display a strong induction of ethylene production and respiration rate at véraison, and the activation of ripening events does not depend primarily on ethylene signaling. Even though the hormonal control of grape berry development is not completely understood, it is established that abscisic acid , brassinosteroids, and ethylene are positive regulators of ripening processes, while auxin delays the initiation of ripening .

In the context of virus–grape berry interactions, dissecting the mechanisms that regulate ripening and plant defenses may provide new opportunities to develop vineyard management strategies to control viral diseases and ameliorate the negative effects on berry quality. In this study, we integrated genome-wide transcriptional profiling, targeted chemical and biochemical analyses, and demonstrated that grapevine red blotch disrupts ripening and metabolism of red-skinned berries. We sampled berries at different ripening stages from vines infected with GRBaV and healthy vines in two vineyards. We identified grape metabolic pathways that were altered in ripening berries because of the viral infection. We determined that GRBaV-induced pathways that are normally associated with early fruit development in berries at late stages of ripening, and suppressed secondary metabolic pathways that occur during normal berry ripening and/ or in response to stress. Using targeted metabolite profiling and enzyme activity analyses, we confirmed the impact of GRBaV on phenylpropanoid metabolism. We identified specific ripening-related processes that were disturbed in GRBaV-infected berries. Remarkably, these processes included alterations in ripening regulatory networks mediated by transcriptional factors, post-transcriptional control, and plant hormones, which lead to berry developmental deffects caused by red blotch.To determine the impact of grapevine red blotch on berry physiology, we studied naturally occurring GRBaV infections in distinct wine grape-growing regions in northern California . We sampled red-skinned grape berries from two different vineyards, one in Oakville and one in Healdsburg . We used multiple vineyard sites to focus on observations consistently made across environments and, thus, to exclude factors associated with specific environmental or cultural conditions. Prior to sampling, vines were screened for the presence of GRBaV and other common grapevine viruses. The appearance of red blotch symptoms on leaves of GRBaV-positive vines and not on those of healthy controls confirmed the initial viral testing. We sampled grape berries from vines that tested positive for GRBaV and negative for other common grapevine viruses. At the same time, we also collected berries from vines that tested negative for all viruses and included them in the study as healthy controls. In order to determine the impact of the disease on berry development and metabolism,procona florida container we collected GRBaV-positive and control berries at comparable developmental stages: pre-véraison , véraison , post-véraison , and harvest . This sampling strategy also aimed to limit confounding effects due to differences in the progression of ripening between berry clusters of GRBaV-positive and healthy vines. In some cases, we observed that GRBaV-positive vines presented grape clusters with evident uneven ripening . Comparisons between berries from GRBaV-positive vines and healthy controls indicated that, at equivalent stages of development, berries affected by red blotch had reduced soluble solids and total anthocyanins in agreement with previous reports on red-skinned wine grapes . Sampled berries were used for genome-wide transcriptional profiling of viral and grape genes. RNAseq was performed using 3–4 biological replicates of each ripening stage, infection status, and vineyard. We first confirmed the presence of the virus in the berries of GRBaV-positive vines by qPCR amplification of viral DNA . Viral activity in the berries was also assessed by quantifying plant-derived mRNA transcripts of GRBaV genes in the RNAseq data. Plant expression of five out of the six predicted genes in the GRBaV genome was detected in all berry samples obtained from GRBaV-positive vines but not in berries collected from the control vines . The most expressed GRBaV genes in the berries corresponded to V1, which encodes a coat protein, and V3 with unknown function. Expression levels of the GRBaV genes appeared to change as berries ripened. However, we could not determine to what extent the progression of ripening or other environmental factors influenced the plant’s transcription of viral genes because their pattern of variation between ripening stages differed in the two vineyards . Expression of 25 994 grape genes was detected by RNAseq across all berry samples. Principal component analysis was carried out with the normalized read counts of all detected genes. The two major PCs, which together accounted for 42.97% of the total variability, clearly separated the samples based on ripening stage, regardless of the vineyard of origin or their infection status . These results indicated that the intervineyard variation was smaller than the ripening effect, and the overall progression of ripening was similar between berries from GRBaV-positive and control vines.

Therefore, we hypothesized that GRBaV infections in berries have altered the expression of particular grape genes and/or molecular pathways, which could subsequently have led to developmental and metabolic deffects.While the PCA described above indicated that overall transcriptome dynamics associated with berry ripening were not perturbed by the infection, the lower levels of soluble solids and anthocyanins in GRBaV-positive berries, particularly later in development, suggested that red blotch may affect specific primary and secondary metabolic processes. We therefore focused the RNAseq analyses to identify grape molecular pathways that were differentially regulated as a result of GRBaV infections. We identified grape genes with significant differential expression due to red blotch by comparing GRBaV-positive and GRBaV-negative berries at each ripening stage and independently for each vineyard. We then looked at the intersection of differentially expressed genes between the two vineyards to identify common responses to red blotch. A total of 932 grape DE genes were found to be consistently down- or up-regulated in infected berries in both vineyards at a given ripening stage, and were classified as GRBaV-responsive genes . On average these GRBaV-responsive genes showed 0.49 ± 0.22-fold changes compared with the healthy controls. Comparing berries at similar ripening stages may have contributed to exclude more dramatic changes in gene expression associated with more pronounced ripening delay due to GRBaV. Key metabolic processes that were suppressed or induced as a consequence of red blotch in ripening berries were identified by enrichment analyses of the functional categories defined by Grimplet et al. in the set of GRBaV-responsive genes . Amino acid biosynthetic pathways were repressed in GRBaV-positive berries, while amino acid catabolic pathways were induced. Changes in carbohydrate metabolism were also observed; in particular, genes involved in glycolysis/gluconeogenesis and starch metabolism had reduced expression in GRBaV-infected berries. The suppression of these pathways may partially explain the lower soluble solids in the GRBaV-positive berries. Genes involved in nucleic acid metabolism, including RNA processing and surveillance, showed higher expression in GRBaV-infected berries. These pathways coincided at véraison with the induction of genes involved in stress responses to virus . RNA metabolic pathways are commonly altered in plants during viral infections and have been related to resistance or susceptibility depending on the particular plant–virus interaction . Red blotch also impacted the transcription of several abiotic stress response pathways. In particular, berries after véraison showed lower expression of genes encoding hypoxia responsive proteins and heat stress transcription factors, among others . A prominent feature of the GRBaV-positive berries was the transcriptional suppression of secondary metabolic pathways, in particular the biosynthesis of phenylpropanoids, stilbenoids, and lignin . Because the lower anthocyanin content observed in the GRBaV-positive berries may have resulted from reduced metabolic flux in the core phenylpropanoid pathway and alterations in flavonoid and anthocyanin biosynthesis, we pursued a deeper evaluation of these pathways using an integrated approach of transcriptional and metabolite profiling coupled to enzymatic analyses.

The rachis, skins and seeds were dried in oven and weighed. The pulp was separated from the juice + pulp with vacuum filtration using a pre-weighed Q2 filter paper . The largest portion of grape juice soluble solids are sugars. Sugars were measured at 25% using a Refractometer PAL-1 . Below ground biomass was measured by pneumatically excavating the root system with compressed air applied at 0.7 Mpa for three of the 12 sampling blocks, exposing two vines each in 8 m3 pits. The soil was prewetted prior to excavation to facilitate removal and minimize root damage. A root restricting duripan, common in this soil, provided an effective rooting depth of about 40 cm at this site with only 5–10 fine and small roots able to penetrate below this depth in each plot. Roots were washed, cut into smaller segments and separated into four size classes , oven-dried at 60 °C for 48 h and weighed. Larger roots were left in the oven for 4 days. Stumps were considered part of the root system for this analysis.In vineyard ecosystems, annual C is represented by fruit, leaves and canes, and is either removed from the system and/or incorporated into the soil C pools, which was not considered further. Structures whose tissues remain in the plant were considered perennial C. Woody biomass volumes were measured and used for perennial C estimates. Cordon and trunk diameters were measured using a digital caliper at four locations per piece and averaged,plastic plant pot and lengths were measured with a calibrated tape.

Sixty vines were used for the analysis; twelve vines were omitted due to missing values in one or more vine fractions. All statistical estimates were conducted in R.The present study provides results for an assessment of vineyard biomass that is comparable with data from previous studies, as well as estimates of below ground biomass that are more precise than previous reports. While most studies on C sequestration in vineyards have focused on soil C, some have quantified above ground biomass and C stocks. For example, a study of grapevines in California found net primary productivity values between 5.5 and 11 Mg C ha−1—figures that are comparable to our mean estimate of 12.4 Mg C ha−1 . For pruned biomass, our estimate of 1.1 Mg C ha−1 were comparable to two assessments that estimated 2.5 Mg of pruned biomass ha−1 for both almonds and vineyards. Researchers reported that mature orchard crops in California allocated, on average, one third of their NPP to harvestable biomass, and mature vines allocated 35–50% of that year’s production to grape clusters. Our estimate of 50% of annual biomass C allocated to harvested clusters represent the fraction of the structures grown during the season . Furthermore, if woody annual increments were considered this proportion would be even lower. Likewise the observed 1.7 Mg ha−1 in fruit represents ~14% of total biomass , which is within 10% of other studies in the region at similar vine densities. More importantly, this study reports the fraction of C that could be recovered from wine making and returned to the soil for potential long term storage. However, this study is restricted to the agronomic and environmental conditions of the site, and the methodology would require validation and potential adjustment in other locations and conditions.

