Horticulture

Using the race 1 resistance phenotypes observed in our studies and California Strawberry Commission production statistics , we discovered that susceptible cultivars have been planted on 49-85% of the acreage in California over the last eleven years . That percentage has hovered between 55 and 59% since 2014. Hence, susceptible cultivars continue to be widely planted in California despite incontrovertible evidence that losses to the disease can be prevented by planting cultivars carrying one of the race-specific R-genes we identified . Over five years of screening plants that were either naturally infected or artificially inoculated with race 1 isolates of the pathogen, we have not observed visible symptoms on cultivars or other germplasm accessions carrying the dominant FW1 allele . Although private sector cultivars were unavailable for inclusion in our studies,seedling starter pot the prevalence of R-genes in publicly available germplasm collections and shared ancestry of public and private sector cultivars worldwide suggests that the same R-genes are widely found in private sector cultivars .

We anticipate that production in California will ultimately shift away from susceptible cultivars, particularly as the incidence of the disease increases and yield losses mount .The cloning and characterization of R-genes underlying race-specific resistance is important for developing an understanding of their function and interactions with the pathogen and building the foundation needed to engineer resistance through genome editing or other approaches . The I genes that confer race-specific resistance to F. oxysporum f. sp. lycopersici in tomato differ in durability and function and provide a model for future studies in strawberry . The I-2 gene had a signifcantly longer life span than the I gene, which was defeated in less than a decade subsequent to deployment . The durability differences of the tomato I genes have been attributed to differences in the dispensability of avirulence genes or mutations in avirulence genes that defeat known R-genes . The life spans of the race-specific R-genes we identified in strawberry are of course unknown; however, the sheer abundance and diversity of race 1 R-genes found in the wild relatives predict that sources of resistance to other races of the pathogen can be rapidly identified and deployed . The earliest reports of resistance to California isolates of the pathogen emerged when the disease initially surfaced in field experiments in California where well known cultivars were being grown .

We have since shown that the resistance phenotypes of those cultivars were mediated by FW1; hence, the FW1 gene has endured for at least 16 years to-date. Our search for diverse R-genes was partly motivated by the need to prepare for the havoc created by the inevitable evolution and emergence of novel pathogen races and inadvertent introduction of foreign races of the pathogen through infected plants or soil . Our findings suggest that the resistant germplasm accessions identified in the present study carry one or more dominant Fusarium wilt R-genes, and that race 1 R-genes are found in a wide range of heirloom and modern cultivars . The latter finding further suggests that race 1 resistance genes are found in domesticated populations worldwide, albeit often at low frequency because they have not been consciously selected, e.g., we previously showed that the frequency of FW1 was 0.16 in the pre-2015 California population and that FW1 originated in Shasta and other cultivars released in the 1930s , 70 to 80 years before Fusarium wilt was first reported in California . The phenotypic and pedigree databases we developed should expedite the identification and incorporation of Fusarium wilt resistance into modern cultivars . Our data suggest that a certain percentage of modern cultivars are bound to fortuitously carry Fusarium wilt R-genes. That was exactly what we discovered in the California population .

The race 1 R-genes we identified in cultivated strawberry are predicted to be a small sample of those found in the wild reservoir of genetic diversity . Our analyses of the pedigree records of heirloom and modern cultivars show that those R-genes were fortuitously introduced through early founders and survived breeding bottlenecks predating the late twentieth century emergence of this disease in strawberry . Their chance survival in individuals that dominate the ancestry of domesticated populations worldwide is noteworthy because artificial selection for resistance to Fusarium wilt was not consciously applied anywhere outside of Australia or Japan until 2015 when we initiated breeding for resistance to California-specific isolates of the pathogen . This suggests that genes conferring resistance to Fusarium wilt were fairly common in the founders, which is what our data showed—52% of the wild octoploid individuals screened in the present study were highly resistant to race 1 and predicted to carry dominant race 1 R-genes . Our analyses of pedigree records further suggest that many of the race 1 R-genes found in heirloom and modern cultivars have flowed through common ancestors and thus could be identical-by-descent . We cast a wide net in our original phenotypic screening experiments because the genetic basis of resistance was unknown before race-specific R-genes were discovered , knowledge was lacking to strategically narrow the search, and the frequency of resistance among accessions preserved in public germplasm collections was unknown. Our phenotypic screens were designed by assuming that we might be searching for a needle in a haystack, primarily because phenotypic screens in tomato and other plant species had shown that genes conferring resistance to Fusarium wilt were uncommon or only found in wild relatives, e.g., the Fusarium wilt R-genes in cultivated tomato were transferred from wild relatives . Although the domestication and breeding histories of tomato and strawberry are quite different, the frequencies of Fusarium wilt resistance among accessions of wild relatives are similar. Fifty-two to 57% of the individuals we sampled from wild populations of F. chiloensis and F. virginiana were resistant to races 1 and 2, which are comparable to the percentages reported for race-specific R-genes in the wild relatives of tomato .Wild relatives could certainly become an important source of R-genes in strawberry breeding going forward; however, the prevalence of R-genes in modern cultivars circumvents the need to introduce alleles from wild relatives or exotic sources, which has often been necessary for the development of Fusarium wilt resistant cultivars in tomato, cotton, and other plants .

The wild relatives of many of the agriculturally important species impacted by this pathogen carry chromosome rearrangements or structural DNA variation that impedes gene flow and the recovery of recombinants, e.g., the tomato I genes have been introgressed from wild relatives with interspecific structural variation that suppresses recombination and causes the persistence of unfavorable alleles through linkage drag . Notably in strawberry, the octoploid progenitors are inter-fertile and have highly syntenic genomes with no known or apparent barriers to gene flow or suppressed recombination in wide crosses . This does not eliminate the linkage drag problem altogether but simplifes the challenge of purging unfavorable alleles introduced by exotic donors in wide crosses . Moreover,round nursery pots cultivated strawberry has emerged from only 250 years of domestication in interspecific hybrid populations between wild relatives and thus has not experienced population bottlenecks on a scale similar to wheat, tomato, and other staples that have undergone 7000 to 10,000 years of domestication.Both high and low acidity titrant solutions HI84532-51 and HI84532-50 were used to titrate high acid and low acid pomegranate juices, respectively. Juice samples were autotitrated with OH- -based titrant solutions to an endpoint pH of 8.1 to calculate the percentage citric acid in solution. One sample was run per fruit juice sample. Solvents, chemicals, and reagents used to measure total phenolics and antioxidant activity of the pomegranate juice were of analytical grade. Solvents used were ethanol and methanol . Chemicals used were gallic acid and 2,2-diphenyl-1-picrylhydrazyl . Reagents used were Folin-Ciocalteu reagent , and sodium carbonate .Juice TP were measured utilizing the Folin-Ciocalteu method as described by Tezcan et al. . For TP, 3 µL of 100% juice supernatant was added to 297 µL 6:4 methanol:deionized water into a 4 mL cuvette. Then 1.5 mL of 10-fold-diluted Folin–Ciocalteu reagent was added and left to react for 3 min. After this reaction period, 1.2 mL of 7.5% sodium carbonate was added to the cuvette and the sample was left in the dark for 90 min. The cuvette was transferred to a Biochrom Ultrospec II spectrophotometer and the absorbance was measured at 765 nm. A standard curve was plotted and the linear equation used to quantify TP in gallic acid equivalents. All results for TP were expressed in mg/L of gallic acid equivalents . Juice TP samples were measured in triplicate and averaged due to the variation within samples. The 10-folddiluted Folin–Ciocalteu reagent was made by measuring 25 mL of 100% Folin–Ciocalteu and pouring it into a 250 mL volumetric flask and bringing up the volume to 250 mL using DI water. The 7.5% sodium carbonate solution was formulated by dissolving 7.5 g of powder sodium carbonate in 100 mL of DI water and mixing with a magnetic stir bar under low heat. All parameters were analyzed with Analysis of Variance . When ANOVA indicated significant differences, post-hoc comparisons were performed utilizing Tukey’s honestly significant difference with an experiment wise error rate of α = 0.05. Relationships between all parameters were analyzed using linear regression , with correlations among parameters determined using general regression with Minitab Software, version 16 .

There were differences among the ten pomegranate cultivars for fruit weight . Phoenicia and Wonderful fruit were among the heaviest, and the fruit weights of these cultivars were significantly different from those of Ambrosia, Eversweet, and Haku Botan. The lightest fruit by weight was Eversweet, and several cultivars had significantly lighter fruit than Wonderful. Fruit weight typically increased over time during both seasons for both a given cultivar and regardless of cultivar. During 2015, fruit weight slightly decreased when pooling weights of all cultivars for that year because some of the heaviest cultivars were not available for that harvest date. To mitigate issues with an unbalanced design, we did not include November fruit in internal fruit quality analysis. Fruit weight was positively correlated with fruit diameter, but not correlated with fruit length. Antioxidant activity was weakly and negatively correlated with fruit weight . There were no other notable or strong correlations for fruit weight. There were differences among the ten pomegranate cultivars for fruit diameter and fruit length . When pooling data, no cultivars had fruit that were significantly larger than Wonderful. For fruit length, fruit of Phoenicia and Wonderful were longest and Eversweet had the shortest fruit of all cultivars. Fruit diameter increased from September to October, but then decreased in November due to lack of availability of large fresh market fruit this late in the season. Most fruit were destroyed by weather, fruit splitting, and pest damage by mid-November for many cultivars, so mean diameter decreased from October to November. Fruit length also increased from September to October, but then decreased in November due to lack of fresh market fruit available from those trees by the end of the season. There were no differences between years for fruit weight, diameter and length when data were pooled to evaluate effect of year on these traits. ‘Ambrosia,’ and ‘Green Globe’ had the heaviest arils, which were significantly different than ‘Wonderful’ . Haku Botan, an ornamental cultivar, had the least heavy arils, which were also significantly lighter than Wonderful. For weight of all arils, ‘Golden Globe,’ ‘Parfianka,’ ‘Phoenicia,’ and ‘Wonderful’ had the greatest weight and ‘Eversweet’ and ‘Haku Botan’ had the least total aril weight on average. ‘Green Globe’ had the highest fruit edible fraction and this was significantly greater than ‘Wonderful,’ having over 15% more edible fraction in the fruit than ‘Wonderful.’ ‘Ambrosia,’ ‘Golden Globe,’ ‘Parfianka,’ and ‘Phoenicia’ also had significantly higher fruit edible fraction than ‘Wonderful.’ ‘Haku Botan’ and ‘Loffani’ had the least fruit edible fraction, with less than 50% of the fruit weight being edible. The only other cultivar with less than 50% fruit edible fraction was Wonderful. There were no notable correlations for weight of 100 arils, weight of all arils, and fruit edible fraction with any other variable. Weight of 100 arils increased from September to October, but then leveled out when comparing October to November . There were significant differences among cultivars for °Brix .

As highly educated individuals tend to be more optimistic than those in less advantaged situations , this may have caused some ceiling effects in our ability to induce optimism. There are also reasons to suspect that the intervention effects may vary depending on socioeconomic status or other sociodemographic factors and that our findings may not be generalizable to a broader population. Most previous research on optimism or related factors like purpose in life and health has found little evidence of effect modification by socioeconomic status or other sociodemographic factors . However, we suspect that limited resources might increase the likelihood of facing barriers that interfere with individuals’ ability to translate their optimism or other facets of positive psychological well-being into action. Limited work has examined these potential sources of effect modification and it will be important for future studies to assess the possibility. Because writing prompts were different in the intervention versus control groups, we cannot be sure that the intervention perse led to higher coder-rated optimism. We did not assess trait characteristics that are likely to attenuate/ exacerbate optimism effects such as levels of learned helplessness or perceived control.

Future study should evaluate how trait characteristics may modify optimism effects. Lastly, our experiment was relatively short,bucket flower and therefore we were unable to determine whether the effects would increase over time or to evaluate how long people would stay with the intervention. Additionally, we did not follow up with participants after they completed the lab visits. As a result, we could not assess if effects of the intervention would persist beyond the study period. One important avenue for future research is to develop a longer optimism experiment with follow up to evaluate the sustainability of the effect. Despite these limitations, our study represents the largest experimental research to date to investigate the effects of induced optimism on physical activity and stress reactivity. Our experimental design allowed us to assess causality from optimism to physical activity and stress reactivity. Additionally, we recruited participants from community-dwelling adults with diverse age, socioeconomic, and racial/ethnic backgrounds. Our research is also among the very few studies that evaluated both participant and coderrated optimism and their effects on physical activity and stress reactivity. The two studies used almost identical writing interventions with different outcomes and were simultaneously conducted at two large research institutions, providing a good example of study replication. In sum, our results suggest that intentional interventions can have beneficial effects on optimism and positive affect, but the size of the effect may not be sufficient to lead to substantial changes in willingness to engage in physical activity and stress reactivity.