Few studies have conducted a thorough evaluation of below ground vine biomass in vineyards, although Elder field did estimate that fine roots contributed 20–30% of total NPP and that C was responsible for 45% of that dry matter. More recently, Brunori et al. studied the capability of grapevines to efficiently store C throughout the growing season and found that root systems contributed to between 9 and 26% of the total vine C fixation in a model Vitis vinifera sativa L. cv Merlot/berlandieri rupestris vineyard. The results of our study provide a utilitarian analysis of C storage in mature wine grape vines, including above and below ground fractions and annual vs. perennial allocations. Such information constitutes the basic unit of measurement from which one can then estimate the contribution of wine grapes to C budgets at multiple scales— fruit, plant or vineyard level—and by region, sector, or in mixed crop analyses. Our study builds on earlier research that focused on the basic physiology, development and allocation of biomass in vines. Previous research has also examined vineyard-level carbon at the landscape level with coarser estimates of the absolute C storage capacity of vines of different ages, as well as the relative contribution of vines and woody biomass in natural vegetation in mixed vine yard-wild land landscapes. The combination of findings from those studies, together with the more precise and complete carbon-by-vine structure assessment provided here, mean that managers now have access to methods and analytical tools that allow precise and detailed C estimates from the individual vine to whole-farm scales. As carbon accounting in vineyard landscapes becomes more sophisticated, widespread and economically relevant, such vineyard-level analyses will become increasingly important for informing management decisions. The greater vine-level measuring precision that this study affords should also translate into improved scaled-up C assessments . In California alone, for example, there are more than 230,000 ha are planted in vines. Given that for many, if not most of those hectares, the exact number of individual vines is known, it is easy to see how improvements in vine-level measuring accuracy can have benefits from the individual farmer to the entire sector.

Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto rootstock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites perse, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, plant health and fruit production,plastic planter pot as well as for those concerned with C storage. The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.High bush blueberries , native to the northeastern United States, are important commercial fruit and are the most planted blueberry species in the world . In the United States, blueberries traditionally have been grown in cooler northern regions; however, the development of new southern cultivars with low chilling-hour requirements has made possible the expansion of blueberry production to the southern United States and California .Blueberry production in California was estimated in 2007 at around 4,500 acres and is rapidly increasing.

Common southern cultivars grown include ‘Misty’ and ‘O’Neal’, but other improved southern high bush cultivars are now being grown from Fresno southward, such as ‘Emerald’, ‘Jewel’ and ‘Star’ . Southern high bush “low-chill” cultivars are notable for their productivity, fruit quality and adaptation , and require only 150 to 600 chillhours, making them promising cultivars for the San Joaquin Valley’s mild winters . Since 1998, we have conducted long-term productivity and performance evaluations of these cultivars at the University of California’s Kearney Agricultural Center in Parlier . North American production of high bush blueberry has been increasing since 1975, due to expansion of harvested area and yields through improvements in cultivars and production systems. In 2005, North America represented 69% of the world’s acreage of high bush blueberries, with 74,589 acres producing 306.4 million pounds . Acreage and production increased 11% and 32%, respectively, from 2003 to 2005. The U.S. West, South and Midwest experienced the highest increases in acreage. In 2005, 63% of the world’s production of high bush blueberries went to the fresh market. North America accounts for a large part of global high bush blueberry production, representing 67% of the fresh and 94% of the processed markets . Blueberry consumption is increasing, which is encouraging increased production. As a result, fresh blueberries are becoming a profitable specialty crop, especially in early production areas such as the San Joaquin Valley . In general, a consumer’s first purchase is dictated by fruit appearance and firmness . However, subsequent purchases are dependent on the consumer’s satisfaction with flavor and quality, which are related to fruit soluble solids , titratable acidity , the ratio of soluble solids to titratable acidity, flesh firmness and antioxidant activity . Vaccinium species differ in chemical composition, such as sugars and organic acids. The sugars of the larger high bush blueberry cultivars that are grown in California are fructose, glucose and traces of sucrose. Lowbush blueberries — which are wild, smaller and grow mostly in Maine — lack sucrose. The composition of organic acids is a distinguishing characteristic among species. In high bush cultivars, the predominant organic acid is usually citric , while the percentages of succinic, malic and quinic acids are 11%, 2% and 5%, respectively. However, in “rabbiteye” blueberries the predominant organic acids are succinic and malic, with percentages of 50% and 34%, respectively, while citric acid accounts for only about 10% . These different proportions of organic acids affect sensory quality; the combination of citric and malic acids gives a sour taste, while succinic acid gives a bitter taste . In addition to flavor, consumers also value the nutritional quality of fresh fruits and their content of energy, vitamins, minerals, dietary fiber and many bioactive compounds that are beneficial for human health . Fruits, nuts and vegetables are of great importance for human nutrition, supplying vitamins, minerals and dietary fiber. For example, they provide 91% of vitamin C, 48% of vitamin A, 27% of vitamin B6, 17% of thiamine and 15% of niacin consumed in the United States . Thedaily consumption of fruits, nuts and vegetables has also been related to reductions in heart disease, some forms of cancer, stroke and other chronic diseases. Blueberries, like other berries, provide an abundant supply of bioactive compounds with antioxidant activity, such as flavanoids and phenolic acids . For example, a study performed in rats showed that when they were fed diets supplemented with 2% blueberry extracts, age-related losses of behavior and signal transduction were delayed or even reversed, and radiation-induced losses of spatial learning and memory were reduced .

As this research field has progressed, it has become apparent that extraction of soil and plant waters for isotope analysis is beset with a number of methodological issues . Soil waters held under different tensions may have different isotopic characteristics: for example, freely moving water sampled by suction lysimeters often shows a much less marked evaporative fractionation signal than bulk soil waters dominated by less mobile storage extracted by cryogenic or equilibration methods . Such differences between extraction techniques may be exacerbated by soil characteristics, such as texture and organic content, which may in turn affect the degree to which water held under different tensions can mix . Similarly, sampling xylem and its resulting isotopic composition has been shown to be affected by methodology. It is usually assumed that methods such as cryogenic extraction isolate water held in xylem, when in fact water stored in other cells may be mobilized to “contaminate” the results . Interpretation of plant-soil water relationships can also be complicated by processes in plants and soils that alter isotopic compositions independently. For example, the spatio-temporal isotopic composition of soil water can change dramatically in relation to precipitation inputs, evaporative losses, internal redistribution and phase changes between liquid and gaseous phases . Moreover,10 plastic plant pots there is increasing evidence that plant physiological mechanisms may affect water cycling and the composition of xylem water . These include effects of mychorrizal interactions in plant roots that may result in exchange and fractionation of water entering the xylem stream .

Research also indicates that as flow in xylem slows, diffusion and fractionation can occur , which may involve exchange with phloem cells . Finally, there is increasing evidence that water storage and release from non-xylem cells may sustain transpiration during dry periods or early in the day , also affecting xylem composition. Thus, there is a need to understand the different timescales involved in uptake processes in the rooting zone, residence times and mixing of water in different vegetation covers . There is also evidence of differences between how such factors affect water movement in angiosperms and gymnosperms, as well as species-specific differences . Clearly, these methodological issues will take some time to address; in the interim there is a need for cautious interpretation of emerging data from critical zone studies in order to improve our understanding.A striking feature of isotopic studies of soil-vegetation systems is a bias to lower and temperate latitudes, with northern latitudes and cold environments being under-represented . Yet, northern environments present particular challenges and opportunities to further advance the growing body of knowledge about plant-soil water interactions. For example, the coupled seasonality of precipitation magnitude and vegetative water demand can be complicated by the seasonality of the precipitation phase. Cold season precipitation that accumulates as snow can replenish soil water in the spring and be available to plants months after deposition . Despite the lack of studies, these areas are experiencing some of the most rapid changes in climate and, as a result, vegetation . The effects of climatic warming on patterns of snow pack accumulation and melt can have particularly marked consequences for soil water replenishment and plant water availability, particularly at the start of the growing season .

Despite the importance of northern environments, remoteness and harshness of environmental conditions result in logistical problems that constrain lengthy field studies and data collection . This study seeks to contribute to the growing body of knowledge about plant-soil water interactions by expanding the geographical representation of sites in cold northern environments. We report the findings of a coordinated project on xylem water isotopic data collection in the dominant soil – vegetation systems of five long-term experimental sites. Isotopic characteristics of soil water have previously been reported for all five sites; this used a comparative approach with, as far as possible, common sampling methods across the sites for a 12 month period . Here, we present xylem water isotopic composition data collected using common methods over the same time period encompassing the complete growing season, and then relate findings to soil water isotopic compositions. The study was conducted at five long-term experimental catchments across the boreal or mountainous regions of the northern latitudes . The catchments were part of the VeWa project funded by the European Research Council investigating vegetation effects on water mixing and partitioning in high-latitude ecosystems . Previous inter-comparison work on this project has examined such issues as changing seasonality of vegetation-hydrology interactions , soil water storage and mixing , water ages and modelling the interactions between water storage, fluxes and ages .At Bruntland Burn, study sites were dominated either by Scots pine or Ericacae species . Dominant vegetation at the Dorset sites was either coniferous trees , Eastern white cedar , Eastern white pine at sites He, Ce, Pw, respectively or deciduous red oaks . At Dry Creek, tree-dominated high elevation locations included Douglas fir and Ponderosa pine .