Further research should aim to refine such interventions to move beyond creating relatively small changes, investigate what factors make such interventions maximally effective, and assess the level of change required to change downstream behavior and other health-related responses. Furthermore, given findings that coder-rated optimism and affect were more strongly associated with physical activity duration and stress reactivity, behavioral measures of these psychological states may provide novel insight into changes occurring below the threshold of conscious awareness or that may not differ noticeably enough to translate into differences in self-reported levels.Fixed nitrogen is often a limiting nutrient for primary productivity in the surface ocean, and consequently influences the dynamics of oceanic carbon sequestration . Nitrogen fixation by marine cyanobacteria is an important source of oceanic fixed nitrogen, adding an estimated 100–200 Tg-N annually to open ocean ecosystems . This nitrogen fixation is often associated with cyanobacterial trichomes or aggregates colonized by heterotrophic bacteria, picoeukaryotes and metazoans . Respiratory activity within these so-called ‘pseudobenthic’ environments can create ephemeral suboxic to anoxic zones, establishing a niche for facultative anaerobes within otherwise oxygenated surface waters . Emerging evidence suggests that denitrification occurs within these anoxic habitats, coupling processes of nitrogen-fixation and loss at the microscale . While initial studies of marine biological nitrogen fixation focused on colonial filamentous Trichodesmium species and symbiotic, heterocystous Richelia species , more recent work has demonstrated the importance of unicellular diazotrophic cyanobacteria from the order Chroococcales . Diazotrophic UCYN have been studied extensively in the global oceans by surveys of the nitrogenase gene nifH diversity, which revealed three phylogenetically distinct clades .

UCYN-A are small , metabolically streamlined, uncultured cyanobacteria that lack the oxygen-producing photosystem II and live as endosymbionts within haptophytes, a lineage of eukaryotic algae . UCYN clades B and C are larger , free-living cyanobacteria and include cultured representatives, such as Crocosphaera watsonii and Cyanothece sp. ATCC51142. Studies of aggregate-associated nitrogen fixation have focused predominantly on Trichodesmium sp. colonies and rafts , or filamentous heterocystous cyanobacterial colonies . However, some Crocosphaera watsonii strains have been observed to produce copious quantities of exopolysaccharides and have been linked to the formation of transparent exopolymer particles . These gel-like particles provide microhabitats for other microorganisms, and thus have the potential to play an important role in marine biogeochemical cycling . Here, we report a new species of uncultured, unicellular cyanobacteria from the order Chroococcales which forms millimeter-sized aggregates together with diatoms and other putatively heterotrophic bacteria. These macroscopic aggregates, which we call “green berries,” are found in the muddy, intertidal pools of Little and Great Sippewissett salt marshes . They are found interspersed with previously described, sulfur-cycling “pink berry” consortia . Using a combination of metagenomic sequencing and ecophysiological measurements, we demonstrate that the green berries are characterized by diazotrophy and rapid rates of photosynthesis and respiration that produce steep oxygen gradients. Heterotrophic bacteria within the green berries are closely related to other marine epiphytic marine strains and encode key genes in the denitrification Pathway.The green berries are found in the same organic-rich, intertidal pools of Little Sippewissett salt marsh on Cape Cod where both multicellular magnetotactic bacteria and pink berries have been previously studied . Though less abundant than the pink berries found in these pools , the green berries form similar irregular ellipsoid aggregates measuring 1–8 mm in diameter, with an average equivalent spherical diameter of 1.7 mm ± 0.1 mm . Green berries were dense and compact aggregates that were typically observed at the sediment-water interface, but were occasionally found to float at the water surface when suspended by bubbles. Microscopic observation of the green berries revealed abundant coccoid unicellular cyanobacteria 5–7 µm in diameter , interspersed with pennate diatoms . Filamentous cyanobacteria were observed occasionally, but were rare compared to the unicellular GB-CYN1 morphotype. A clear, extracellular matrix coated these aggregates of phototrophic cells, and was colonized by a variety of smaller bacteria .

GB-CYN1 exhibited absorption maxima at 620, 660, and 680 nm corresponding to thepresence of phycocyanin,cut flower bucket allophycocyanin and chlorophyll a, respectively. Sequencing of 18S rRNA genes from the green berries indicated that the eukaryotic community was predominantly made up of two different pennate diatom species related to Navicula cari strain AT-82.04c and Amphora pediculus strain AT-117.11 . These same diatom species were also the dominant eukaryotic 18S rRNA gene sequences recovered from pink berry aggregates, though diatoms were more abundant in green berries than in pink berries, as observed by microscopy and the relative abundance of 16S rRNA chloroplast sequences . Bacterial 16S rRNA gene sequences amplified from the green berries were dominated by sequences related to either diatom chloroplasts or Chroococcales unicellular cyanobacteria . Unassembled metagenomic sequence reads assigned to rRNA sequences and protein-coding regions support the observed abundance of Chroococcales , but did not recover comparable proportions of diatom chloroplasts . FIGURE 2 | Comparison of green berry bacterial diversity estimates from16S rDNA PCR amplified clones library with unassembled Roche 454 metagenomic sequence reads. Taxonomic assignment of metagenomic reads matching ribosomal RNA reads was conducted using the M5RNA database in MG-RAST . A similar taxonomic assignment was conducted with metagenomic reads matching protein coding sequences in the M5NR database . Note that the 16S rRNA clone library abundance data for the Bacillariophytawas obtained from diatom chloroplasts sequences, which are likely present in multiple copies in the cell and thus not directly comparable to metagenomic 18S rRNA sequences for this group . The overall bacterial community structure of the green berries was significantly different from coexisting pink berry consortia . Some abundant taxa from the pink berries co-occurred in the green berries as rare OTUs, such as the purple sulfur bacterial species Thiohalocapsa sp. PB-PSB1 , and a Winogradskyella species . The persistence of these distinct, co-occurring pink and green berry consortia suggests that the process of macroscopic aggregation enables niche partitioning between oxygenic and anoxygenic phototrophs in these marsh pools. Most of the non-cyanobacterial sequences in the green berry consortia are related to aerobic and facultatively anaerobic marine heterotrophs from the Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria . Many of these sequences were most closely related to environmental 16S rRNA sequences associated with aggregates of oxygenic phototrophs. Examples of such habitats included phytodetrital aggregates collected from euphotic and hadal environments , and epiphytes of marine macroalgae . The occurrence of related phylotypes in such environments suggests that taxa may be well adapted to an attached lifestyle, degradation of photosynthate, and the fluctuating oxygen conditions in an aggregate environment. Metagenomic data indicate that the orders Rhizobiales and Rhodobacterales of the Alphaproteobacteria are abundant in the green berry consortia. While these groups were rarely detected in the PCR-based 16S rRNA survey, we have previously observed this same PCR bias from the 8F primer during studies of the pink berry consortia . We find the abundance of these clades in the green berries particularly interesting as they include lineages of marine denitrifying bacteria.

For example, pelagic Rhizobialeshave been linked to denitrification when found in association with macroscopic Trichodesmium sp. aggregates in oxic waters bordering oxygen minimum zones .The cyanobacterial 16S rRNA gene sequences from the green berries grouped into two closely related OTUs , GB-CYN1a and GB-CYN1b, that can be confidently placed in the order Chroococcales . The GB-CYN1 monophyletic cluster formed a clade basal to the UCYNA clade . Using 29 concatenated single-copy phylogenetic marker genes assembled from the metagenome , we reconstructed a phylogenetic tree that placed the GB-CYN1 within a clade including Crocosphaera watsonii and Cyanothece sp. ATCC 51142 as a sister taxa to “Candidatus Atelocyanobacterium thalassa” isolate ALOHA . A phylogenetic tree inferred from nifH gene sequences reveals that the near full-length nifH gene recovered from the GB-CYN1 metagenomic data affiliated with the UCYN-B clade, and was most closely related to Cyanothece sp. 8801/8802 and Crocosphaera watsonii . We conclude that the observed discordance between 16S rRNA, concatenated, and nifH gene phylogenies involving species such as Cyanothece sp. 8801, Gloeothece sp. KO68DGA, and the cyanobacterial endosymbiont of Rhopalodia gibba is most likely due to lateral gene transfer of the nifH gene. Lateral transfer of nifH has been observed in many other species, including mat-forming filamentous cyanobacteria . A full suite of nitrogenase genes were found in the green berry metagenome and were consistently assigned to GB-CYN1, indicating the metabolic potential for nitrogen fixation characteristic of other members of the UCYN A-C clades . Diazotrophy in the green berries was detected in whole aggregates by acetylene reduction. In two separate experiments , we measured rates of 11 and 20 nanomoles acetylene reduced per hour per milligram of aggregate dry weight . These rates are comparable, though faster than the rates of 3–6 nmol mg−1 hr−1 reported in macroscopic aggregates of filamentous cyanobacteria from Bogue Sound, North Carolina . Rates measured from actively growing Cyanothece cultures were two orders of magnitude larger than the rates in the green berries. The total aggregate elemental composition was analyzed using elemental analyzer isotope ratio mass spectrometry . The mean observed C:N ratio in the green berries, 7.1 ± 0.6 , falls within range of the Redfield molar ratio . This observed C:N ratio is higher than that the ratio of 5.4 ± 0.4 observed in similarly large, anoxic, diazotrophic Nodularia spumigena aggregates from the Baltic Sea . Cultures of Crocosphaera watsonii exhibit wide diel fluctuations in C:N ratios as a result of temporal partitioning of carbon and nitrogen fixation activities . Our samples, collected in the late afternoon on a 14 h light/10 h dark photoperiod, are comparable to reports of C:N = 7 from C. wastsonii at similar late afternoon times in a 16 h light/8 h dark photoperiod . Future studies investigating the temporal partitioning for such activities in the green berry aggregate would be informative to elucidate the dynamics of carbon and nitrogen flow in the consortia.

Most enzymes involved in phenylpropanoid metabolism are encoded by large gene families. There is also high redundancy among these genes, which ensures the functional integrity and plasticity of the phenylpropanoid-related pathways . Therefore, to test the hypothesis that the red blotch-induced transcriptional changes had an actual impact on phenylpropanoid metabolism, we measured the activity of key enzymes and the abundance of compounds involved in these pathways . We detected significant reductions in activity of seven enzymes that catalyze important steps in the core phenylpropanoid, stilbene, flavonoid, and anthocyanin biosynthetic pathways due to GRBaV infections of berries at three ripening stages . In addition, the first enzyme committed to flavone and flavonol biosynthesis, flavonol synthase , had significantly lower activity at post-véraison and harvest stages . Red blotch altered the accumulation of 17 compounds that result from the phenylpropanoid metabolism and two compounds upstream of this pathway,procona London container shikimic acid and gallic acid . Most of these compounds showed significantly lower abundance in the GRBaV-positive berries compared with the controls at later stages of ripening.

The main anthocyanins present in grape berries: malvidin-3-O-glucoside, petunidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargodin-3-O-glucoside, and cyanidin-3-O-glucoside, were significantly reduced by red blotch at harvest. Gallic acid, sinapic acid, and quercetin also showed lower abundance in infected berries. Few exceptions to this general suppression of phenolic accumulation were the accumulation of the precursor shikimic acid, which significantly increased in infected berries at harvest, and of resveratrol that showed significantly greater accumulation at véraison and postvéraison. Additional experiments are necessary to understand the accumulation of these two metabolites in the presence of GRBaV: preliminarily, we can hypothesize that the higher abundance of resveratrol is due either to a restriction of subsequent enzymatic steps in stilbene metabolism for which this compound is a substrate, or to the enzymatic hydrolysis of resveratrol glycosides or stilbenoid dimers previously synthesized. The integrated analysis of transcriptomic, metabolite, and enzyme activity data supported a general repression of the core and peripheral phenylpropanoid pathways, which are normally triggered in red-skinned berries throughout ripening and in response to stress . These results suggest that GRBaV infections disrupt secondary metabolic pathways by altering the regulation of berry ripening processes and/or signaling mechanisms related to plant defense.

Interestingly, GRBaV infections seemed to have a more a pronounced impact on enzymatic activities and metabolite accumulation than on the expression levels of the genes in the pathway, which in general displayed small fold change differences between healthy and infected samples. This observation further confirms the importance of evaluating metabolic perturbations at multiple regulatory levels.Understanding how plants respond to external stimuli in the field is crucial to improve agricultural traits under naturally fluctuating conditions. Most studies on plant–pathogen interactions are performed with model organisms in the greenhouse or laboratory, which reduce the confounding effects of the environment, but also challenge the reproducibility of the results in the field . Compatible plant–virus interactions in perennial woody crops are complex due to the presence of multiple and systemic infections, tissue and developmental stage-specific responses, differences between species and cultivars, and the combination of biotic and abiotic factors during the crop season . The application of a system biology approach to study red blotch under multiple vineyard conditions allowed us to explore grapevine responses to GRBaV infections in real agronomic settings and to characterize the influence of viral activity on berry physiology. In almost all viral diseases occurring in the vineyard, the virus is distributed systemically throughout the grapevine. Once introduced in the host, viral particles move rapidly within the vascular tissue towards sink tissues and establish infections . Although we detected the presence of GRBaV in vegetative and berry tissues during growing and harvest seasons, symptoms of red blotch were only evident after véraison, which suggests that the disease onset is mostly dependent on grapevine phenology and not necessarily linked to viral accumulation.

Similar observations have been made during grapevine leafroll disease, supporting the hypothesis that the appearance of viral disease symptoms in the vineyard may result from the interaction between pathogen and host cellular factors at specific phenological stages . Whether GRBaV is able to modulate its infection strategy as a function of plant development and/ or grapevines have distinct responses to red blotch throughout the season remains to be resolved. GRBaV shares several similarities with geminiviruses, including a small single stranded DNA genome that encodes six potential proteins . Because of their limited protein-coding capacities, geminiviruses rely heavily on host cellular machinery and interact with an assortment of plant proteins and pathways to promote infection . Although three of these genes have been assigned putative functions in viral DNA replication and coat formation , their specific functions in pathogenesis are yet to be elucidated. Our results indicated that the developmental stage of the berries may not influence the expression of GRBaV viral genes and that host factors could play a more critical role in the establishment of successful infections. Red blotch symptoms in grape berries include abnormal chemical composition and asynchronous ripening in the clusters , both reflecting alterations in host metabolic homeostasis and developmental processes. Interestingly, we determined that GRBaV infections induced some processes associated with early fruit development in berries at late stages of ripening, while repressing pathways involved in fruit ripening . Abiotic stress responses to hypoxia and temperature were also suppressed by red blotch in grape berries. These observations may imply that the virus, as a biotrophic pathogen, could redirect host metabolic processes to sustain higher energy demands due to viral replication and at the same time suppressing ripening related events and responses to stress , potentially counterproductive to viral infections. However, further evidence using infected berries from different grape cultivars and environmental conditions will be necessary to understand how both grapevine genotype and environment may influence the disease outcome. It is also important to consider that the effects of GRBaV infections on berries may not be comparable with those occurring in vegetative tissues; for instance, photosynthetic pathways are generally inhibited during viral infections of grape leaves . GRBaV infections restricted the biosynthesis and accumulation of phenylpropanoids and derivatives, which preferentially accumulate after véraison in red-skinned berries. These secondary metabolites function as antioxidants and phytoalexins to protect the berries against a variety of stresses, and are important contributors of berry quality parameters, such as color, flavor, and aroma . The inhibition of phenylpropanoid metabolism appears to be a hallmark of viral infections in red-skinned berries, as has previously been reported in leaf roll-affected berries . In particular, the anthocyanin biosynthetic pathway was greatly impaired as a result of leaf roll and red blotch, correlating with the reduced coloration observed in GRBaV infected berries from several red-skinned cultivars . In this study, we demonstrated that GRBaV infections compromised the regulation of ripening by: suppressing specific ripening events; altering the expression patterns of transcription factors that control the transition from the growth to ripening phases and the activation of ripening pathways ; and causing hormonal imbalances. Most of the alterations in the ripening regulatory networks occurred in infected berries at véraison, the same developmental time when red blotch symptoms initiated,cut flower transport bucket suggesting a link between mechanisms involved in the control of grapevine responses to viruses and berry development. Similar observations were made in leaf roll-affected berries, which presented lower expression levels of MYB transcription factors at late stages of ripening . Plant hormones modulate ripening processes and stress responses in grape berries. Red blotch had a profound impact on ABA, ethylene, and auxin pathways. ABA is considered the triggering signal of berry ripening, since its accumulation coincides with véraison and ABA responsive transcription factors have been implicated in the activation of ripening processes . Particularly, the induction of anthocyanin biosynthesis in a variety of grape cultivars has been linked to ABA-mediated signaling pathways . Our results indicate that alterations of ripening events in the GRBaV-infected berries, including the reduced anthocyanin content at late stages of ripening, could be a direct result of lower ABA levels at véraison.

ABA has been shown to stimulate ethylene-mediated pathways in berries, and together both hormones appear to regulate the progression of ripening . GRBaV infections also affected genes involved in ethylene biosynthesis and responses , which could further account for the antagonistic effect of red blotch on ripening events and immune responses. In contrast, red blotch promoted auxin-mediated pathways, known to suppress berry ripening. Auxins play crucial roles in the early development of grape berries and are known to inhibit ripening processes by delaying ABA-triggered processes . The mechanisms by which grapevine viruses cause disease in ripening berries need to be investigated. GRBaV may actively interfere with the regulation of berry ripening by hijacking the plant post-transcriptional control. We identified elements of the host post-transcriptional machinery that were misregulated in GRBaV-infected berries. Post-transcriptional regulation intersects plant immune responses, developmental transitions, and hormone signaling . Plant viruses, in particular geminiviruses, encode multiple silencing suppressors that interfere with host siRNA production and alter plant DNA methylation and miRNA pathways, causing developmental deffects . A previous study on leafroll indicated that the viral infection caused up-regulation of Dicer-like genes in ripening berries . Therefore, the relationship between the viral-induced repression of host RNA silencing processes and the post-transcriptional regulation of ripening should be addressed from the perspective of compatible grapevine– virus interactions.In contrast with a sculptor, who can control his piece from creation to the final version viewed by the public, a composer requires a performer in order to reach his audience. A composer’s finished product, the score, relies on the conventions of music notation to transmit information to the performer, therefore depending on external interpretation by another to yield the final product. The notation of the piece, the compositional process of the composer, the style of the music, and the composer’s background are some of the factors that influence how the piece will evolve and how the performer will engage with the score. Six works for solo cello will be examined in this paper: Plainsound-Litany by Wolfgang von Schweinitz, Another Secular Calvinist Creed by Andrew McIntosh, Sweet Bay Magnolia with Berry Clusters by Wadada Leo Smith, For Stephanieand another anxiety by Nicholas Deyoe, and to a lesser extent, Sequenza XIV by Luciano Berio. These works have been selected for discussion because of my concentrated work on the learning, performing, and recording of each piece. Although it will not be discussed in detail later in this paper, Berio’s Sequenza XIV serves as an inspiring model of composer-performer collaboration. Written for Rohan de Saram in 2002, the piece contains elements of rhythmic cycles of the Kandyan drumming that Saram taught to Berio. This piece serves as an artifact of the meaningful musical relationship of this composer and performer, showing the possibility of a composer’s work to reflect the voice of the dedicatee. My involvement with Deyoe, McIntosh, Smith, and Schweinitz predates the recording project. Four ofthe pieces were written for me: both works by Deyoe, the McIntosh, and the Smith, whereas the Berio and Schweinitz were written for other cellists. In this paper I will discuss to what extent each of these pieces was written specifically for me, focusing on both abstract and concrete examples of how this influenced the final composition. I will also discuss the methods of interaction and collaboration between each composer and me, focusing on how the collaborative process influenced my own interpretation of the work. Finally, I will investigate how these variations in the composer’s inspiration for each piece, the final piece itself, and my interpretation of each piece shape the listening experience for the audience.Composed in 2004, Plainsound-Litany was neither written for nor premiered by me. As a result, my engagement with the score began years after Schweinitz finished the piece and my role was not as collaborator but as interpreter. My desire to learn Plainsound-Litany stemmed from my appreciation of Schweinitz’s music, the knowledge that he enjoyed working with performers and my own interest in exploring a solo cello work written in just intonation. I began learning this piece in 2009, being only the fourth cellist to embark on this process. The piece uses a unique style of notation that functions as an aide to the learning process for the performer, a guide to phrasing, and as a framework for the creation of a concentrated, shared ritual of listening for the performer and audience.

Some studies have shown that the effects of consuming whole foods are more beneficial than consuming compounds isolated from the food, such as dietary supplements and nutraceuticals. Because fruit consumption is mainly related to visual appearance, flavor and antioxidant properties, we decided to evaluate fruit quality attributes, antioxidant capacity and consumer acceptance of the early-season blueberry cultivars currently being grown in California. We characterized the quality parameters of six southern high bush blueberry cultivars grown in the San Joaquin Valley for three seasons , and evaluated their acceptance by consumers who eat fresh blueberries.For the quality evaluations at UC Kearney Agricultural Center, we used three patented southern high bush blueberry cultivars — ‘Emerald’ , ‘Jewel’ and ‘Star’ , and three non-patented cultivars — ‘Reveille’, ‘O’Neal’ and ‘Misty’. The plants were started from tissue culture and then grown for two seasons by Fall Creek Farm and Nursery in Lowell, Ore. Before planting these cultivars in 2001, the trial plot was fumigated to kill nut grass . Because blueberries require acidic conditions, the plot’s soil was acidified with sulfuric acid,30 litre plant pots which was incorporated to a depth of 10 to 12 inches with flood irrigation, resulting in a pH ranging from 5.0 to 5.5.

The plants were mulched with 4 to 6 inches of pine mulch and irrigated with two drip lines on the surface of the mulch, one on each side of the plant row. Irrigation frequency was two to three times per week in the spring and daily during June and July. The emitter spacing was 18 inches , with each delivering 0.53 gallon per hour of water acidified with urea sulfuric acid fertilizer to a pH of 5.0. The plot received an application of nitrogen in the first season, as well as in subsequent growing seasons. The rate was 80 pounds nitrogen per acre at planting, 60 pounds the second year, 90 pounds the third year and 120 pounds the fourth year. Annual pest control was limited to one application of Pristine fungicide in February for botrytis management, and two or three herbicide treatments of paraquat . In year three, the plants received one insecticide treatement of spinosad for thrips management. Twenty-eight plants per cultivar were planted in a randomized block design using seven plants per block as an experimental unit, replicated in four rows. Rows were spaced 11 feet apart, with the plants in the rows spaced 3 feet apart, with a space of 4 feet between plots. Fruit was harvested at times when it would have been commercially viable if it had been in a commercial field. Fruit from each of the seven plant blocks was harvested and a composite sample of 80 random berries per each replication was used for quality evaluations.Berries were randomly selected from each replication for quality evaluation at the first harvest time for each respective season .

During the 2007 season, in addition to the initial quality evaluations, harvested berries were stored at 32°F in plastic clam shells, and measured for firmness 15 days after harvest and for antioxidant capacity 5, 10 and 15 days after harvest. Three replications per cultivar were measured for each quality parameter. The initial firmness of 10 individual berries per replication was measured with a Fruit Texture Analyzer . Each berry was compressed on the cheek with a 1-inch flat tip at a speed of 0.2 inch per second to a depth of 0.16 inch and the maximum value of force was expressed in pounds force . Sixty berries per replication were then wrapped together in two layers of cheesecloth and squeezed with a hand press to obtain a composite juice sample. The juice was used to determine soluble solids concentration with a temperature-compensated handheld refractometer and expressed as a percentage. Twenty-one hundredths of an ounce of the same juice sample was used to determine titratable acidity with an automatic titrator and reported as a percentage of citric acid. Some samples that had a high viscosity were centrifuged with a superspeed centri-fuge at 15,000 rpm for 5 minutes, in order to get liquid juice for soluble solids concentration and titratable acidity measurements . The ratio of soluble solids concentration to titratable acidity was calculated.Antioxidant capacity was measured in the 2005 and 2007 seasons. Eighteen hundredths of an ounce of berries per replication was used to determine the level of antioxidants by the DPPH free-radical method . Samples were extracted in methanol to assure a good phenolic representation, homogenized using a polytron and centrifuged for 25 minutes.