Mid-elevation sites had a mixture of similar trees plus shrubs including Sagebrush . Low elevation sites had no trees, but a variety of additional shrubs including Bitter brush , Chokecherry , Yellow willow and Water birch . At Krycklan, Norway spruce and Blueberry were present at site S04 about 4 m away from a stream, while Scots pine and Blueberry were the dominant species at the upslope site S22 about 22 m from the stream. The Wolf Creek sites, RP in the riparian zone and PL located on a relatively dry plateau, were vegetated by birch and willow shrubs . Prevailing soil textures at the sites varied from loam to silty sands . Soil characteristics are described in detail by Sprenger et al. . Briefly, these are podzolic soils at Bruntland Burn, Dorset and Krycklan, loamy sand at Dry Creek, and Wolf Creek had considerable amounts of organic matter in the upper soil layers. At Dry Creek, shrub and tree roots extend through the soil column, which ranges from ~10 cm to ~120 cm thick. Ponderosa pine roots may extend into fractured bedrock. The rooting depths are limited to the upper 15 cm for the heather sites at Bruntland Burn and to 50 cm depth for trees at Krycklan and Dorset. Rooting depths at Wolf Creek and Bruntland Burn are largely within the top 30 cm with smaller fractions to 50 cm. At each site, plants and surrounding soils were sampled concurrently for isotope analysis following a common sampling protocol . Depending on the nature of the soil cover,plastic pots large the maximum depth of sampling varied from -20 cm at BB to -70 cm at Dry Creek . Sampling took place at 5 cm intervals for Bruntlad Burn, Dorset, and Krycklan with two to five replicates for each depth. At Dry Creek, sampling was done at -10, -25, -45, and -70 cm with two to four replicates. Sampling depths at Wolf Creek varied between -2 and -40 cm with one to three replicates. Daily soil moisture data based on continuous soil moisture measurements at 10 or 15 cm soil depth were available for each soil water sampling location at Bruntland Burn, Dry CReek, Krycklan, and Wolf Creek. Only weekly manual soil moisture measurements were available for Dorset, and daily soil moisture data were derived from soil physical modelling . The volumetric soil moisture data were used to assess the hydrologic state on the sampling days. Plant samples from trees with a diameter > 30 cm were taken horizontally with increment borers at breast height . Retrieved plant xylem cores were directly placed in vials without bark and phloem. Shrub vegetation was sampled by clipping branches. These were immediately placed in vials after the bark was chipped off or left on . All vials were directly sealed with parafilm and immediately frozen until extraction was conducted at Boise State University, Boise, Idaho, USA. There were five replicates for each species and day at the sites in Bruntland Burn, Krycklan, Dorset. At Wolf Creek, the number of replicates varied between two and five and there were always four replicates for each sampling campaign at the Dry Creek sites. In total, 1160 xylem water samples were collected; 831 for angiosperms and 329 for gymnosperms . Dates of sample events varied at each site, but included the end of the growing season/senescence, pre-leaf out the following year, post leaf out, peak growing season and senescence . Precipitation was sampled daily or on an event basis at Bruntland Burn and Krycklan. Daily to fortnightly precipitation sampling was conducted at Dorset, Dry Creek, and Wolf Creek. Melt water was sampled from lysimeters at Krycklan, Dorset, Dry Creek and Wolf Creek during several snow melt events, while snowfall seldom occurred over the study year at Bruntland Burn . Various measures were taken to prevent evaporation of collected precipitation, including paraffin oil and water locks prior to transfer to the laboratory. The long-term groundwater signal was assessed at all sites, apart from Dorset, using several sampling campaigns of springs and wells tapping the saturated zone over the last few years .

There were no nearby wells from which to sample the regional groundwater at Dorset, which is found well below the surface in the granitic gneiss and amphibolite bedrock.Source water apportionment of plant xylem: To quantify the potential source of vegetation water from different soil depths and over a range of time periods, a modification of the ellipsoid method was utilized for the gymnosperms and angiosperms at soil depths in 10 cm increments up to 40 cm. All soil samples deeper than 40 cm were lumped together. The 40 cm cut off was chosen due to fewer sites sampling below 40 cm and a large decrease in the temporal resolution of sampling which could otherwise skew results. Due to soil water fractionation resulting in deviation from the local meteoric water line, the data are not well represented in an ellipsoid shape such as that employed in Amin et al. . Therefore a minimum polygon area was used to encompass the data points. Plant water and soil water data from the five sites are plotted in Figure 2. For both soils and xylem, the sites occupied partially overlapping regions showing a general gradient from highly isotopically depleted at Wolf Creek, the coldest of our sites in Canada, to the more isotopically enriched waters at Bruntland Burn at the temperate/boreal transition in Scotland. For each site there was a substantial range of variability in soil and xylem water isotope composition over the course of the sampling year. Most soil and xylem samples plotted below the GMWL, although xylem waters were generally more 2 H-depleted at each site, which was also evident from the lc-excess data . Samples from Dry Creek and, in particular, Wolf Creek showed the greatest divergence from the GMWL. These two sites slightly obscured an otherwise clear relationship between plotting position along the GMWL and the mean annual temperature gradient through Krycklan, Dorset and Bruntland Burn. Despite this, the isotopic ratios of δ2 H and δ18O in soils and xylem water correlate positively with air temperature, annual precipitation and aridity index, and negatively with elevation and to a lesser extent latitude . At all sites, substantial isotopic differences were apparent between xylem and soil water isotopes, and between angiosperms and gymnosperms . Soil waters at each site generally tracked precipitation and snowmelt inputs being more 2 H- and 18O-depleted in winter/spring and more enriched in summer; evidence of evaporative fractionation was also most evident in the more 2 H- and 18O-enriched summer soil water samples. The soil water data are shown relative to the sampling dates for each site in Figures S2 to S6 in the Supplementary material; also see Sprenger et al. for more detail. Soil water δ2 H data were significantly different from precipitation at Dry Creek, Dorset and Wolf Creek, while soil water δ18O differed from precipitation at Bruntland Burn and Dorset . Bruntland Burn, Krycklan and Dorset showed the greatest visual deviation of xylem δ2 H samples from soil water, while the most southern site, Dry Creek, and the most northern site, Wolf Creek, showed smaller differences between the xylem and soil water isotopes for δ2 H . However, at all sites the δ2 H characteristics of both angiosperms and gymnosperms were significantly different from soil water . Angiosperm xylem water δ18O at all sites, apart from Krycklan, was significantly different from soil water δ18O; whereas significant differences for gymnosperms were apparent only for Dorset and Bruntland Burn.

Data were analyzed using linear mixed models and generalized linear mixed models with the GLIMMIX procedure of SAS . The normal or log‐normal distribution and identity link function were used for continuous response data, the Poisson distribution and log link function for count data, and the binary distribution and logit link function for binary data. Inverse link functions were used to transform predicted means back to the original units of measure for presentation. P-values for multiple mean comparisons were adjusted with the Holm‐Tukey method. Potential relationships between vegetative and rooting attributes were analyzed using linear regression . The cultivars with high rooting percentage were Desertnyi, Eversweet, Golden Globe, Haku Botan, Ki Zakuro, Loffani, Nochi Shibori, Parfianka, Phoenicia, and Wonderful. Ambrosia and Green Globe had lower rooting percentages compared to other cultivars in both experiments. These data are consistent with other studies on rooting of pomegranate cuttings. Karimi et al. reported significant differences among cultivars for several growth attributes. Highest rooting percentages previously reported were 90% by Singh et al. , 86.7% by Polat and Caliskan , and 96.8% by Owais . Hussain et al. reported a mean percentage of 75.6% for hardwood cuttings propagated under different environmental conditions. Overall, research results indicated that pomegranate is readily propagated by hardwood cuttings,blueberry container size but there are exceptions, with certain genotypes more difficult to root than others.

Green Globe rooted poorly in both experiments, with rooting approximately 25% that of most other cultivars evaluated. According to Kennedy , Mae is a cultivar in the USDA NCGR collection that also roots poorly using hardwood cuttings, but no published data confirm this report. Dry root mass data are generally in agreement with Owais , who reported a maximum dry root mass of 0.223 g for hardwood cuttings. In our experiments, a gel formulation of IBA was used to stimulate rooting. Gel formulations are used less commonly in commercial propagation than liquid or powder formulations, but acceptable rooting was obtained using 3 g×L -1 IBA for all cultivars except Green Globe. In contrast, Owais utilized very high concentrations of IBA, up to 12 g×L -1 , and found that cuttings treated with 6 or 9 g×L -1 IBA had the highest rooting percentages compared to control cuttings or cuttings treated with 3 or 12 g×L -1 IBA. Karimi et al. reported that rooting percentages of pomegranate were lower for cuttings treated with 1 g×L -1 IBA when compared to non-treated cuttings. Some of these concentrations of IBA are higher than what is typically used for pomegranate in commercial practice. Our subsequent experiments with higher concentrations of IBA did not increase rooting percentages of ‘Ambrosia’ and ‘Green Globe,’ with ‘Ambrosia’ and ‘Green Globe’ having rooting percentages of 38.5% and 0%, respectively, for dormant hardwood cuttings treated with 9 g×L -1 IBA. If difficult-to-root cultivars are chosen for nursery propagation, further research on propagation methods will be needed.The factors causing one pomegranate cultivar to be more successful at rooting than another have not been confirmed.

Some research suggests that endogenous auxins explain cultivar differences in propagation. Auxins can regulate gene expression in plant tissues and endogenous auxin concentrations are under genetic control . According to Levin , ‘Wonderful’ and ‘Desertnyi’ are related, with ‘Wonderful’ being one of the parents used to hybridize ‘Desertnyi,’ with the parentage being x ‘Wonderful.’ Cuttings of both ‘Wonderful’ and ‘Desertnyi’ rooted at 96.9%. Whether rooting success is a heritable trait in pomegranate is unknown. There were significant differences in vegetative growth attributes between Wonderful and other cultivars in both experiments. Owais recommended hardwood cuttings of pomegranate be made 15 to 20 cm long and 6 to 7 mm in diameter. Our 10- cm cuttings rooted better than the 20-cm hardwood cuttings used by Owais , suggesting that dividing the longer cuttings in half not only produced twice as many cuttings, but also perhaps better rooting. In both of our experiments, stem diameter had no effect on any attribute measured and many cuttings were less than 6 mm in diameter. This indicated that more cuttings of smaller diameter may be used, but with the same propagation success. Wonderful, the industry standard, and nine other fruiting and ornamental cultivars of pomegranate rooted very well using hardwood cuttings treated with a gel formulation of 3 gL -1 IBA. Also, differences in certain vegetative growth attributes, such as plant height and branching, could be detected early in the production of some cultivars. This is the first known study to evaluate rooting and vegetative growth of dormant hardwood cuttings of Wonderful and other important cultivars from the USDA NCGR pomegranate germplasm collection. Further research is needed to discover methods to enhance the propagation success of the cultivars with poor rooting.

Global food security is threatened by climate change, which includes increasing temperatures and other unpredictable changes in weather patterns potentially damaging to agricultural systems . For example, long-term drought in California has caused significant economic losses to farmers . Drought in California has driven farmers to engage in crop abandonment, stress irrigation, the replacement of water-intensive crops to alternative crops , and most recently, using secondary water resources often available from urban or suburban wastewater systems. To lessen the impacts of climate change, it has been proposed that crop diversification may reduce food system vulnerability . Challenges posed by drought and water scarcity issues have led physiologists and breeders to focus on water-use efficiency in agriculture . Improving production efficiency and drought tolerance through cultivar selection has been proposed in citrus Prunus species , dates , and coffee , among others. Because tree crops have some degree of variability of physiological traits among genotypes in the same species, it is useful to study diversity within crop species to determine if there are cultivars that use water more efficiently or are as productive in cropping system conditions of high abiotic stress. Pomegranate is touted as a drought tolerant crop, especially once established , and it is being evaluated in California as an alternative tree crop species to replace or supplement more water-intensive species, such as avocado, citrus and almond , all three of which face pest and disease threats of their own. Historically, pomegranate has been grown in California for hundreds of years, even before statehood, starting with the Spanish missionaries who came over from Spain in the 1700s and planted mongrel seed from Spain in their gardens. Pomegranate was a minor crop in California until the 21st Century, when planting area increased by tenfold within twenty years and its value reached over $200 million annually. The body of knowledge regarding differences in pomegranate physiology, establishment, and site/climate effects among cultivars conserved in germplasm is limited. This limitation is a barrier to commercial growers and nurseries adopting new or forgotten cultivars that have traits with the potential to decrease water use and increase consumer demand for the fruit. Breeders also benefit from having germplasm phenotyped so that efforts to utilize molecular tools to map quantitative trait loci within breeding populations can be pursued for crop improvement. The pomegranate variety collection located at the United States Department of Agriculture – Agricultural Research Service National Clonal Germplasm Repository in Davis, CA, conserves about 200 genotypes of pomegranate in Winters, CA,growing raspberries in containers many of which have consumer-friendly phenotypic traits not seen in ‘Wonderful,’ the industry standard . Experiments have demonstrated differences in morphology and vegetative growth traits during the propagation phase of the present study . Studies have shown that there can be differences among cultivars for many agriculturally-relevant physiological traits of pomegranate in other collections, including transpiration rate, stomatal conductance, water-use efficiency, photosynthetic rate and chlorophyll content . The objectives of this research were: 1) to evaluate eleven unique pomegranate cultivars for field performance in semi-arid and coastal Mediterranean climates to determine plant establishment rates and site effects on reproductive biology; and 2) to evaluate four pomegranate cultivars for field performance in coastal versus inland agroecosystems to determine if there are differences among genotypes for physiological traits that would be conducive to commercial crop production in drought conditions.

The sites were located at the Agricultural Experiment Station of the University of California, Riverside, CA and on private land in Somis, CA . Riverside, CA is a semi-arid climate with hot, dry summers and cool winters and Somis, CA is a coastal Mediterranean climate with warm, dry summers and wet, cool winters. At Riverside, the mean annual precipitation of the area is 262 mm and mean high temperatures are 28⁰ and 36⁰ C for June and August, respectively. Mean temperature lows are 13⁰ and 18⁰ C for June and August, respectively and 5⁰, 6⁰, and 7⁰ C for December, January and February, respectively. The soil is a sandy loam with good drainage and was previously an established lemon grove. At Somis, mean annual precipitation of the area is 468 mm and mean high temperatures are 26⁰ and 28⁰ C for June and August, respectively. Mean temperature lows are 13⁰ and 14⁰ C for June and August, respectively and 5⁰, 5⁰, and 6⁰ C for December, January and February, respectively. The soil at Somis is a heavy sandy clay that was previously an established avocado grove. All trees were growing under natural light, outside in field conditions with drip irrigation three times per week to replace water lost to soil evapotranspiration as determined by California Irrigation Management Information System weather stations in Riverside and Ventura counties. Trees were grown under conventional commercial management practices and fertilized in spring with urea and potassium sulfate, totaling 31.75 kg N, and 34 kg K per year, respectively, over approximately 0.81 ha. All experimental trees were followed during their first four years of development and were located on the inside of the grove, with at least one border tree acting as a buffer to reduce the edge effect.Two pomegranate cultivar trials were utilized for this study. The cultivars utilized in the study were Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, Haku Botan, Loffani, Parfianka, Phoenicia, a proprietary cultivar and Wonderful . Cultivars were either cultivars bred for coastal sites and those that are typically grown in inland sites. Coastal cultivars included Ambrosia, Eversweet, Golden Globe, Green Globe, and Loffani. Inland cultivars included Desertnyi, Haku Botan, Parfianka, Phoenicia, and Wonderful. For the proprietary cultivar included, it is unknown whether it is a coastal or inland cultivar. All plants were sourced from the National Clonal Germplasm Repository, Davis, CA, and propagated via dormant hardwood cutting at the same time in winter of 2012 and. All trees included were mature and most cultivars were bearing commercial loads of fruit by the fourth year. For physiology trait evaluations, the healthiest tree in each of three blocks were selected among 15 trees per cultivar in the trial, which was planted in a randomized complete block design. Wonderful is the industry standard in many countries and was chosen as a control in the cultivar field trial. Wonderful is a widely-grown commercial cultivar that originated in Florida, it accounts for approximately 90-95% of production in the USA. It is a highly vigorous, thorny tree that has high yield with red fruit that have red arils with moderate seed hardness and a sweet-tart flavor. The growth habit is willowy, with a tendency to sucker at the base of the tree. There exists limited quantitative field establishment or physiology data for Wonderful and all other cultivars in this study. Trunk diameter was measured in cm with a digital caliper in spring of each year, with the measurement taken approximately 15 cm above the soil level. Tree canopy diameter was measured in-row and between rows to determine how quickly trees were growing into each other and into the rows. Tree height and in- and between row spacing measurements were taken with a folding rule. Tree canopy area was calculated with an equation reported by Serfontein et al. , where “S” is canopy area, “a” is tree height, “b” is skirt radius and “e” is eccentricity of an ellipse.During fruit development , an infrared gas analyzer was used to measure maximum rates of net CO2 assimilation , stomatal conductance , and transpiration during the morning hours. Morning light availability ranged from 1500-1600 µmol m-2 ·s-1 photosynthetic photon flux density . Photosynthesis measurements for three days were pooled for the four cultivars, which occurred on 29 July 2016, 4 August 2016, and 7 September 2016 for Riverside and 25 July 2016, 31 July 2016, and 31 August 2016 for Somis.

Recommendations or prescriptions must be simple, understandable and easy to implement in daily life by most patients. In the Appendix, we provide a leaflet as a guide for education and counselling in the field of hyperkalaemia for patients and professionals. In our experience the use of educational tools such as brochures with food images and inserts with “traffic light” colors can be useful during counselling to summarize some essential aspects of nutritional therapy and to help patients remember instructions. Several foods within the various food groups have been color-coded. The color green identifies foods that can be safely consumed even in hyperkalaemia, orange means foods that can be consumed with caution in hyperkalaemia, while in red are foods that should be avoided in hyperkalaemic patients if possible. This results in 3 columns, which allow users to build 3 kinds of diets characterized by mild, moderate or severe potassium restriction , recommended in the case of mild, moderate or severe chronic hyperkalaemia, respectively . In addition, we also provide several pragmatic suggestions meant to facilitate the application of potassium restricted diets in daily life . Obviously this tool, such as any other visual and practical tool, can only help in achieving excellent results if it is used within nutritional education programs appropriately applied by skilled and well trained health professionals who adapt the nutritional intervention to the individual patient. As we described above, the leaflets report a selection of foods from various food groups. It is possible that some foods are missing but boxes can be filled with other foods as needed,plastic potting pots to adapt the tool to patients’ habits, culture, traditions and needs. It is a great honor to receive the 2014 Cooley-Mead Award.

I feel especially honored when I review the names of those who have received this award in past years. Three are former Harvard teachers and colleagues of mine——Fred Bales, Alex Inkeles, and George Homans; many are old friends whose work I have long admired——Larry Bobo, Bernie Cohen, Gary Fine, Hal Kelley, Jane Piliavin, and Sheldon Stryker. The first Cooley-Mead Award was given to Muzafer Sherif in 1979. Had the award’s inception been two decades earlier, I am confident that Samuel Stouffer , a mentor and role model for me, would have also been a recipient. He was elected president of the American Association of Public Opinion Research in 1952 and the American Sociological Association in 1953. And he is certainly one of the truly great and influential social psychologists in sociology’s history. His generation followed that of Charles Horton Cooley , George Herbert Mead , W. I. Thomas , and other founders of American sociology in the early development of social psychology. Together with Paul Lazarsfeld and Rensis Likert, Stouffer developed the probability survey into a refined research instrument for all the social sciences. He was also the vital leader of three of the major social science projects of the mid-twentieth- century: Gunnar Myrdal’s An American Dilemma, for which Stouffer kept the study going when Myrdal returned to Sweden when his nation was threatened by Germany’s Wehrmacht; The American Soldier series ,which Stouffer organized and directed on Army morale throughout World War II; and the survey study of McCarthyism published at the height of that dark period in American political history. This third book, Communism, Conformity and Civil Liberties , although six decades old, remains a model of how to analyze large-scale survey data and how to write about it simply and succinctly. Stouffer’s political courage was involved in this last work that undercut Senator Joseph McCarthy’s claim that the nation was highly concerned about Communists in government. In 1952, The FBI.’s J. Edgar Hoover received notice from a secret Harvard informant who claimed that, of all people, “Professor Talcott Parsons was probably the leader of an inner group” of Communist sympathizers at Harvard!