The supernatant was analyzed against the standard, Trolox, a water-soluble vitamin E analogue, and reported in micromoles Trolox equivalents per gram of fresh tissue .An in-store consumer test was conducted on ‘Jewel’, ‘O’Neal’ and ‘Star’ blueberry cultivars in 2006, and on the six blueberry cultivars studied in 2007, using methods described previously . The fruit samples were held for 2 days after harvest at 32°F prior to tasting. One hundred consumers who eat fresh blueberries, representing a diverse combination of ages, ethnic groups and genders, were surveyed in a major supermarket in Fresno County. Each consumer was presented with a sample of each blueberry cultivar in random order at room temperature, 68°F . A sample consisted of three fresh whole blueberries presented in a 1-ounce soufflé cup labeled with a three-digit code. At the supermarket, the samples were prepared in the produce room out of sight from the testing area. For each sample, the consumer was asked to taste it, and then asked to indicate which statement best described how they felt about the sample on a 9-point hedonic scale . Consumers were instructed to sip bottled water between samples to cleanse their palates. Consumer acceptance was measured as both degree of liking and percentage acceptance, which was calculated as the number of consumers liking the sample divided by the total number of consumers within that sample . In a similar manner, the percentage of consumers disliking and neither liking nor disliking the sample was calculated.Quality attributes such as soluble solids concentration, titratable acidity, soluble-solids-totitratable-acidity ratio and firmness were significantly different among cultivars and seasons . There was wide variability in soluble solids concentration among cultivars. ‘Reveille’ had the highest average value of the 2005 to 2007 seasons, followed by ‘Misty’ , ‘Emerald’ and ‘Star’ . ‘Jewel’ and ‘O’Neal’ had the lowest soluble solids concentration within this group. Titratable acidity within cultivars was less variable, and only ‘O’Neal’ had a significantly lower average value than the rest of the tested cultivars. Titratable acidity varied from 0.70% to 0.80% within this group with the exception of ‘O’Neal’. Cultivars segregated into three groups based on their soluble-solids-to-titratable-acidity ratio. Because of its low titratable acidity, ‘O’Neal’ had the highest ratio, while ‘Jewel’ had the lowest ratio due to its high titratable acidity. The rest of the cultivars formed an intermediate group in which the soluble-solids-to-titratableacidity ratio ranged from 17 to 20.3. ‘Jewel’ and ‘O’Neal’ also had the lowest firmness , while ‘Reveille’ and ‘Misty’ had the highest . ‘Emerald’ and ‘Star’ were significantly different than these two groups,25 liter pot plastic forming an intermediate group . Quality attributes were also significantly affected by the season. Soluble solids concentration across all cultivars was highest in 2007 and lowest in 2006, while titratable acidity was highest in 2006. Soluble-solids-to-titratableacidity ratio and firmness were significantly higher in 2007 than the other years. There was a significant interaction between cultivar and season for all these quality attributes . The lowest soluble solids concentration was 10.8% in 2006 for ‘O’Neal’ and the highest was 15.8% for ‘Reveille’ in 2007. During this 3-year period, all of the cultivars yielded soluble solids concentrations higher than 10%, which has been proposed as a minimum quality index for blueberries . Titratable acidity was similar among cultivars in these three seasons except for ‘O’Neal’ in 2007, which reached 0.3%, and ‘Jewel’ and ‘Emerald’ in 2006 with about 1.0%. ‘O’Neal’ and ‘Reveille’ had the highest soluble-solids-to-titratableacidity ratio, followed by the rest of the cultivars with ratios from 11.4 to 20.6. During this 3-year period, ‘Jewel’ and ‘O’Neal’ were the softest cultivars, and ‘Misty’ and ‘Reveille’ the firmest. Antioxidant capacity was significantly different among the cultivars but not between seasons .

There was a wide variability of TEAC within cultivars. ‘Misty’ had the highest average TEAC followed by ‘Reveille’ and ‘Emerald’ . ‘Star’ , ‘O’Neal’ and ‘Jewel’ had the lowest TEAC within this group. Like the rest of the quality attributes, there was a significant interaction between cultivars and seasons for antioxidant capacity . Storage of the six blueberry cultivars at 32°F for 15 days did not affect either antioxidant capacity or firmness, except for ‘O’Neal’ and ‘Misty’, whose firmness was reduced slightly but not significantly .Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds . The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios . Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening . Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown . Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality. However, the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance .

Xylem water isotopic characteristics differed between angiosperms and gymnosperms at some sites. For δ2 H, they were significantly different for Krycklan, Bruntland Burn and Dry Creek, while for δ18O they were different for Dorset and Dry Creek . Snowmelt plotted on the LMWL and was more depleted for 18O than almost all measured soil and xylem waters, although a substantial number of xylem samples at Dry Creek, Krycklan and Wolf Creek were more depleted in 2 H but plotted off the LMWL . Similarly, at the four sites where groundwater samples were collected, the mean isotopic composition of groundwater fell on the LMWL but plotted towards the more depleted end of the range of soil water samples. This reflects the generally higher recharge of groundwater by depleted water following the spring melt at Dry Creek, Krycklan, and Wolf Creek ; and during winter rainfall at Bruntland Burn . Isotopic composition of groundwater at all sites showed limited temporal variation, indicating the volume of annual recharge is small relative to groundwater storage. Groundwater was generally more strongly depleted in 18O than xylem waters forboth angiosperms and gymnosperms,black plastic nursery pots although at each site a substantial proportion of xylem samples were more depleted in 2 H.

The minimum boundary polygon analysis quantifies the degree to which xylem water for both angiosperms and gymnosperms overlaps bulk soil water sources at different depths. The use of the spatially bulked data for soils and vegetation at each site was necessary to provide a sufficient number of samples for the development of encompassing polygons. This may lead to larger estimated polygon areas and a greater estimated overlap of soil and xylem water, although the effect is much less marked than for the ellipse method of Amin et al. . This may provide insight into the sources of xylem water, although the proportion that cannot be ascribed to soil water sources is equally informative regarding the need to hypothesise and identify other causal reasons. Distinct inter-site differences emerged in terms of the overall overlap of xylem and soil water isotopic composition . For Bruntland Burn, soil water had a 77% overlap for angiosperms, but only 6% for Gymnosperms. At Dorset, like Bruntland Burn, angiosperms showed a much higher degree of overlap than gymnosperms . At Dry Creek, almost all xylem water in both angiosperms and gymnosperms overlapped soil water at almost all profile depths. Of all sites, Krycklan had the lowest degree of overlap with only 27% for angiosperms and 0% of gymnosperms . Finally, while Wolf Creek had only angiosperms present as willow and birch shrubs, a 99% overlap between xylem water and soil water was evident. The depth dependent overlap of xylem and soil water isotopic composition showed differences between depths, with higher overlap tending to be in shallow soil depths for most sites. For Bruntland Burn, there was 72% and 55% overlap between angiosperms and soil water at 0-10 cm and 10-20 cm depths, respectively, but only 9% and 3% for gymnosperms.

Dorset was the only site with the greatest overlap occurring in deeper soil, with overlaps of 34%, 28%, 24%, 59% and 31% for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. The gymnosperms at Dorset had a similar deviation to that of angiosperms, with much smaller overlaps of 4%, 7%, 8%, 18%, and 7%, for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. Depth-dependent overlap of soil and angiosperms at Dry Creek was high through all soil layers with the greatest overlap in the near-surface soils . Gymnosperms at Dry Creek had a similarly high overlap of 78%, 55%, 86%, 72% and 86 % for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. At Krycklan, the upper two soil depths had approximately the same overlap for angiosperms , with a moderate decrease to 12% in the 20-30 cm soils. Wolf Creek angiosperms showed a high overlap in the upper two soil depths with a more substantial decrease in deeper soils .The general patterns of the pooled data sets for the entire study year mask differences in the degree to which seasonal variations in the isotopic composition of xylem water can be ascribed to soil water data collected on the same day or integrated over increasing monthly time windows to capture antecedent conditions. However, as described in section 2.2, soil water boundary polygons for increased averaging periods can also be calculated to estimate the overlap relative to xylem.

The bulk soil overlaps are summarised for angiosperms and gymnosperms . Depth dependent overlaps are shown in Figure S7 and Figure S8, respectively. At Bruntland Burn, a longer time window of soil water isotopes explained a greater degree of variation in xylem water isotopic composition for angiosperms . Bulked soil water samples collected on the same day provided 80% and 87% of overlap in spring and autumn, respectively, but only 4% in summer. Increasing this window to 3 months increased overlap to 90%, 38% and 87% in spring, summer, and autumn, respectively. The spring and summer bulked soil and xylem water overlap increased to 100% and 58%, respectively, with a 6 month window. For gymnosperms, same day sampling provided no overlap in spring and summer, and only 7% in autumn . For a 3 month window, overlap increased to 20% in spring, but only 3% in summer and 7% in autumn. For a 6 month window, the autumn overlap increased to 13%. There were marked seasonal differences between angiosperms and gymnosperms at Dorset. For angiosperms, bulked soil and xylem water overlapped for same day sampling 100% in spring, 0% in summer and 20% in autumn . This increased to 20% in summer for a 3 month averaging window and 47% in summer for a 6 month average. The overlaps were much lower for gymnosperms; same day sampling showed bulked soil and xylem water overlaps of only 13% in spring, 2% in summer and 7% in autumn . The respective increases were to 13%, 4% and 15% using a 3 month window; and 13%, 9%, and 15% using a 6 month window. For Dry Creek angiosperms, same day bulked soil water sampling provided 34% overlap with xylem water in spring, 78% in summer and 30% in autumn. For a 3 month sampling window, overlaps increased to 34% in spring, 81% in summer, 73% in autumn; and for a 6 month window respective overlaps were 80%, 81% and 86% . This implies xylem water in angiosperms, especially in spring , is reflecting bulked soil water integrated over longer periods, including the previous growing seasons. Similar patterns were evident for gymnosperms at Dry Creek, with same day samples overlapping with xylem water by 35% in spring, 92% in summer, 40% in autumn. Overlaps using a 6 month window were 78%, 93%, and 80%, respectively. The 3 month window values were intermediate . Of all sites, the vegetation at Krycklan showed the least overlap with bulked soil water, and this changed little with sampling period . Same day sampling for angiosperms showed only 27%, 3% and 0% overlap for spring, summer and autumn. Values increased slightly for bulked soil sampling over the preceding 3 months to 27%, 13%, and 0% for the three seasons, but remained constant for the 6 month window . There was no overlap with any time window for gymnosperms . Only angiosperms were sampled at Wolf Creek, and the severe winter conditions allowed analysis only for summer and autumn. A 52% overlap was evident in summer and 89% in autumn for same day sampling.

This increased to 64% and 97%for 3 month wand 6 month windows, respectively . Unsurprisingly, sw-excess values of individual soil water samples plotted around 0 ‰ throughout the year . This gave confidence that the sw-excess is an appropriate metric to describe the potential water source,greenhouse pot since individual soil water samples deviated relatively little from the regression through all soil water samples. Plant sw-excess was usually <0 ‰, indicating that xylem water was generally more depleted in 2 H compared to soil water. At Bruntland Burn, Dorset and Krycklan, swexcess was more negative for gymnosperms than for angiosperms. The deviation from sw-excess of 0 ‰ occurred generally under lower soil moisture conditions. At Bruntland Burn, angiosperms had a similar sw-excess to soils in most sampling periods, apart from the start of the study period in October 2015 and the following summer, when it dropped in July, August and September before recovering in September 2016. Differences for gymnosperms were more pronounced and only close to the soils in winter and early spring. Similar patterns were evident for Dorset, although the differences from sw-excess were greater for both plant groups. In general, summer saw the greatest isotopic difference between xylem and soil waters. For Krycklan, angiosperms occasionally showed sw-excess closer to 0 ‰, although the timing was generally limited to early summer after snowmelt. Gymnosperms were closer to soils at this time too, although both plant groups deviated from the soils with the approach of autumn. The S4 site at Krycklan also had the wettest soil conditions. At Dry Creek, differences between angiosperms and gymnosperms were less pronounced and gymnosperm values were usually less negative than for angiosperms. Both gymnosperms and angiosperms periodically reached sw-excess of 0 ‰, although the timing was not as consistent as at the other sites. It was striking that the Dry Creek site with the greatest similarity between soil and xylem waters was also the driest, with the lowest soil water content. At Wolf Creek, angiosperm sw-excess was usually close to the soil water sw-excess with the exception of May 2016, which was at the end of winter when shrubs were not active. The xylem waters sampled in this study provided a series of snap-shots of plant water over the course of the growing season at five northern experimental catchments. This resulted in an unusually rich comparative data set allowing a meta-analysis of inter- and intra-site similarities. Some clear findings emerged from this inter-comparison, though there remain many unanswered questions. The close link to soil water at each site was apparent from the similar positions of xylem water when plotted in dual isotope space . However, for most sites, much of the xylem water tracked towards lower δ2 H and δ18O plotting below the meteoric water line and below the soil water samples. The swexcess was shown to be a helpful metric to describe the dynamics of the deuterium offset of xylem waters compared to soil water. For some sites, there was much less or no overlap for gymnosperms or some angiosperms . The results also showed seasonal variations in xylem composition at most sites, although this differed . The plotting positions of xylem water from angiosperms and gymnosperms were quite distinct at some sites, despite some overlap. Apart from Dry Creek, gymnosperms at most sites were more offset from both the LMWL and soil waters compared to the angiosperms. The operationally-defined boundary polygon analysis provided an objective way of comparing the distribution of the soil and xylem data from the five sites . It is notable that the sites with greatest general overlap between all sampled angiosperm xylem waters and soil waters are characterised by smaller shrubs and trees . That said, larger trees at Dorset also showed quite a high degree of overlap, especially for more depleted, potentially snowmelt-recharged water sources earlier in the growing season. In contrast, Vaccinium at Krycklan showed little overlap. However, the physiology of smaller plants, with shorter rooting systems, lower internal storage and more rapid water throughput rates may at least partly explain the greater coherence between xylem water and soil water. Indeed, previous ecohydrological modelling experiments at Bruntland Burn by Kuppel et al. and calibrated only on hydrometric data, found quite good agreement between simulated and observed soil water and xylem δ2 H values in angiosperm using the spatial distributed EcH2O-iso model. Conversely, the same model failed to simulate the xylem isotopes in gymnosperms . The polygon analysis at most sites also seemed to indicate that overlaps between soil and xylem waters reflected integrating effects of water sources across the rooting zone, which at most sites was relatively shallow . This is consistent with the conclusions of Amin et al. for northern sites in their global meta-analysis that found isotopic evidence that cold region plant water was sourced from shallower depths compared to more temperate and arid regions.

Two leaves per tree and a minimum of three trees per cultivar were analyzed with the infrared gas analyzer. All leaves were collected and leaf area was quantified with a leaf area meter to normalize the photosynthesis data . Only the most recently fully-formed, sun-exposed leaves were selected for this study. Cuvette temperatures were allowed to vary with field conditions. Leaves were measured in a chamber that provided 1500 µmol m-2 ·s-1 . Instantaneous water-use efficiency was calculated as A/E and intrinsic water-use efficiency was calculated as A/gs. The difference between these two traits is that intrinsic water-use efficiency corrects for differences in vapor pressure deficit while instantaneous water-use efficiency does not. This is important because knowing if climate affects water-use efficiency is useful for cultivar selection. Predawn and midday stem water potential measurements were taken for each day of photosynthesis data collection. For both predawn and midday water potential, nonactively growing shoots were covered with a plastic bag for 10 min before being pruned, placed in a sealed plastic bag and kept in a cooler bag until transferred to a cool, lit indoor environment for plant moisture stress measurements with a pressure chamber . For afternoon stem water potential measurements,raspberry container size canopy-shaded non-actively growing shoots were covered with a plastic bag for 10 min before being pruned.

Flowers were counted during peak bloom, defined as 90% or more of the flowers being open in the grove, which was typically in April and early May of each year . Male flowers, as identified by their signature bell shape in pomegranates, were counted along with the female flowers, which were identified by their easily recognizable vase shape. For reasons unknown, ‘Haku Botan’ flowers had morphological differences between sites that made determining male and female difficult at Somis, the coastal site. Male to female flower ratio was calculated by dividing number of male flowers by the number of female flowers. Total number of flowers was determined by adding male flowers with female flowers. Number of fruit was determined by counting fruit after fruit set, which was determined in the months of July and August. All variables were analyzed with Analysis of Variance . When ANOVA indicated significant differences, post-hoc comparisons were performed utilizing Tukey’s honestly significant difference with an experiment wise error rate of α = 0.05. Relationships between all variables were analyzed using linear regression , with correlations among parameters determined using general regression with Minitab Software, version 16 . For the purposes of this work, the R 2 value is the percentage of variation in one variable that is explained by the variation in the correlated variable. Regression models were fit to determine differences in slope coefficients and constants among variables using regression analysis. Trees at both sites established at varying rates, with low tree mortality for some cultivars and no tree mortality for others . By year four, trunk diameter was significantly different between sites , with Riverside having 7.6 cm trunk diameter on average and Somis having an average of 6.4 cm trunk diameter.

Every year, trunk diameter was larger at Riverside compared to Somis. There were significant differences among cultivars for trunk diameter at Somis, but not at Riverside . ‘Desertnyi’ had a relatively thin trunk at Somis, which was significantly lower than ‘Wonderful.’ Canopy area was larger at Somis , with Riverside having 34.1 m2 canopy area on average and Somis having an average of 37.4 m2 canopy area . There were significant differences among cultivars at each site . At Riverside, Desertnyi, Eversweet, and Loffani had the lowest rate of establishment, with these cultivars having the least vigor as measured by vegetative growth when compared with the other cultivars in the study. At Somis, four cultivars grew at a significantly higher rate than the others. The faster establishing cultivars at the coastal site were Ambrosia, Haku Botan, Proprietary cv. and Wonderful. Of the remaining cultivars at Somis, Desertnyi, Eversweet and Golden Globe were the slowest growing cultivars during establishment. Results for in-row and between-row establishment are similar . There were significant differences between sites and among cultivars for relative chlorophyll content. Riverside had higher leaf relative chlorophyll content than Somis , with average values of 47.7 and 42.4 SPADs for Riverside and Somis, respectively. ‘Haku Botan’ had the highest relative chlorophyll at both sites . At Riverside, Haku Botan had the greenest leaves of all cultivars according to SPAD readings.

Haku Botan was significantly greener than Loffani at Riverside and significantly greener than all cultivars except for Green Globe and Proprietary cv. at Somis. Of all cultivars at both sites, Loffani had notedly low relative chlorophyll content at Riverside. There were significant differences among cultivars and between sites for tree height . Somis had the tallest trees , and had mean tree height 231.4 cm compared to 182.7 cm at Riverside. The tallest cultivars at Riverside were Haku Botan, Phoenicia, Proprietary cv. and Wonderful, which were significantly taller than Desertnyi and Eversweet. At Somis, Ambrosia, Golden Globe, Haku Botan and Proprietary cv. were tallest, and these cultivars were significantly taller than Desertnyi and Eversweet. Results for in-row and between row canopy diameter were similar to those of canopy area. Trees at Somis established and grew larger than trees at Riverside . Values for in-row and between row canopy diameter were similar for a given cultivar, demonstrating uniformity of in-row and between-row growth. There were also significant differences among cultivars at each site, with Desertnyi and Eversweet smaller than most other cultivars at both sites. The pomegranate cultivars were actively photosynthesizing and transpiring during morning hours at both sites during all days of data collection . There were significant differences among cultivars for photosynthesis at Riverside but not at Somis. ‘Haku Botan’ had significantly lower rates of A , gsand Ethan ‘Wonderful’ during the morning hours. There were no other differences detected for photosynthetic variables among the four cultivars investigated. The effect of site on pomegranate physiology was significant. Riverside had significantly higher rates of photosynthesis than Somis, with mean 15.0 and 12.7 µmol CO2 m-2 ·s-1 for Riverside and Somis, respectively . For A, rates at Riverside were about 15% higher on average than Somis . Rates of gs at the two sites were similar, with Riverside and Somis having 0.107 and 0.097 mol H2O m-2 ·s -1 , respectively. Rates of E were about 34% higher on average at Riverside than at Somis . Mean rates of E were 2.45 mmol H2O m-2 ·s-1 for Riverside and 1.61 mmol H2O m-2 ·s-1 for Riverside. There was no difference in intrinsic water-use efficiency among cultivars or between Riverside and Somis. There were significant differences between sites for instantaneous water-use efficiency, with Somis having significantly higher instantaneous water-use efficiency than Riverside . ‘Wonderful’ had significantly higher rates of A·E-1 at Riverside than at Somis, and ‘Wonderful’ had significantly higher rates of A·E-1 than ‘Eversweet’ at Somis and ‘Haku Botan’ at Riverside . There were no significant differences among cultivars or between sites for predawn stem water potential . Predawn stem water potential was significantly less negative than midday stem water potential . There were significant differences between sites and among cultivars for midday stem water potential . Somis had less negative midday stem water potentials than Riverside ,raspberry plant container with Riverside having a mean stem water potential of -2.31 MPa and Somis having a mean stem water potential of -1.53 MPa for midday measurements. ‘Haku Botan’ had less negative stem water potential than ‘Parfianka’ at Riverside . There were no differences in stem water potential among cultivars for midday measurements at Somis. There were positive and negative correlations between physiological variables for morning, afternoon and for data pooled for the two times of day. The relationship between A and gs was positively correlated and statistically significant for photosynthesis measurements for pooled data from both sites at Riverside and at Somis . There was also a positive correlation between A and E for pooled data from both sites , at Riverside and at Somis . There were site differences for precocity . Riverside had significantly higher precocity than Somis , with Riverside having 138.6 flowers on average and Somis having 28.7 flowers on average by year four.

For each cultivar at both sites, number of male flowers was greater than number of female flowers . There were significant differences among cultivars at both sites for number of male flowers and number of female flowers. However, there was no evidence for significant differences among cultivars at either site for male to female flower ratio. There were also significant differences among cultivars at both sites for total number of flowers and total number of fruit. Riverside had significantly more fruit than Somis in the first three years, but not for year four. This investigation provided evidence that some cultivars are more productive than others during the first four years of establishment. In Somis, the most precocious and productive cultivars in this investigation were Green Globe and Wonderful. ‘Wonderful’ was rather productive at both sites. Haku Botan was the only ornamental “double flower” cultivar in the trial and its flowers were difficult to sex at Somis. Observationally, ‘Haku Botan’ had larger flowers at the Somis, the coastal site. This phenotype made it difficult to sex the flowers and thus only total number of flowers was determined for this cultivar at that site. Usually, pomegranate flowers are easy to sex based on their shape, with bellshaped flowers being males and vase-shaped flowers being female. This distinction was not possible at Somis during the four years of this study. The objectives of this research were 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 to evaluate four pomegranate cultivars in coastal versus inland agroecosystems to determine if there were differences among genotypes for physiological traits that would be conducive to commercial crop production in drought conditions. The purpose was to phenotype these accessions for grower suitability and to aide in future breeding endeavors for crop improvement and germplasm diversity enhancement. Our findings indicate that many of the cultivars established at the same rate as Wonderful, but others did not. Namely, ‘Desertnyi’ and ‘Eversweet’ established much slower at both sites. ‘Eversweet’ was known to be less vigorous among rare fruit cognoscenti and ‘Desertnyi’ has been known to have trouble with canopy architecture at the germplasm repository. Our findings provide evidence that these two cultivars may not be the best choices to grow in a conventional spacing for the Californian pomegranate cropping system, unless perhaps rootstocks are employed that alter their growth rate. Literature on rootstocks is limited, so more research is needed to determine if these two cultivars would benefit from being grafted on a more vigorous rootstock. If these cultivars were to be chosen for commercial production due to their soft seeded arils and ornamental fruit, a possible method for commercial production would be to reduce spacing to 2-3 m and/or grow them in a trellis system. For at least one of the two sites, all four cultivars evaluated for physiological traits in these cultivar studies displayed eco-physiological traits suitable for commercial production if the industry standard, Wonderful, is used as the standard for comparison. However, ‘Wonderful’ performed better at Riverside than at Somis for A, which may indicate that ‘Wonderful’ may be better planted for commercial production inland than in coastal regions. Photosynthesis values obtained for ‘Wonderful’ at Riverside were different than those reported in Greece , but similar enough to suggest the possibility that differences in climate or cultural practices between the two sites were the source of this discrepancy. We found evidence that there are differences among cultivars within a site for physiological traits including stomatal conductance, transpiration and midday water potential. Values for physiological traits were similar to previous investigations into pomegranate physiology , a topic which is limited in the literature. Differences were also detected between sites, with higher rates of assimilation and transpiration at Riverside compared to Somis. There was a significant difference between sites for instantaneous water-use efficiency, with Somis having more efficient water use than Riverside. This indicates that some pomegranate cultivars may use less water for commercial production on the coast, but more studies must be carried out on irrigation scheduling to determine if these cultivars can be grown with less water on the warm coast of Southern California compared to hotter, semi-arid climates inland.

Drawing on his small-town Methodist boyhood, he knew the words to many of the hymns and was thoroughly familiar with the Old Testament quotations that laced the heated sermon. But the Biblical distortions that flowed from the pulpit upset him. “My God,” he said in a too loud voice to both of us, “has this guy got a racket! He takes selected Biblical passages out of context, twists them around, and draws crazy conclusions from them!” In the small hall, many present must have heard him, and Ernie and I were worried that he had blown our cover. But we should have never underestimated Stouffer. Upon leaving the church at the close of the service, he shook the minister’s hand warmly. He told him that the sermon had recalled his childhood, and pointedly suggested integrationist Biblical passages from the New Testament that he then proceeded to cite verbatim. Even this hardened segregationist melted under Stouffer’s charm to our surprise and relief. Stouffer was an inspiring but unorthodox teacher. Instruction from him was invariably informal and empirical. Intensely engrossed in his work, Stouffer taught by example.

You followed him around from office to computing room and back, absorbing as best you could his excitement,blueberry plant pot expertise and “feel” for survey research and analysis. To this day, I have never lost the sense of excitement and curiosity in analyzing survey data instilledby these memorable occasions. If a member of his graduate seminar on survey analysis would offer an interesting hypothesis, he would often leap from his chair and exclaim, “Let’s test it!” At which point he would lead the entire class to the machine room and start stuffing the survey data cards into the old IBM 101 counter, sorter, and printer——the leading machine for social science in the early 1950s. While he was intensely focused on his work, Stouffer often displayed a robust, even puckish, sense of humor. When Gordon Allport lost money on his blueberry crop at his summer retreat in Maine, Stouffer began to call him, to Allport’s amusement, “the blueberry king!” Stouffer loved his family, his work, baseball, Mickey Spillane mysteries, and Shakespeare. His favorite Shakespearian citation from King Henry IV has Glendower asserting that he could “call spirits from the vasty deep.” But, Hotspur retorts, “Why, so can I, or so can any man; but will they come when you do call for them?” When Stouffer called for them from survey data, the answers always came. Stouffer resisted sociology’s fondness for “grand theory.” Consistent with his emphasis on empirical research, he believed in close-to-the-data reasoning and middle level concepts. For example, he advanced the concept of “intervening opportunities” to account for migration flows . Thus, to understand the massive African American migration up the Mississippi River to Chicago in the early twentieth century, one has to take into account not only the distance to Chicago but the intervening opportunities offered by Memphis, St. Louis, and other communities along the migration route.