The informant alleged that the new Department of Social Relations had turned into a dangerous left-wing center as a result of “Parsons’ manipulations and machinations.” Based on this dubious “evidence,” Hoover authorized the Boston FBI. to initiate a security investigation of Parsons. Because he knew such suspected “Communists” as Parsons, Stouffer was then denied access to classified documents. Infuriated, Parsons in turn immediately prepared an affidavit in defense of Stouffer. “This allegation is so preposterous,” Parsons wrote, “that I cannot understand how any reasonable person could come to the conclusion that I was a member of the Communist Party or ever had been.” To Stouffer, he wrote, “I will fight for you against this evil with everything there is in me: I am in it with you to the death.” Stouffer soon responded. The Republican Methodist from small-town Iowa not only wrote Communism, Conformity and Civil Liberties soon after this episode, but he also successfully faced off a McCarthyist inquisition held in Boston. He arranged for his former doctoral students who were Roman Catholic nuns and priests, replete in their habits and collars, to sit ostentatiously behind him at the hearing. That proved too much for the nominally Catholic McCarthy; he abruptly ended the session. Stouffer’s many key contributions are not remembered today as fully as they deserve. So I devote this paper to recalling his life and achievements and to one of his most lasting social psychological conceptual contributions—relative deprivation. Stouffer was born and grew up in the small town of Sac City in western Iowa. In 1900, the year of his birth, Sac City boasted only 2,079 people, and today it has virtually the same population. His father was the publisher of the local newspaper, and he initially planned to join him in journalism. He majored in English at the tiny Methodist college, Morningside, just sixty miles to the west in Sioux City. Upon graduation, he set off for Harvard University where he earned his masters degree in English before returning to his father’s newspaper. His path to sociology began with a chance meeting that he once described to me in detail. One summer in the 1920s, he took his family to the Wisconsin lake country for a vacation. Restless and sociable as always, Stouffer quickly made acquaintance with other vacationers staying at nearby cabins. One of these neighbors was Edward Ross, the famous University of Wisconsin sociologist who at the time was one of the most cited writers in the discipline . Ross responded by giving him a copy of his then-popular social psychology textbook . Stouffer read it voraciously, became fascinated with social science, and wanted to know how one became a sociologist. Ross encouraged him to apply to a PhDprogram in the subject. With characteristic decisiveness, Stouffer soon applied to the University of Chicago’s sociology department. He was accepted, but to pay for his training he had to work long hours at the Chicago Tribune as a reporter.

Years later, he would attribute his writing style more to the Tribune than to the formal style of academia. “I’ll never be thought of as a great sociologist,” he liked to joke, “I’m not a theorist and everyone can understand every damn word I write!” Stouffer studied at Chicago with two leading American pioneers in quantitative techniques for social science——William Ogburn in sociology and Lewis Thurstone in psychology. After receiving his PhD in 1930, he went to the University of London to learn more about the rapidly developing field of statistics. There he spent a postdoctoral year with R. A. Fisher and Karl Pearson——world leaders in statistics. Ogburn and Fisher in particular greatly influenced him,raspberry container growing and he often mentioned them in lectures and seminar discussions. His rigorous body of work consistently reflects the training he received from these teachers. His important statistical contributions include the Htechnique for making Guttman cumulative scales practical , and Stouffer’s Z, a straightforward method for combining probabilities . Stouffer’s remarkable energy and single-minded devotion to his research are legendary. I could tell dozens of stories of his exploits, but I will restrict myself to a few revealing anecdotes. In 1948, national political surveys were put in bad repute by predicting that Harry Truman would lose the presidential election to Thomas Dewey. Fred Mosteller, the statistician, loved to tell the story of this survey debacle. He wondered how Stoufferwould react, but he should have known. Early the next morning, he saw Stouffer plowing at top speed across Harvard Yard to Emerson Hall, fired up to begin a major study as to why the surveys had gone wrong. Stouffer was a heavy cigarette smoker. But, consistent with his intense style, he was extremely careless about his cigarette ashes. He regularly allowed the ashes to drop without noticing them. As might be expected, this habit had unfortunate consequences. At regular intervals, small paper fires would spring up from ashes falling on his desk while he worked preoccupied as ever. Usually, he took these incidents in stride. He would pop out from his office and calmly ask his frightened secretary to hand him the fire extinguisher. But once I witnessed a different reaction. He sprang from his office with uncharacteristic alarm as smoke poured out. What made the difference this time was that it was his annual tax returns that were going up in smoke! In the fall of 1957, I returned to Harvard as an assistant professor of social psychology in Harvard’s then-titled Social Relations Department. I audited Stouffer’s famous survey analysis graduate seminar. In September 1957, the racial desegregation crisis at Little Rock, Arkansas’s Central High School burst forth on the very same day the seminar held its first meeting. Stouffer rushed excitedly into the seminar room. He looked around the room at the students waiting for the seminar to begin. “Campbell,” he muttered, “and you, Pettigrew—you are the two Southerners around here. I want you to leave right away for Little Rock and study what is going on down there!” The late Ernest Campbell, later of Vanderbilt University, was a visiting scholar at Harvard for the 1957–1958 academic year. We had never met, but we were the only two native Southerners within Stouffer’snear reach. He wanted us to leave on a plane that night; but we persuaded him that our wives might need to know where we were going and what we were doing. However, we did leave on a plane two days later. Wild as it may sound, there was a point to this hurried approach to field research. Like the good newspaperman he started out to be, Stouffer wanted to strike while the iron was hot. He strongly believed in gathering field data while events were unfolding, not months later after sharpening and leveling of people’s memories had taken place. His initial guidance was to start by interviewing a group in Little Rock that was caught in cross-pressures. Such a group, he assured us, would lay bare the underlying dynamics of both sides of the conflict. Ernie and I decided that the white ministers of modern Protestant denominations represented the ideal target group. Most of these men were racially far more liberal and open to school integration than their congregations. However, a major problem arose from the fact that promotions in the ministerial profession are heavily influenced by how successfully one raises money and enlarges the flock. And these critical resources were under the control of their conservative congregations. As our later publications detailed , these cross-pressures did indeed lead to intense situations in which the ministers often had to choose between their beliefs and their careers. Stouffer did not want us to have all the fun. He soon joined us for a few days to see how we were doing. When we told him what we had learned thus far, he took a special interest in the fundamentalist Protestant ministers. These men were not at all in conflict. They preached against racial integration at every opportunity to the delight of their pro-segregationist congregations. They held racial segregation to be ordained by God. Their popular actions drew members away from some of the modern denominations, which further heightened the pressures on the liberal ministers. Given his special interest, we took Stouffer to a Sunday evening service at a small church led by one of Little Rock’s most fervent segregationist ministers. Stouffer soon made it obvious that he was far more comfortable in this setting than we were.

Flavor chemical concentrations in refill fluids often exceeded concentrations permitted in other consumer products. These data support the regulation of flavor chemicals in EC products to reduce their potential for producing both cancer and non-cancer toxicological effects.Adverse health effects have been linked to electronic cigarette use in prior experimental studies on cells, animals, and humans, case reports,and Internet posts. The recent epidemic of “electronic cigarette or vaping product use associated lung injury” has further heightened concerns about the safety of ECs. The Centers for Disease Control and Prevention suggested that poor quality counterfeit and black-market products are linked to some EVALI cases10 and further recommended that vaping products not be used until the causes of EVALI are determined. 11 We have previously shown that some EC refill fluids contain very high concentrations of some flavor chemicals and that the presence of some flavor chemicals at high levels is significantly correlated with cytotoxicity. Although flavor chemicals have not been directly linked to EVALI, we did previously conclude that the high concentrations of flavor chemicals used in some EC refill fluids may cause adverse health effects. While many flavor chemicals in EC products are GRAS for ingestion; their safety has not been evaluated for inhalation. 16 Some EC products have flavor chemical concentrations that far exceed those acceptable for ingestion, for example, we have found cinnamaldehyde in one product at 343 mg/mL. Most prior studies on EC flavor chemicals have been done using products purchased in one country, often the USA,square pot and have generally focused on identification only. In this study, all products were manufactured by one company, and purchases were made in four different countries.

We compared the flavor chemicals in each product to determine: if there were variations in content and concentration with country, if products were cytotoxic, if specific flavor chemicals contributed to cytotoxicity, if any flavor chemicals or co-constituents were present in high enough concentrations to be a risk factor for cancer and how flavor chemicals in the current study compared to those we have examined previously. Mouse neural stem cells are sensitive to EC refill fluids, are amenable to highthroughput screening, and are an excellent model for neurological development. mNSC werecultured in Nunc T-25 tissue culture flasks containing growth medium prepared using methods previously described.For the MTT experiments, cell concentrations were determined using a BioMate 3S Spectrophotometer -based standard curve, and single cells were plated at 1500 cells/well in 96-well plates. For live-cell imaging in a BioStation CT , mNSC were seeded at 5000 cells/well in 24-well uncoated culture plates and allowed to attach overnight before imaging. Seeding densities were adjusted to achieve ~80-85% confluency at the end of the experiments. Human bronchial epithelial cells , which are often used in inhalation toxicology studies, were cultured in bronchial epithelial cell growth medium using protocols previously described.At 80% confluency, cells were harvested and plated at 3500 cells/well in pre-coated 96-well plates for the MTT assay.Direct effects of EC refill fluids or authentic standards of flavor chemicals on mitochondrial reductases were evaluated in concentration-response experiments that included untreated wells to control for vapor effects. After seeding and overnight attachment, cells were either treated with 0%, 0.001%, 0.1%, 0.03%, 0.1%, 0.3%, and 1% refill fluids solutions or 10 fold dilutions of the actual concentration of authentic standard solution made up in culture medium.

All treatments were incubated for 48 hours at 37 ºC. After treatment, 3–2,5- diphenyltetrazolium bromide reagent was added to wells and incubated for 2 hours at 37ºC. Solutions were removed from wells, and 100 µl of dimethyl sulfoxide were added to each well to solubilize formazan crystals. Absorbance readings were taken against a DMSO blank at 570 nm using an Epoch microplate reader . For each variable tested, three independent experiments were performed.For non-invasive analysis of cell morphology, motility, and survival, live-cell imaging was performed using a 10x phase contrast objective in a BioStation CT using automatic Z-focus. After attachment, mNSC were treated with refill fluid solutions at 0.1%, 0.3% and 1% made up in culture medium. Images were taken at 5 – 8 regions in each well once every 2 hours for 48 hours to collect time-lapse data for analysis. Evaluation of mNSC confluency, morphology, and survival was compared in control and treated groups using CL Quant software . For GC/MS analysis, each sample was analyzed twice, and the means were plotted using Prism software . For the MTT assay, data were normalized to the negative control , and treatment groups were expressed as percentages of the negative control. IC50s were computed using the log inhibitor vs. normalized response-variable slope in GraphPad Prism, and IC70s were evaluated visually. Statistical significance in the MTT assay was determined using a one-way analysis of variance , and when there was significance, treated groups were compared to the untreated control. In the live-cell imaging assay, significance was evaluated using a two-way ANOVA in which the variables were time and treatment. The number and concentrations of flavor chemicals in 105 refill fluids were evaluated . Each refill fluid was grouped into a product flavor category and compared for variability based on country of purchase. Refill fluid categorization was done according to flavors and types on the manufacturer’s website . Products are sorted from lef to right in Figure 1 in order of decreasing total concentrations of flavor chemicals.