The most famous illustration of Stouffer’s talent for middle-range concepts comes from his most celebrated work—the monumental World War II American Soldier studies . Stouffer devised relative deprivation as a post hoc explanation for the well-known anomalies from these studies. Two examples became especially famous. He and his wartime colleagues found that the military police were more satisfied with their slow promotions than the air corpsmen were with their rapid promotions. Similarly, African American soldiers in southern camps were more satisfied than those in northern camps despite the fact that the racist South of the 1940s remained tightly segregated by race. These apparent puzzles assume the wrong referent comparisons. Immediate comparisons, Stouffer reasoned, were the salient referents: the military police compared their promotions with other military police——not air corpsmen whom they rarely encountered. Likewise, black soldiers in the South compared their lot with black civilians in the South——not with black soldiers in the North who were out of view. Satisfaction is relative, he held, to the available comparisons we have. Relative deprivation became a major social science concept, because social judgments are shaped not only by absolute standards but also by standards set by social comparisons . A comparative approach to explain his American Soldier puzzles came readily to Stouffer. Comparable ideas had been advanced by both Karl Marx and Alexis de Tocqueville . Moreover, between the world wars, the theory of general relativity won acceptance in physical science while cultural anthropology firmly established a relativity perspective in social science. Hyman was the first to reflect this emphasis in social psychology with his introduction of reference group theory. Following World War II, comparative ideas abounded in American social science. In economics, for example, another Harvard professor introduced a related concept in the same year that Stouffer introduced RD. Duesenberry fashioned “the ratchet effect” to help explain consumer behavior.

Following closely Stouffer’s usage of the concept, we can define relative deprivation as a judgment that one or one’s ingroup is disadvantaged compared to a relevant referent, and that this judgment invokes feelings of anger, resentment and entitlement. In addition to the fundamental feature that the concept refers to individuals and their reference groups, note that there are three critical components of this definition that are frequently overlooked. Individuals undergoing RD experience in turn three psychological processes: 1) they first make cognitive comparisons, 2) then cognitive appraisals that they or their ingroup are disadvantaged, and finally 3) that these disadvantages are seen as unfair and arouse angry resentment. If any one of these three requirements is not met, RD is not operating . Defined in this manner, relative deprivation is a social psychological concept par excellence. It postulates a subjective state that shapes emotions, cognitions, and behavior. It links the individual with the interpersonal and inter group levels of analysis. It melds easily with other social psychological processes to provide more integrative theory——a prime disciplinary need . Moreover, RD challenges conventional wisdom about the leading importance of absolute deprivation. And it has proven useful in a wide range of areas, —as I shall review.The concept of relative deprivation has now won wide acceptance in criminology , economics , and throughout the social sciences. To be sure, two social scientists have used the concept extensively without referencing any work whatsoever of social psychology . Indeed, it has recently even appeared in a popular book, David and Goliath, by the journalist, Malcolm Gladwell ——although, unfortunately, it was grossly misused in this bestseller. After Stouffer introduced RD, Merton enlarged the idea within a reference group framework. Building on this framework, Davis provided a formal mathematical model of RD. This work led me to point out that RD was but one of a large family of concepts and theories that employed relative comparisons in both sociological and psychological social psychology. From sociology,plastic gardening pots this theoretical family embraces Hyman’s and Merton’s reference group theory, Lenski’s concept of status crystallization, Blau’s concept of fair exchange, and Homans’ concept of distributive justice. From psychology, these social evaluation ideas include Walster, Walster, and Bersheid’s equity theory, Festinger’s social comparison theory, and Thibaut and Kelley’s concept of comparison level. Many theories in social psychology burn hot and then suddenly cool. But RD and related ideas have simmered slowly on a back burner for two-thirds of a century. First, Runciman broadened the RD construct by his invaluable distinction between egoistic and fraternal RD. People can believe that they are personally deprived or that a social group to which they belong and identify is deprived . Feelings of GRD should be associated with group-serving attitudes and behavior such as collective action and outgroup prejudice, whereas IRD should be associated with individual-serving attitudes and behavior such as academic achievement and property crime. This is a crucial point for RD theory and research which I shall call “the fit hypothesis.” During the 1970s, Suls and Miller edited a volume that offered a host of interconnections between these comparative concepts and such other disciplinary concerns as causal attributions. Albert focused on temporal comparisons in an influential article. The following decade witnessed further progress. Mark and Folger introduced their referent cognitions model of RD, and a major work edited by Olson, Herman, and Zanna offered additional connections between social comparison theory and RD.

A volume edited by Walker and Smith continued these advances by linking RD theory with attribution, social identity, selfcategorization, and equity theories as well as procedural justice and counterfactual thinking processes. To be sure, not all work on RD has proven useful. In political science, Gurr wrote a widely cited book, Why Men Rebel. The volume largely ignores social psychological work on the subject, and the fact that RD is a phenomenon of individuals ——not societies. Gurr employed such gross macro-level measures of RD as economic and political indices of whole societies. It is a macro-level study that does not involve the micro-level of Stouffer’s conception of RD. Why Men Rebel uncovered some intriguing findings, but it is not a RD study. As I shall discuss later, criticism of this work in the social movement field mistakenly cast RD as having little value. A classic ecological fallacy occurs when macro-level findings are placed within a RD micro-level framework . That is, micro-level phenomena, such as the RD of individuals, are being erroneously assumed from macrophenomena. This mistake is often seen in loose statements made about individual voters from aggregate voting results. It is a fallacy because macro-units are too broad to determine individual data, and individuals have unique properties that cannot be inferred from macro data. In his famous volume on Suicide, Durkheim fully understood this issue. Although he could not obtain individual data, he strove to obtain data from ever-narrowing reporting districts to reduce the bias. Indeed, the central thrust of RD theory is that individual responses are often different from that which is expected of the macro-category. For instance, given contrasting comparisons, the rich can be dissatisfied and the poor content—just the opposite from that which their macro-income characteristics would indicate. Complicating the issue further, more advantaged members of disadvantaged groups often engage in protest actions rather than the most disadvantaged . Although not the most objectively deprived group members, they are the most likely to make subjective social comparisons with members of advantaged groups in part because they are more likely to have contact with them . As we shall see, the ecological fallacy has seriously hurt the development of RD theory in sociology.In short, RD makes the claim that absolute levels of deprivation of individuals— much less collective levels of deprivation – only partly determine feelings of dissatisfaction and injustice. Imagined alternatives, past experiences, and comparisons with similar others also strongly influence such feelings . Relative deprivation describes these subjective evaluations by individuals. The RD concept offers social scientists an elegant way to explain numerous paradoxes . For example, RD explains why there is often little relationship between objective standards of living and satisfaction with one’s income . Thus, the objectively disadvantaged are often satisfied with receiving low levels of societal resources, while the objectively advantaged are often dissatisfied with high levels of societal resources . RD models suggest that the objectively disadvantaged are frequently comparing themselves to others in the same situation or worse, while the objectively advantaged are frequently comparing themselves to those who enjoy even more advantages than they possess. Thus, the RD concept has inspired a vast international and cross-disciplinary literature. Yet the concept’s initial promise as an explanation for a wide range of social behavior remains unfulfilled. Some investigations strongly support RD models . But other studies do not . In response to these inconsistencies, previous literature reviews have sought to clarify the theoretical antecedents and components of the concept , or to dismiss its value altogether . Two fundamental problems of the RD literature help to explain these persistent discrepancies in results. First, in The American Soldier, Stouffer did not measure RD directly; rather, as noted earlier, he inferred it as a post hoc explanation for a series of surprising results. This failure to initiate a prototype measure has led to literally hundreds of diverse and often conflicting measures that have bedeviled RD research ever since. Indeed, many of the measures throughout the social scientific research literature purporting to tap relative deprivation do not meet the basic features of the concept just outlined.

For MOEs below 10,000, cancer risk needs to be considered. We calculated MOEs for β-myrcene, hydrocoumarin, estragole, and pulegone based on an available BMD that caused a 10% increase in tumor incidence in animal models and NOAELs and a user consumption of 3.4 or 5 mL of fluid/day for a body weight of 60 kg 15,24,28-30 . The MOEs for β-myrcene and hydrocoumarin were >10,000 in all samples , indicating a low cancer risk. In contrast, some products had pulegone and estragole concentrations that were well below 10,000, meaning there is a cancer risk associated with these products . Q Menthol and Two Apple had extremely low MOEs. In the current study, concentrations of the flavor chemicals were averaged and plotted as a function of their frequency . The dominant flavor chemicals separated into three groups. Ethyl maltol and triacetin were most frequently, followed by vanillin, corylone, menthol, ethyl vanillin, benzyl alcohol, and ethyl lactate, while carvone, furaneol, and isobutyl alcohol were infrequently used. The individual dominant flavor chemicals were compared across our current and two previous studies . Twenty-seven dominant flavor chemicals were identified in the present study bringing the total number across our three studies on refill fluids to 37 .12,14 Of these,planting blueberries in pots five flavor chemicals were used in at least one product at > 1 mg/mL in all three studies .

Ten dominant chemicals -3-Hexen-1-ol, corylone, ethyl acetate, ethyl butanoate, ethyl vanillin, furaneol, and isoamyl acetate were in two of the three studies, and 13 were found only in the current study. Other chemicals present in only one study of our prior studies at > 1 mg/mL included acetoin, allyl hexanoate, linalool, strawberry glycidate_A and _B, 14 and benzaldehyde, cinnamaldehyde, ethyl cinnamate, and panisaldehyde.12This study is the first to identify and quantify the flavor chemicals in refill fluids manufactured under one brand and purchased worldwide. In general, the flavor chemicals and their concentrations were similar in duplicate bottles of refill fluids from each country. One bottle of “Apple” from Kansas, USA was an exception in that it had twice the total concentration of flavor chemicals than “Apple” bottles purchased at other locations , These differences may be due to instability or reactivity of the flavor chemicals in these products, mislabeling, human error in compounding, or the use of different batches of ingredients during production at plants in Italy and China. While some of the “Ritchy” refill fluids that we previously purchased in Nigeria were counterfeits, 17 all the products in the current study were manufactured by Ritchy LTD. Generally, the flavor chemicals and their concentrations were similar irrespective of the country of purchase. One of our objectives was to determine which flavor chemicals are used frequently in refill fluids and to establish their concentration ranges by amalgamating data from our prior and current studies. We categorize flavor chemicals as “dominant” when they are 1 mg/ml or higher.

Dominant chemicals are likely added intentionally to create the desired flavor profile. Chemicals at low concentrations may be added intentionally or may be co-constituents of the dominant flavors. For example, pulegone, a potential carcinogen, 31 is often found at low concentrations in menthol-flavored products, but it is not likely added intentionally during manufacture. One hundred thirty-seven flavor chemicals were quantified in our prior12,14 and current studies . These refill fluids represent a convenience sample, 12 the most popular flavors in southern California vape shops, 14 and products manufactured by one company and sold worldwide . Of the 137 flavor chemicals identified in the three studies, 37 were present at concentrations > 1 mg/ml and were distributed among the studies . This number of flavor chemicals reinforces our earlier conclusions that a relatively small number of flavor chemicals are used in the manufacture of a broad range of EC refill fluid products. In contrast to our prior studies, triacetin was the most frequently used flavor chemical in the current LIQUA study, where it exceeded 44 mg/mL in one product. In all studies, esters were the most used chemical class with terpenes, ketones, alcohols, and aldehydes also identified. The five dominant flavor chemicals in our three studies have also appeared in products analyzed in other labs, supporting the conclusion they are commonly used. Most products have at least one flavor chemical that is > 1 mg/ml. Tobacco-flavored products are sometimes an exception, having few flavor chemicals at low concentrations. The LIQUA “Ry4 Tobacco” product was unusual in having four dominant flavor chemicals. Products that are a single flavor, such as menthol, peach, or cinnamon, often use one dominant flavor chemical to create the desired profile . An exception would be LIQUA “Two Apple” which had four dominant flavor chemicals. Products with names that obscure the flavor profile, such as Dewberry Cream14 or Cheesecake , often use multiple dominant flavor chemicals to create a more complex profile.