Based on our target analyte list, the total number and concentration of flavor chemicals varied among products. Two “Q American Blend Tobacco” products did not have any chemicals on our target analyte list. Total flavor chemical concentration and number in original LIQUA flavors were high in “Two Apple” and “Peach” , “Mints,” and Two Mints” , and “RY4 Tobacco” , and “Sweet Accelerator” , and Cheesecake . Within the mint/menthol groups, the total concentration of flavor chemicals varied with “Mints” , having over twice the total concentration of the other products . In all these products, total flavor chemical concentration was > 10 mg/mL, and the total number of flavor chemicals was > 10. In contrast, low total concentrations of flavor chemicals were found in various categories . Based on the duplicate samples we processed, the total number of flavor chemicals and their concentrations were similar in most products with the same flavor name irrespective of country of origin . However, there were some exceptions, such as “Apple” , which was purchased in different cities within the USA and had different flavor chemical concentrations. The concentrations of flavor chemicals across all products ranged from 0.001 – 44.3 mg/mL . All products with ≥ 10 mg/mL in total flavor chemicals contained 3-9 dominant flavor chemicals , and the most frequently occurring were ethyl maltol, triacetin, corylone, ethyl vanillin, vanillin, and menthol . When comparing flavor chemical concentrations across duplicate products purchased in different countries, concentrations of specific chemicals were generally similar . However, we did find some differences. For example, the concentration of corylone was about five times lower in the “Peach” product purchased in the UK than in those from the two US sites and China. Moreover, for “Ry4 Tobacco”, the concentrations of corylone and furaneol varied with the location of purchase. The frequency of occurrence of the 126 flavor chemicals is shown in Figure 3a and Supplemental Figure 2. In descending order of frequency,blueberries in containers the most frequently used flavor chemicals, which appeared in at least 30 products, were triacetin , ethyl butanoate , ethyl maltol , γ-decalactone and δ-decalactone , hydroxyacetone , vanillin and ethyl acetate , 3-Hexen-1-ol and linalool , corylone , and phenethyl alcohol . Less frequently used flavor chemicals that appeared in fewer than 6 products are shown in Supplementary Figure 2. Using publicly available safety information, 22 flavor chemicals were grouped according to their potential to cause harm . Most of the flavor chemicals identified were either “irritants” or “harmful” . At the same time, two were “irritant and dangerous to the environment” , 2 were “harmful and dangerous to the environment” , and one was “toxic” . Additional information on flavor chemicals less frequently used is included in Supplementary Figure 2. Esters, terpenes, and ketones were the most abundant chemical classes .The cytotoxicity of 16 refill fluids that contained at least one flavor chemical ≥ 1 mg/mL and total flavor chemical concentrations ≥ 10 mg/mL is shown in Figure 4. The MTT assay, which evaluates the metabolic activity of mitochondria, was performed using mNSC and BEAS-2B cells after 48 hours of exposure to dilutions of refill fluids in submerged culture. Absorbances that are lower than the untreated controls indicate that the treatment decreased mitochondrial reductase activity. Cytotoxic refill fluids and their inhibitory concentrations at 70 % and 50 % are shown in Figure 4 and Table 2.

“Two Apples” and “Ry4” were the most cytotoxic refill fluids and duplicates from multiple countries produced similar results in the MTT assay. When cytotoxicity was observed, the mNSCs were generally more sensitive to the effects of the refill fluids than the BEAS-2B cells. Even though “Cheesecake,” “Peach,” “Mints” and “Honeydew” contained relatively high concentrations of flavor chemicals, they produced little to no response in either cell type in the MTT assay. Non-invasive analysis of mNSC growth was performed using time-lapse images of cells taken over 48 hours. “Two Apples” from Nigeria and China, and “Ry4” from the USA and China inhibited cell growth in a concentration-dependent manner irrespective of country of origin . In the treatment group with “Two Apples,” 2-way ANOVA revealed statistical significance as early as 12 hours and 20 hours for cells treated with EC refill fluid solutions at 1% and 0.3% . The effect observed when cells were treated with 0.1% solutions was statistically different from the control starting at 34 hours . Micrographs show images taken at 0, 24, and 48 hours. Compared to the untreated group, 0.3 and 1 % concentrations inhibited cell growth early in the experiment . The effects of “Ry4 Tobacco” on mNSC growth at 1% and 0.3% were similar with p values < 0.0001 starting at 10 hours . 0.1% differed significantly from the control beginning at 20 hours . Peach did not significantly alter growth in any treatment . To evaluate the effects of authentic standards of flavor chemicals individually and as mixtures, BEAS-2B cells were treated with concentrations of specific flavor chemicals that were dominant in “Peach” and “Mint” . Inhibitory concentrations at 70 % and 50 % which are indicators of cytotoxicity23 are shown in Table 3. Individually, triacetin , corylone , and γ-decalactone at the concentrations found in “Peach” would be cytotoxic to BEAS-2B cells. However, when combined, there was no effect in the MTT assay . Similarly, the concentrations of triacetin and carvone in “Mint” are high enough to induce significant cytotoxic effects individually, but when combined, the mixture was non-cytotoxic .Regression analysis was performed to determine if cytotoxicity correlated with total flavor chemical concentrations , the total number of flavor chemicals , and the concentration of individual flavor chemicals . The correlations were grouped into 3 categories: high , moderate , and low . Cytotoxicity was strongly correlated with total flavor chemical concentration for mNSC and moderately correlated for BEAS-2B cells . The relationship between the total number of flavor chemicals and cytotoxicity was moderate for BEAS-2B and not correlated for mNSC . The concentrations of six flavor chemicals were high to moderately correlated with cytotoxicity for both cell types . Although carvone was not very cytotoxic in the MTT assay, its concentration did correlate with cytotoxicity for mNSC cells, but not for BEAS-2B cells . Triacetin concentrations, which were high in “Peach” flavored products, were not correlated with cytotoxicity for BEAS-2B cells or mNSC .Some refill fluid chemicals are known or probable carcinogens. The Margin of Exposure approach aids risk managers in prioritization and is used by the FDA and other expert groups to access the cancer risk of food additives. 24-27 The MOE is the ratio of a reference point for an adverse effect to the estimated daily intake or exposure of a chemical in humans. Reference points obtained from experimental or epidemiological data based on dose-response curves include the BenchMark Dose , the No Observed Adverse Effect Level , or the Low Observed Adverse Effect Level .

We report a panel of 30 features, including 27 bacterial features with discriminatory ability to detect NAFLD-cirrhosis using a Random Forest classifier model. In a derivation cohort of probands, the model has a robust diagnostic accuracy for detecting NAFLDcirrhosis, confirmed in a validation cohort of relatives of proband with NAFLD-cirrhosis . This study provides evidence for a fecal-microbiome-derived signature to detect NAFLDcirrhosis.NAFLD is the most prevalent cause of chronic liver disease worldwide . NAFLDcirrhosis represents the most severe stage of the disease, carries a significant risk of hepatocellular carcinoma , and is consistently identified as the most important predictor of liver-related morbidity-mortality in NAFLD . However, non-invasive, accurate and easy-to-perform modalities for early detection of NAFLD-cirrhosis remain a major unmet need in the field. Overthe last decade, the gut-liver axis has emerged as a pivotal component of NAFLD and represents a potential source of non-invasive biomarkers for the detection and stage of liver disease . Limited data are available regarding the diagnostic accuracy of a stool microbiome derived signature for detecting NAFLD-cirrhosis. We previously demonstrated that first-degree relatives of probands with NAFLD-cirrhosis have a high risk of AF . However, factors associated with progression towards NAFLDcirrhosis among families remain obscure. Although earlier studies reported familial aggregation of NAFLD and NAFLD-related cirrhosis ,square pots and demonstrated that both liver steatosis and fibrosis are heritable , known genetic risk only accounts for ~10-30% of the variance observed in NAFLD .

This suggests an additional role for environmental factors, which predominate over genetic factors in shaping the human gut-microbiome . Heritability of gut-microbiome features has been reported in twins studies, but limited data exist regarding the similarity of gut-microbiome composition among family members, and whether similar microbiomes associate with disease traits especially in the entire spectrum of NAFLD including NAFLD-cirrhosis.Using a uniquely phenotyped twin and family study design including well-characterized participants with and without NAFLD, assessed using MRI-proton-density-fat-fraction for quantifying hepatic steatosis and MR-elastography for quantifying liver fibrosis , we examined familial correlation of gut-microbiome composition and tested whether a non-invasive stool-microbiome-derived signature accurately detects NAFLD-cirrhosis. This cross-sectional analysis included 203 well-characterized, prospectively recruited participants, encompassing the entire spectrum of NAFLD divided into three groups paired with their first-degree relatives. Subjects included 26 probands with NAFLD-cirrhosis and 37 of their first-degree relatives, 18 probands with NAFLD without advanced fibrosis and 17 of their first-degree relatives, and 54 non-NAFLD normal controls and 44 of their firstdegree relatives. The detailed derivation of the study cohort is shown in Figure 2.S1. The detailed demographic, biochemical, imaging data of the probands and first-degree relatives stratified by the metabolic and liver phenotype of the probands are provided in Supplementary Table 1 and Supplementary Table 21 , respectively.We identified a significant familial correlation of the gut-microbiome composition involving shared housing.