Interestingly, LIQUA “Peach” and “Q Pina Colada” have very similar flavor chemicals with triacetin being the dominant flavor chemical in both. Presumably, some of the flavor chemicals with lower concentrations contribute to the taste and enable the users to distinguish between the two flavors. In general, the total concentration and the total number of flavor chemicals in LIQUA “Q” and “HP” products were lower than in the regular LIQUA products. The concentrations of flavor chemicals in some LIQUA products were higher than those typically used or permitted in other consumer goods, such as fragrances and food. Triacetin, ethyl maltol, and corylone were used at concentrations averaging 6 mg/mL, 4 mg/mL, and 2 mg/mL, respectively . While triacetin should not exceed 2% in cosmetics for external use, 38 its concentration in LIQUA “Mint” was 4.4% . Ethyl maltol concentrations in edible products and cosmetics should not exceed 0.015%. However, LIQUA concentrations were 0.015% or higher in 60% of the products containing ethyl maltol, with one product containing 2.6%. These concentrations exceed the MTT NOAEL for ethyl maltol. Ethyl maltol has been linked to free radical formation, 41 which could increase the cytotoxicity of these products. Likewise, the maximum average concentration of corylone in chewing gum for example, is 0.015 mg/mL, 22 while in some LIQUA refill fluids, concentrations ranged between 0.03 to 10.2 mg/mL. Flavor chemicals that were not dominant may also have significant health effects, including the potential to cause cancer with chronic use. Hydrocoumarin , a derivative of coumarin which is prohibited in human food increased kidney and liver neoplasms in male rats and female mice, respectively.β-myrcene is a naturally occurring acyclic monoterpene which increased kidney and liver neoplasms in male rats and mice, resulting in its prohibition in food. Because the MOEs for hydrocoumarin and β-myrcene in LIQUA products were >10,000, they do not appear to present a cancer risk to EC users. In contrast, the MOEs for both pulegone and estragole were far below 10,000 in some LIQUA products, consistent with cancer risk. The “Q” version of refill fluids, which are Ritchy’s higher quality products, had the lowest MOEs, indicating that more expensive products are not necessarily safer. Pulegone levels in other EC products have likewise produced MOEs below the safe threshold. Pulegone, a naturally occurring oxygenated monoterpene, is a major constituent of pennyroyal plant oil extracts and several other mint plants and has been classified as a type 2B carcinogen by the International Agency for Research on Cancer. Estragole a naturally occurring chemical found in spices, plants, and essential oils,blackberries in containers is a rodent hepatocarcinogen at high doses. While the Joint FAO/WHO Expert Committee on Food Additives concluded further research is needed to assess the risk of estragole to humans, the European Medicines Agency recommended keeping exposures to the lowest levels possible.Other flavor chemicals that are not carcinogens may cause health effects, even at low concentrations. Diacetyl and cinnamaldehyde were less frequently found in LIQUA products than in our other studies and ranged in concentration between 0.005 – 0.057 mg/mL and 0.003 – 0.112 mg/mL, respectively. While probably not added intentionally, diacetyl causes bronchiolitis obliterans in humans, and cinnamaldehyde is highly cytotoxic in vitro, having IC50s within the LIQUA range when tested in the MTT assay with human embryonic stem cells and human pulmonary fibroblasts .Cinnamaldehyde also inhibits ciliary beating in bronchial epithelial cells and impairs innate immune function.Triacetin, the most frequently used flavor chemical in the LIQUA products, ranged in concentration from 0.005 to 44.333 mg/mL, a concentration significantly higher than triacetin in our other EC studies.

Triacetin is a clear, colorless, oily GRAS human food and cosmetic additive that produces eye and skin irritation in humans but is non-toxic in animals when administered orally or dermally. While triacetin has relatively low cytotoxicity in vitro, 14 upon heating, it produces acetic acid, which catalyzes the formation of acrolein, formaldehyde hemiacetals, and acetaldehyde from propylene glycol and glycerol. We are currently determining if triacetin increases the concentrations of reaction products in LIQUA aerosols. While our cytotoxicity data is based on refill fluids, other factors may affect results when heated aerosol are used. For example, additional chemicals that can be toxic, such as 2, 3 butanedione, acetaldehyde, formaldehyde, and acrolein, may form upon heating and could alter cellular responses. In addition, 100% of the flavor chemicals may not transfer to aerosol so that users are exposed to lower concentration than those in the fluids. These factors notwithstanding, in one study that compared refill fluids and aerosols, the cytotoxicity of the fluids accurately predicted that of the aerosols in 74% of the samples when one EC devise was tested. The cytotoxicity of refill fluids generally correlates with the concentration of cytotoxic flavor chemicals, and this was observed in the current study for “Two Apples” and “Ry4 Tobacco” in the both the MTT and cell growth assays. These products contained high concentrations of ethyl maltol, benzyl alcohol, ethyl vanillin, and corylone, which were themselves directly correlated with cytotoxicity in the MTT assay. In contrast, “Peach,” with high levels of triacetin , was not cytotoxic in the MTT or proliferation assays, even though a concentration of triacetin lower than 20 mg/ml was cytotoxic when tested individually as an authentic standard. This observation may be explained by the fact that three of the “Peach” chemicals that were cytotoxic individually produced no effect when tested in a mixture . A similar neutralizing effect was observed when carvone and triacetin were combined . Both mixtures in Figure 6 contained high concentrations of triacetin, which may decrease cytotoxicity in mixtures or the presence of solvents. Previously, a similar unexpected decrease in cytotoxicity was observed when benzyl alcohol, which was cytotoxic by itself, was used in a refill fluid. This type of antagonism usually occurs when the chemicals in a mixture interact with each other to inhibit uptake or interaction with a target. Antagonism appears to be rare in EC refill fluid mixtures; however, it should be studied further as “Peach” aerosols may be cytotoxic due to reaction products formed during heating. The MTT assay measures mitochondrial reductase activity and is widely used to evaluate mitochondrial function and cell health.The inhibition of cell growth by “Two Apples” and “RY4 Tobacco” may have occurred due to the reduction in ATP levels by poorly functioning mitochondria. Although not measured in this study, disruption of mitochondrial function can lead to increases in reactive oxygen species, inflammation, altered expression of genes in the electron transport chain, abnormal Ca2+ elevation, and glutathione depletion. These changes underlie diseases of the respiratory system including chronic obstructive pulmonary disease, asthma, and lung cancer.The use of flavor chemicals and their concentrations may differ for refill fluids made by other companies. In addition, it is possible that LIQUA products had additional flavor chemicals that were not on our target list. In summary, flavor chemicals in LIQUA products were generally similar in all countries of purchase. The flavor chemicals on our target list varied in total flavor chemical concentration and the number of flavor chemicals per product in 103 of the refill fluids we analyzed. No target and non-target flavor compound was detected in two tobacco flavored refill fluids . Twenty seven flavor chemicals were dominant , and triacetin was the most frequently used, often at high concentrations. Thirty-seven chemicals not identified in our prior work were present in LIQUA products.

Participants were excluded from the study if they met any of the following criteria: significant alcohol intake for at least 3 consecutive months over the previous 12 months or if the quantity of alcohol consumed could not be reliably ascertained; clinical or biochemical evidence of liver diseases other than NAFLD ; metabolic and/or genetic liver disease ; clinical or laboratory evidence of systemic infection or any other clinical evidence of liver disease associated with hepatic steatosis; use of drugs known to cause hepatic steatosis for at least 3 months in the last past 6 months; history of bariatric surgery; presence of systemic infectious illnesses; females who were pregnant or nursing at the time of the study; contraindications to MRI ; any other condition which, based on the principal investigator’s opinion, may significantly affect the participant’s compliance, competence, or ability to complete the study.All participants underwent a standardized clinical research visit at the UCSD NAFLD Research Center. A detailed history was obtained from all participants. A physical exam, which included vital signs, height, weight,raspberry container and anthropometric measurements, was performed by a trained clinical investigator. Alcohol consumption was documented outside clinical visits and confirmed in the research clinic using the Alcohol Use Disorders Identifications Test and the Skinner questionnaire.

A detailed history of medications was obtained and no patient took medications known or suspected to cause steatosis or steatohepatitis. Other causes of liver disease and secondary causes of hepatic steatosis were systemically ruled out using detailed history and laboratory data. After completion of the earlier described elements of the history and physical examination, participants had a comprehensive fasting laboratory including metabolic and liver assessment previously described in references .VCTE was performed by a trained technician, using the FibroScan® 502 Touch model . VCTE measurement was obtained in the supine position with the right arm fully adducted by scanning the area of abdomen at the location of the right liver lobe during a 10 seconds breath hold. Participants were asked to fast at least 3 hours prior to the exam. The details of VCTE assessment have been previously described in references . The threshold used for the classification of cirrhosis was VCTE > 11.8 kPa as previously determined in reference . Among the first-degree relatives of proband with NAFLD-cirrhosis, 11 did not have an MRE assessment due to contraindication and the presence of advanced fibrosis was determined using a VCTE threshold> 11.8 kPa as previously determined in reference . Liver biopsy was not used for hepatic fat content and fibrosis assessment of controls and first-degree relatives as they were asymptomatic with no suspected liver disease and therefore performing a liver biopsy would have been unethical. A non-invasive, accurate quantitative imaging method was used to estimate liver fat and fibrosis.

We have previously shown that MRIPDFF is a highly precise, accurate, and reproducible non-invasive biomarker for the quantification of liver fat content . In addition, MRE is the most accurate, currently available, noninvasive quantitative biomarker of liver fibrosis . MRE has been shown to be have excellent diagnostic accuracy in differentiating between normal liver and mild fibrosis and between non-advanced fibrosis and advanced fibrosis .Participants were considered to have NAFLD-related cirrhosis if they had NAFLD according to the definition above, and have biopsy proven cirrhosis . We have previously validated that a liver stiffness cut point of >3.63 kPa on MRE provides an accuracy of 0.92 for the detection of advanced fibrosis, and it is the most accurate non-invasive test for the diagnosis of advanced fibrosis . Advanced fibrosis among first-degree relatives was determined by either imaging evidence of nodularity and presence of intraabdominal varices or other evidence imaging evidence of portal hypertension or liver stiffness assessment with MRE threshold ≥ 3.63 kPa or if MRE were not performed using transient elastography assessment with VCTE threshold ≥ 11.8 kPa. DNA extraction and 16S rRNA amplicon sequencing were done using Earth Microbiome Project standard protocols and previously described in references . In brief, DNA was extracted using the Qiagen MagAttract PowerSoil DNA kit as previously described. Amplicon PCR was performed on the V4 region of the 16S rRNA gene using the primer pair 515f to 806r with Golay error-correcting barcodes on the reverse primer. Amplicons were barcoded and pooled in equal concentrations for sequencing.

The amplicon pool was purified with the MO BIO UltraClean PCR cleanup kit and sequenced on the Illumina MiSeq sequencing platform. Sequence data were demultiplexed and minimally quality filtered using the QIIME 1.9.1 script split_libraries_fastq.py, with a Phred quality threshold of 3 and default parameters to generate per-study FASTA sequence files .To build a model capable of distinguishing samples belonging to NAFLDcirrhosis from those of non-NAFLD-controls, we developed a custom machine learning process that employed Random Forest analysis . The set of input features for model building consisted of 16S sequences and patient metadata features. Features from stool microbiome data consisted of the number of 16S sequences and the patient metadata consisted of age, gender and BMI. The first step in building an RF model consisted of training RF and then selecting features with the most important score > 0.005 in a second step. The final random forest model included the 27 bacterial features and important patient metadata for a total of 30 predictive features. Patients’ demographic, anthropometric, clinical, and biochemical characteristics were summarized. Categorical variables were shown as counts and percentages, and associations were tested using a chi-squared test or Fisher’s exact test. Normally distributed continuous variables were shown as mean , and differences between groups were analyzed using a two-independent sample t- test or Wilcoxon-Mann-Whitney test. Statistical analysis of cohort characteristics were performed using SPSS 25.0 . A two-sided p-value <0.05 was considered statistically significant.Cyrielle Caussy: study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, approved final submission. Anupriya Tripathi: data analysis and figure generation, interpretation of data, drafting of the manuscript, critical revision of the manuscript, approved final submission. Gregory Humphrey: microbiome sequencing data generation, approved final submission Shirin Bassirian: patient visits, data collection, critical revision of the manuscript, approved final submission. Seema Singh: patient visits, data collection, critical revision of the manuscript, approved final submission. Claire Faulkner: patient visits, data collection, critical revision of the manuscript, approved final submission. Emily Rizo: patient visits, data collection, critical revision of the manuscript, approved final submission. Lisa Richards: patient visits, critical revision of the manuscript, approved final submission. Michael R. Downes: analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, approved final submission. Ronald M. Evans: analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, approved final submission. David A. Brenner: study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, obtained funding, study supervision, approved final submission. Claude B. Sirlin: study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, obtained funding, study supervision, approved final submission. Zhenjiang Zech Xu: data interpretation,growing raspberries in container critical revision of the manuscript, approved final submission. Rob Knight: directed microbiome sequencing and data analysis, critical revision of the manuscript, approved final submission.