The gut-microbiome profile showed significant correlation within related pairs compared to random-unrelated pairs at the level of the phyla Figure 2.1a and at the level of 16S tag sequences Figure 2.1b. In our analyses at the phylum level, this familial correlation was mainly driven by significant correlation of Bacteroidetes and Actinobacteria between related individuals. Furthermore,phylogenetic dissimilarity assessed by unweighted UniFrac distances among related pairs was significantly lower than in random-unrelated pairs . When stratified by the liver phenotype of the proband, the phylogenetic dissimilarity remained significantly lower among nonNAFLD controls and relatives and probands with NAFLD without AF and relatives compared to unrelated pairs, while no significant difference was observed among probands with NAFLD-cirrhosis and relatives Figure 2.1c. These results suggest that familial gut-microbiome similarities are independent of mild/moderate liver phenotype but are impacted by severe stages of liver disease. Finally, related individuals with shared-housing had a lower phylogenetic dissimilarity than those who did not share housing Figure 2.1d. These results confirm a strong impact of the environment in the familial similarity of the gutmicrobiome and demonstrate that shared-housing is a major determinant that should be controlled for in study designs assessing the microbiome in liver disease. The gut-microbiome profile of NAFLD-cirrhosis was first assessed in a derivation cohort including the 3 groups of probands encompassing the entire spectrum of NAFLD. As shown in previous studies , α-diversity as measured by Faith’s phylogenetic diversity decreased with increase in liver damage severity . The β-diversity was lower among individuals with moderate liver damage compared to nonNAFLD controls , whereas it was higher among individuals with severe liver damage compared to probands with moderate liver damage  Figure 2.2b.

This suggests an hourglass signature of disease severity in the gutmicrobiome, with an initial decrease in phylogenetic diversity associated with a moderate stage of the disease that progress towards a phylogenetic dispersion in individuals with severe stages of disease such as NAFLD-cirrhosis. Further investigations with larger sample sizes are needed to determine whether this phylogenetic dispersion reflects a distinct profile among NAFLD-cirrhotic patients, and whether it is associated with specific NAFLD-cirrhosis related outcomes.Several taxa were differentially abundant in NAFLD-cirrhosis compared to non-NAFLD controls. At the genus level, the NAFLD-cirrhosis group was enriched with Streptococcus and Megasphaera, whereas Bacillus and Lactococcus were enriched in the non-NAFLD controls Figure 2.2c. Source data are provided as a Source Data file2 . Species belonging to the family Enterobacteriaceae and the genera Streptococcus and Gallibacterium were the most enriched inNAFLD-cirrhosis, while Faecalibacterium prausnitzii and species belonging to the genus, Catenibacterium and the families Rikenellaceae, Mogibacterium, Peptostreptococcaceae were enriched in non-NAFLD controls. These results are consistent with the study performed by Ponziani and colleagues in an Italian cohort showing higher Enterobacteriaceae and Streptococcus in NAFLD-cirrhosis with and without HCC. In addition, it confirms a shift towards more Gramnegative microbes in advanced fibrosis stages, as previously reported in NAFLD . A stool-microbiome signature accurately detects NAFLD-cirrhosis. A Random Forest model comprised of 30 features identifies probands with NAFLD-cirrhosis. The bacterial features most important for predicting NAFLD-cirrhosis are shown in Figure 2.3. In a derivation cohort of probands, the model had a robust diagnostic accuracy, with an AUROC of 0.92 after cross-validation for detecting NAFLD-cirrhosis Figure 2.4a. The diagnostic accuracy of the model was then confirmed in a validation cohort of first-degree relatives of proband with NAFLD-cirrhosis with good diagnostic accuracy, with an AUROC of 0.87 for the detection of advanced fibrosis with a high negative predictive value of 91.6% Figure 2.4b. . In addition, we performed sensitivity analyses in another validation group enriched with mild to moderate stage of NAFLD including probands with NAFLD without AF. The diagnostic accuracy of the model was confirmed and yielded a very good diagnostic accuracy with an AUROC of 86% Figure 2.S2.In conclusion, using a unique twin and family study design including well-characterized participants with and without NAFLD, we identified a specific stool-microbiome-derived signature of NAFLD-cirrhosis that yielded a robust diagnostic accuracy for the detection of NAFLD-cirrhosis. Hence,large plastic pots this conveniently assessed microbial biomarker presents an adjunct tool to current invasive approaches to determine the stage of liver disease. We previously demonstrated that a microbial biomarker can detect AF in biopsy-proven NAFLD . The fundamental difference between the previous study and the present study is the clinical context of use of the gut-microbiome signature. In the present study, the clinical question is to accurately differentiate using a non-invasive gut-microbiome signature who among the first degree relatives have advanced form of NAFLD and who are unaffected in a general setting as opposed to a liver clinic setting. The context of use is critical for biomarker development as suggested by the BEST Guidelines by FDA. In order to address this clinically important question, this study leverages 2 distinct levels of innovations. 1. Study design: This innovative study leverages from a unique prospectively recruited case-control study design. This cross-sectional analysis included 203 well-characterized participants, encompassing the entire spectrum of NAFLD divided into three groups paired with their first-degree relatives. 2. Discovery of shared housing effect: the familial cohort design enabled us to discover the effect of shared housing on the gutmicrobiome signature related to NAFLD-cirrhosis. This unique familial cohort study design led us to a new discovery that shared housing had a dominant effect on microbiome. This novel effect would not be apparent from previous study in NAFLD among unrelated individuals. Hence, this study is novel due to its clinical context of use, study design, and co-housing effect on gut microbiome in families with NAFLD-cirrhosis. We acknowledge the following limitations of this study. This is a single-center study performed in a center with expertise in clinical investigation of NAFLD with advanced MRI-based phenotyping, and the generalizability of the findings in other clinical settings remains to be established. 16S rRNA sequencing may not have captured additional insights associated with the disease status available at the species or strain level. Finally, the association does not suggest causality, and additional studies are needed to assess whether these microbial species impact gut permeability and/or induce NAFLD progression through cross-talk between serum metabolites and the liver .

However, the strengths of the study include a prospective study design, detailed phenotyping of participants using the most accurate non-invasive imaging modalities available, and assessment of accuracy using AUROC in both a derivation and validation cohort. Further multi-center studies including a larger number of individuals are needed to validate the clinical utility of the proposed microbiome-derived signature to detect NAFLD-cirrhosis. This is a cross-sectional analysis of a prospective family cohort study of participants from the Familial Cirrhosis cohort and Twins and Family cohort who were participating in a biobank initiative and prospectively recruited at the University of California at San Diego NAFLD Research Center between December 2011 and December 2017. All participants underwent a standardized exhaustive clinical research visit including detailed medical history, physical examination, and testing to rule out other causes of chronic liver diseases , fasting laboratory tests at the University of California at San Diego NAFLD Research Center . Participants also underwent an advanced magnetic resonance examination including magnetic resonance imaging proton-density-fat-fraction and magnetic resonance elastography at the UCSD MR3T Research Laboratory forthe screening of NAFLD and advanced fibrosis . Participants from the Familial Cirrhosis cohort also underwent an ultrasound-based vibration controlled transient elastography assessment using a FibroScan. At the time of each research visit, patients provided stool samples. These were collected and immediately stored in a -80*C freezer. Written informed consent was obtained from every participant.Probands and first degree relatives had to be at least 18 years old. Probands were required to have a documented diagnosis of NAFLD-cirrhosis either by liver biopsy or by documented imaging evidence by a protocol-specified criterion. First-degree relatives with written informed consent who did not meet any exclusion criteria were included in the study. Exclusion criteria included: regular and excessive alcohol consumption within 2 years of recruitment ; use of hepatotoxic drugs or drugs known to cause hepatic steatosis; evidence of liver diseases other than NAFLD, including viral hepatitis , Wilson’s disease, hemochromatosis, alpha-1 antitrypsin deficiency, autoimmune hepatitis, and cholestatic or vascular liver disease; clinical or laboratory evidence of chronic illnesses associated with hepatic steatosis, including human immunodeficiency virus infection , celiac disease, cystic fibrosis, lipodystrophy, dysbetalipoproteinemia, and glycogen storage diseases; evidence of active substance abuse, significant systemic illnesses, contraindication to MRI, pregnant or trying to become pregnant, or any other condition which, in the investigator’s opinion, may affect the patient’s competence or compliance in completing the study.The study included 140 participants from the Twin and Family study corresponding to 100 twins and 40 siblings or parents-offspring. The non-NAFLD controls included 54 probands and 44 first degree relatives and the group with NAFLD without AF included 18 probands and 17 first-degree relatives of community-dwelling controls either twin, sib-sib or parent-offspring pairs. These twin, sib-sib, and parent-offspring pairs were prospectively recruited and they reside in southern California. Twins without evidence of NAFLD and advanced fibrosis were considered as non-NAFLD control and twins with evidence of NAFLD without evidence of advanced fibrosis and their twin pair were randomly assigned as proband or first-degree relatives. The study was approved by the UCSD Institutional Review Board number 111282. Patients were included if they were twins, siblings or parent-offspring at least 18 years old, willing and able to complete all research procedures and observations. For each twin pair, a detailed assessment of twin ship status or dizygotic was obtained. The majority of twin-pairs were diagnosed by their physician as either MZ or DZ by genetic testing. Furthermore, twin-ship status was confirmed by using a previously published questionnaire .

Response surface methodology with the multi-response statistical technique was applied to optimize a yogurt formulation. The chemicals used in the experiments, including Folin-Ciocalteu reagent, analytical standards of tannic acid, and 2,2-diphenyl-1-picrylhydrazyl were purchased from Sigma-Aldrich . Sodium carbonate was obtained from Fisher Scientific . Milk powder was from the local market. Yogurt starter consisting of S. thermophilus and L. bulgaricus was purchased from Natural Probiotic Selection The manufacturing process was modified from El-Said et al. . To study the effects of protein and phenolic addition, A modified response surface methodology with the central composite design was applied with 5 levels of protein content and phenolic extract to form a 130g mixture. 2 times of water was used to extract the phenolic content from pomegranate peel. Protein was incorporated from skim milk powder and boiled water was added to make up 130g of weight. After homogenization, the mixture was fermented at 37 ºC for 36 h. For each response, linear, 2FI, and quadratic models were built. Models of the highest adjusted-R2 value without aliasing were selected, and all the responses could be extrapolated by linear models. Linear effects of protein content were significant on all response variables, while that of extract content were only related to TPC ,snap clamp firmness and DSA . The corresponding coefficients along with respective p-values were listed in Table 4.4. 3D response surface graphs were generated to visualize the interaction effects of protein and extract content on GSY characteristics . TPC increased with lower protein content and extract addition. This finding was in line with previous research.

Trigueros et al., incorporated pomegranate juice into yogurt and observed the polyphenolprotein interaction. After formulation, their PGY contained 40% of juice and presented 241.44 mg GAE/L of TPC, which meant 85.35% of the theoretically expected. They also evaluated the TPC of PGY permeate after 1-day storage and concluded a TPC of 111.92 mg GAE/L. indicating nearly 54% of the TPC remained interacting with milk proteins. An increase in DSA was observed with higher protein and extract content. The same pattern was found in the study carried out by Jiménez et al., As expected, syneresis decreased with enhanced protein content, which led to a dense yogurt matrix micro-structure and enhanced denaturation of whey protein . Usually, lower syneresis equals to longer shelf-life. However, a grainy texture should be avoided when enriching the milk base with high protein. pH and all the texture properties were positively correlated with protein content while not affected by the extracted content. S. thermophilus and L. bulgaricus in yogurt starter were able to produce exopolysaccharides during fermentation and improve yogurt texture. According to Sodini et al., , higher solid content was correlated with stronger EPS interaction with casein, therefore a stronger texture could be formed. ectin is widely found in the middle lamella layers between plant cells , forming a primary cell wall during plant growth and development . It is a family of heterogeneous polysaccharides consisting of α-1,4-Dgalacturonic acid , L-rhamnose , D-galactose , L-arabinose , and other 13 different monosaccharides through 20 different linkages . The pectin backbone primarily consists of D-GalA residues linked at α-1,4 positions. Based on the abundance of side chains, pectin can be divided into “smooth”, homogalacturonan, and “hairy” regions, namely rhamnogalacturonan I, rhamnogalacturonan II, xylogalacturonan, and apio-galacturonan .

A comparison of these regions is listed in Table 5.1 . The backbone unit, GalA, can be partially esterified with a methyl group or converted into the carboxylic acid amide with ammonia. . Based on the degree of methyl-esterification and acetylation, pectin can be divided into high-methoxyl pectin , low-methoxyl pectin , and amidated pectin as shown in Table 5.2.Besides the aforementioned significant industrial benefits, pectin has functional properties as dietary fiber, prebiotics, and fat replacer, as well as in antiglycation, antioxidant, and antibacterial. As a viscous soluble fiber, pectin is associated with lowering blood serum total cholesterol and low-density lipoprotein cholesterol , without changing high-density lipoprotein cholesterol . By increasing satiety, pectin can help weight control by reducing food consumption. . Due to its complex structure, pectin is a common drug formulation agent for oral delivery, in the form of tablets, gels, hydrogels, beads, aerogels, coated, and Pectin is considered a safe food substance through various government food agencies. Even though its usage and composition are regulated under different food additive laws, the Food and Drug Administration of the U.S. considers it GRAS. At the Joint FAO/WHO Expert Committee Report on Food Additives and in the European Union, no numerical acceptable daily intake has been set, as pectin is considered safe. Its abundant benefits stimulated great demands and the global pectin market size was valued at USD 964.1 million in 2015 . Polyphenol compounds can bind with the cell wall polysaccharides to strengthen the structure . In return, CPSs can interact with polyphenols to modify their bio-accessibility, bio-availability, and bio-efficacy . Liu et al., summarized multiple research that has been done to investigate the binding effects between pectin and polyphenol. Noncovalent bonds are dominant in the polyphenol-pectin interactions, such as hydrogen bonds, hydrophobic interactions, electrostatic interactions.

These weak bonds are sensitive to the intrinsic properties of polyphenol and pectin , as well as environmental conditions . Considering the binding property of pectin and polyphenols, a co-extraction of pectin and polyphenol should be considered because of the following advantages: Boosted stability of polyphenol and overall antioxidant activity; and Enhanced unique and beneficial characteristics for developing novel food products. As for stability, Oszmiański et al. proposed a cooperative hydrogen bond or hydrophobic interactions existed between the oxygen atom of pectin and the phenolic hydroxyl group. A natural pectin coating is speculated as less loss of polyphenols was observed due to the limited oxygen contact. The strength of pectin-polyphenol interaction is mainly determined by the non-covalent bonds, including hydrogen bonds, ionic bonds, and hydrophobic forces. Regarding the quality improvement, Hayashi et al., found the astringency of gallate-type polyphenol was reduced by the addition of pectin. A similar finding was reported in persimmons . Pectin polymer degradation is a fundamental step of extraction. The common degradation approaches include chemical hydrolysis, heating, radiation, and enzymatic reaction. Acid hydrolysis on pectin was one of the most conventional chemical degradation methods. Thibault et al. studied the mild acid hydrolysis of apple, beet, and citrus pectin. Inorganic solvents, including HCl and HNO3, have been widely used in the industry. However, they raised concerns in food applications and were difficult to recover, thus causing undesired pollution and high processing cost . Novel technologies with reduced pollution are in demand. Physical technologies, such as the non-thermal process, were superior due to their high efficiency, reduced pollution and cost, and ease of control. Ultrasound as a novel non-thermal technology has been widely applied in food industries,plastic gutter including extraction, preservation, emulsification, filtration, and cutting . Successful ultrasound applications in pectin extraction include citrus , apple , and sweet potato . Ultrasound extraction combined with acid hydrolysis was reported to further increase the pectin yield. Muñoz-Almagro et al. studied the pectin degradation under ultrasound power of 400 W in the presence of nitric acid and citric acid . The combined approach achieved higher pectin degradation than ultrasound or acid individually. In the meantime, no significant differences were found between using CA and nitric acid under ultrasound. Zhang et al., concluded the same findings when comparing the combined extraction with CA and hydrochloric acid. Since CA is a common food-grade ingredient that increases polyphenol stability , the technique of combining ultrasound and CA should be considered to extract pectin along with stable polyphenol. Enzyme extraction is another option for non-thermal pectin recovery. It relies on the enzymes that selectively degrade the cell wall composition to release the pectin, such as cellulases, hemicellulases, and proteases . Compared with traditional thermal processes, enzyme extraction can achieve selective catalysis reaction , reduce the solvent needed or increase higher yield with the same solvent . However, the major challenges for industrialization include high cost and high sensitivity to processing parameters . Besides TPY, DSA, and pectin yield, several other parameters were also evaluated to investigate the quality change of co-extraction through the extraction process at different temperatures, including galacturonic acid content, degree of methoxylation , and acetylation of pectin. GalA content was analyzed by hydrolyzing with sulfuric acid . 2 mg of pectin was mixed with 0.5 mL of DI water and 3 mL of concentrated sulfuric acid. After 15 mins of hydrolyzation in boiling water, the solution was cooled down in an ice bath. The solution was further mixed with 0.1mL of 0.15 % carbazole ethanol, incubated at 25 °C for 30 mins, and diluted 5 folds. The absorbance at 530 nm was recorded and compared with standard GalA. DM and DA are two major indicators of pectin accessibility.

Following the method modified by Wang et al. , DM and DA were quantified based on the molar percentage of methanol or acetic acid content from the GalA content. 5 mg of pectin was reacted with 0.5 mL of 0.2 mM CuSO4 for saponification, while 0.25 μM of isopropanol was added as internal standard. After 1 hr of incubation at 4 °C, the mixture was centrifuged at 8000 rpm for 5 min and adjusted to a pH of 2. HPLC system with C-18 column and refractometer function was applied for analysis, where the isocratic elution was with 4 mM sulfuric acid solution at a flow rate of 0.8 mL/min. A demonstration of major peaks, including methanol, acetone, and isopropanol, is shown in Figure 5.4. To speculate the chemical bonds involved in the co-extract, Fourier Transform Infrared spectroscopy analysis of the mixture was recorded by a Nicolet 6700 FTIR spectrometer at the absorbance mode with the frequency ranging from 4000 to 400 cm−1 and a resolution of 4 cm−1 . Box-Behnken design is an effective response surface methodology to evaluate the main effects of among multiple factors. Levels of variables and corresponding response results are shown in Table 5.5. Regression coefficients and statistic parameters for each model are exhibited in Table 5.6. Significant fitting was found in all responses , suggesting the effectiveness of regressions. TPY varied from 4.26 to 19.59% and was favored by higher ultrasound intensity, solvent ratio, extraction time, pH, and lower temperature. DSA was elevated from 1.12 g/g to 7.47 g/g at higher ultrasound intensity and solvent ratio but with lower extraction time, pH, and temperature. The results were consistent with the research from Pan et al. , which studied the effects of continuous and pulsed ultrasound-assisted on pomegranate polyphenol extraction using 25 °C water. They observed a positive relationship between phenolic yield and ultrasound intensity as well as extraction time. Acidic pH promoted the dissolve of phenolic acid compounds, until to a limit that the polyphenol compounds will be degraded into other molecules . In some conventional extraction research, high temperature tended to increase the TPY, due to the chemical reactions subjected to the heat treatment, including polymerization and the release of polyphenol compounds . However, Çam & Hışıl and Sood & Gupta concluded the negative correspondence of TPY towards high temperature due to heat-degradation, which resonated with the results hereby. Pectin yield demonstrated a linear positive dependence relative to ultrasound intensity, extraction time, and temperature, as well as acidic pH. Ultrasound treatment and high temperature stimulated the de-polymerization of pectin from the cell wall . Moorthy et al. utilized a 130W ultrasonic device on an Indian variety of pomegranate peel and obtained up to 24.18% of pectin yield. They also concluded the optimal pectin extraction pH to be 1.6 since, at this condition, the acidic hydrolysis of the insoluble pectin constituents into soluble pectin reached maximum pectin recovery. However, a lower pH value beyond 1.6 could cause the aggregation of pectin, therefore, contributing to a lower pectin yield. Pereira et al. researched pectin extraction from dried pomegranate peel using citric acid and yielded from 2.81% to 8.74. Similar responses of pectin yield to the extraction temperatures , times , and pH were concluded. The energy usage for the co-extraction was also recorded to guide the ultrasound-assisted extraction process design. After determining the major factors for co-extraction, pH 1.6 and ultrasound intensity of 90% were selected for the co-extraction kinetic study. Co-extraction using citric acid was conducted at 2, 10, 20, 30, 60, and 90 mins with 3 temperature levels: 25, 55, and 85 °C. For comparison, HCl at pH 1.6 was also utilized as a solvent to co-extract at 85 °C.