Rohit Loomba: study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, obtained funding, study supervision, approved final submission. The co-authors listed above supervised or provided support for the research and have given permission for the inclusion of the work in this dissertation.Obstructive sleep apnea is a common disorder characterized by episodic obstruction to breathing due to upper airway collapse during sleep. OSA has been associated with adverse cardiovascular and metabolic outcomes, although data regarding potential causal pathways are still evolving. As O2 and CO2 affect the ecology of the gut microbiota and the microbiota has been shown to contribute to various cardio-metabolic disorders, we hypothesized that OSA alters the gut ecosystem which exacerbates the downstream physiological consequences. Here, we model human OSA and its cardiovascular consequences using Ldlr-/- mice fed a high-fat diet and exposed to intermittent hypoxia and hypercapnia . The gut microbiome and metabolome were characterized longitudinally and seen to co-vary during IHH. Joint analysis of microbiome and metabolome data revealed marked compositional changes in both microbial and molecular species in the gut. Moreover, molecules altered in abundance included microbe-dependent bile acids, enterolignans and fatty acids, highlighting the impact of IHH on host-commensal co-metabolism in the gut. Thus, we present the first evidence that IHH perturbs the gut microbiome functionally, setting the stage for understanding its involvement in associated cardio-metabolic disorders.Intestinal dysbiosis marks various cardiovascular diseases comorbid with OSA. It has not been systematically studied if dysbiosis due to hypoxic stress in OSA is causally linked to these comorbidities. We take advantage of a longitudinal study design and paired ‘-omics to investigate correlations in microbial and molecular dynamics in the gut to ascertain the contribution of microbes on intestinal metabolism. We observe microbe-dependent changes in the gut metabolome that will guide future research on unrecognized mechanistic links between gut microbes and comorbidities of OSA. Additionally, we highlight novel, non-invasive biomarkers for OSA-linked pathologies. Obstructive sleep apnea afflicts nearly 12% of the adult population in the USA with a cost burden of nearly $149.6 billion, according to a recent study commissioned by the American Academy of Sleep Medicine . Timely diagnosis and treatment of OSA improves not only sleep and cognitive function but also management of comorbid cardiometabolic diseases . Therefore, identifying downstream consequences of OSA would aid in development of effective treatment modalities, reducing overall health care utilization. OSA is marked by changes in oxygen and carbon dioxide-inspired concentrations which impacts the gut microbial community . Since the gut microbiota play a key role in metabolism of dietary precursors including lipids, cholesterol and choline, it impacts the cardiometabolic health of the host . Gut dysbiosis has already been linked to an array of metabolic disorders such as hypertension, T2 diabetes, hepatic steatosis and atherosclerosis . Additionally, previous work has identified specific gut bacteria to be significantly correlated with plasma cholesterol and apolipoprotein levels . Thus, probing this commensal ecosystem may provide a valuable avenue of investigation to understand the mechanism of pathogenesis of cardiovascular consequences of OSA. In this study, we investigated the taxonomic and molecular alterations in gut microbiome that potentially mediate the interplay between OSA and related CMDs.We used atherosclerosis-prone adult mice fed high-fat diet enriched in cholesterol and milk fat to evaluate atherosclerosis risk in OSA. We previously demonstrated that IHH increases atherosclerosis plaque formation in this model . As episodic hypoxia and hypercapnia mimic the changes in blood gases that occur in OSA-driven downstream consequences , these mice were exposed to IHH or air and examined longitudinally for 6 weeks . Fecal samples, representative of the gut ecosystem, were collected at baseline and twice each week thereafter, and the microbiome and metabolome were profiled using 16S rRNA amplicon sequencing and LC-MS/MS-based untargeted mass-spectrometry, respectively. These data were processed to obtain relative abundances of microbial and molecular species per sample , which were used for comparing OSA-mimicking and control mice. First, we performed principal coordinate analysis on the microbiome and metabolome feature tables to identify major factors driving the clustering of samples. Figure 3.1 shows the PCoA plotted against time to visualize the dynamics of clustering based on gut microbiome ; Figure 3.1a and metabolome; Figure 3.1b,c as duration of IHH-exposure increases. Here, the first fecal sample represents the baseline gut composition before animals were switched to a HFD. There is a rapid shift in both microbial and molecular composition due to HFD alone, consistent with similar previous findings. Moreover, starting from a highly congruent gut composition, IHH-exposed mice significantly diverge from controls with increasing exposure duration . This demonstrates that prolonged IHH-exposure cumulatively perturbs the gut microbiome and metabolome. We tested the relationship between the two omics datasets by superimposing the principal coordinates computed from microbiome and metabolome data ; Figure 3.1d. The ordination spaces are correlated , and changes in metabolome and microbiome of samples within the treatment groups over time are proportional, suggesting microbe-dependent changes in intestinal metabolism on chronic OSA.We then tested for specific microbes and metabolites that changed with OSA.

To specifically understand the effects of different parameters on co-extraction, phenolic concentration could be a better indicator for kinetic study, as it excluded the evaporation of solution induced by high heat. Phenolic concentration was the highest in CA+85 °C group and the lowest in CA+25 °C group . Citric acid was a more effective extraction solvent compared with HCl, but the temperature was the dominating factor, like HCl, 85 °C achieved a higher equilibrium concentration than CA+55 °C . These values were lower than the results from Pan et al. , who applied ultrasound for polyphenol extraction in pomegranate peel using water and observed equilibrium concentration up to 2.8g/L. This indicated that acidic pH might interfere with the releasing of certain polyphenol components in source materials, such as phenolic acid. DSA varied from 3.09 g/g to 5.66 g/g. No significant trend was observed at different conditions. GalA content ranged from 47.78% to 83.20% and increased with higher temperature and longer extraction time. Pereira et al. applied conventional heating extraction of pomegranate peel pectin using citric acid and received similar GalA content . Citric acid-extracted pectin tended to have a higher level of GalA than the HCl-extracted one since citric acid could selectively extract pectin chains rich in homogalacturonans,growing blueberries which further released GalA . It is an important pectin property for applications in the industry.

The initial DM depended on the material intrinsic properties and DM reached an equilibrium of de-methylation at different processing conditions . As shown in the current study , the degree of methylation varied from 53.31% to 79.66%. For each condition, no significant change of DM over-extraction was observed. CA-extracted pectin had higher DM compared with the HCl-extracted ones , as HCl is a strong acid and could induce higher de-methylation. It was similar to research findings from Muñoz-Almagro et al. , who observed a lower DM in nitric acid-extracted pectin. Except for extraction with CA at 25 °C, other extractions showed the tendency of decreasing DM relative to higher extraction time and temperature, which was in agreement with Andersen et al. . Pereira et al. researched CA extraction of pectin in oven-dried pomegranate, at a temperature of 70 to 90 °C and pH 2 to 4 for 40 to 150 mins. They observed a set of DM ranging from 47.18 to 71.45% , while pectin yield and DM were negatively correlated in CA-extracted extracts from pomegranate peel. The current study demonstrated the same tendency. The percentage of GalA that is acetylated at positions O-2 and O-3 could be expressed as DA. DA is a major factor in pectin emulsifying properties. Hereby, DA ranged from 6.19% to 10.68% and slightly decreased with higher temperature and longer extraction time. The results were lower than research from Zhuang et al. , who found 12% to 15% of DA in 3 Chinese var. of pomegranate peel. Shakhmatov et al. also obtained a high-DM and highDA pectin from lyophilized pomegranate Punica granatum. However, the DA in the current study was still higher than that of some common pectin sources, including citrus and apple .

This indicated pomegranate peel had the potential to be applied as an effective emulsification agent, which was in agreement with the study from X. Yang et al., . Figure 5.10 Comparison of extraction rate reciprocal of polyphenol concentration and pectin yield from pomegranate peel over different extraction times using citric acid and HCl at different temperatures . It exhibited the second-order model for coextraction of polyphenol content and pectin yield in the linearized form. The kinetic parameters, including initial extraction rate , extraction rate constant , and equilibrium phenolic concentration , were determined by plotting t/PCt vs. extraction time . With a high coefficient of determination and great fitting of experimental data , these second-order models were sufficient to describe co-extraction kinetics. This suggested that there could be two phenomena involved in co-extraction, including the dissolution and degradation of polyphenol and pectin . Theoretically, the k parameter should increase with higher temperature as heat accelerate the mass transfer . However hereby when increasing the temperature, the extraction rate constant dropped while the equilibrium level elevated for both polyphenol concentration and pectin yield. This finding was consistent with research from Patil & Akamanchi , who extracted camptothecin from Nothapodytes nimmoniana using 76.4 to 191 Wcm−2 of ultrasound at 30 to 60 °C. Goula also extracted oil from pomegranate seeds using 130W ultrasound from 20 to 80 °C and observed a similar trend. In all these studies, the k parameter was negatively correlated to an equilibrium level. It was speculated that during ultrasound-assisted extraction, multiple processes could jointly affect the extraction rate and result in various responses. Hypothesis-driven research has led to many scientific advances, but hypotheses cannot be tested in isolation: rather, they require a framework of aggregated scientific knowledge to allow questions to be posed meaningfully. This framework is largely still lacking in microbiome studies, and the only way to create it is by discovery- and tool-driven research projects. Here we describe the value of several such projects from our own laboratories, including the American Gut Project, the Earth Microbiome Project , and the knowledge base-driven tools GNPS and Qiita. We argue that an investment of community resources in these infrastructure tasks, and in the controls and standards that underpin them, will greatly enhance the investment of hypothesis-driven research programs.

Microbiome research is making dramatic progress, with thousands of papers now published each year linking specific microbes and/or host-microbe co-metabolites to specific diseases, physiological properties, or environmental parameters. Much of this research is performed in a traditional, hypothesis-driven way, or at least presented as a rational reconstruction that fits this model, much as Darwin re-wrote much of his discovery-driven work as hypothesis driven to increase its respectability under the influence of contemporary philosophers of science such as William Whewell . However, it should be noted that hypothesis-driven science was not always so respectable — Isaac Newton famously wrote “Hypotheses non fingo”, or “I feign no hypotheses”, in an essay appended to the second edition of the Principia— so the tradition of modifying how science is framed in order to meet respectability criteria dates back at least 300years. In any case, what can be framed as a singular hypothesis suffers important limitations based on what we can measure, and what we already know.One constant in microbiome research has been that most factors that we would intuitively suspect to drive differences in the microbiome are of minor importance. For example, although long-term dietary changes have a major effect on the microbiome, short-term changes don’t. Similarly, sex has a very limited impact on microbiomes across the human body and has a much weaker effect than many other variables such as age and the time of year the sample was collected . Perhaps more surprisingly,square plant pots factors such as temperature and pH have a much smaller impact on environmental microbiomes than salinity , and even the saline vs. non-saline difference is much smaller than the host-associated vs free-living difference . Samples from different sites of the same person’s body can be more different from one another in terms of their overall microbial communities than radically different free-living microbial communities, such as soils versus oceans . Differences of this magnitude can also occur within the gut of a single person, with sufficiently large perturbation . As a consequence, it is easy to incorrectly frame hypotheses, especially when supervised ordination and classification techniques are used in experiments with many confounding variables. For example, suppose that for mouse experiments we don’t know that cage effects are important in the microbiome , then we profile the microbiomes in each of two cages of each of two different genotypes of mice. Our results are likely to be driven by which pair of cages happens to resemble each other more closely. If the variable of cage is not measured, or not tested in an unsupervised model, we might never know that our results are driven by this important confounding variable! There may be many more important confounding variables that we are not yet aware of, so longitudinal studies with meticulous metadata annotation will be crucial for defining which environmental factors matter. This is especially important in the context of clinical samples, where single data points are often collected and obtaining contextual information in retrospect is exceedingly difficult . Similarly, a frequent practice is to discard unannotated microbes or unannotated molecules, focusing on the subset of microbes or molecules that can be matched to an existing database. Because databases of both microbes and molecules are heavily biased , the entities that actually best discriminate among classes of samples may be lost in the analysis: often, only 60% of sequences and 2% of molecular features from an untargeted metabolomics experiment can be annotated by existing references . However, a rational reconstruction of why the annotatable microbes or molecules are plausible can always be developed by creative scientists looking to respond to their reviewers’ criticism that their manuscript is “too descriptive”.

An important metaphor in science and information visualization is the idea of the map, whether of real spaces or of abstract spaces. Indeed, as data volumes increase, it is frequent that the field moves from tests of hypotheses among sites, to tests of these hypotheses with replicates at each site, to spatially or temporally explicit sampling, to detailed spatial maps. This progression has already occurred in 16S rRNA amplicon-based microbiome studies over the past decade , and has increasingly been taking place in mass spectrometry-based metabolome studies over the past four years . The value of spatial maps is so self-evident that the results are often cursed by obviousness. For example, the finding that metabolomes cluster by individual, as revealed by principal coordinates analysis , is interesting . However, the finding that a given molecule such as lauryl sulphate covers one individual, but is absent from the other individual is obvious , especially when you know that individual subject A uses a stereotypically gendered product such as Nivea for Men, which is the source of the molecule . How such personal lifestyle influences the microbiome is not known; it is also not known how even some basic parameters such as, skin temperature, skin pH, amount of sebum influences the microbial communities on the skin. Similarly, the finding that samples from four individuals differ to a statistically significant extent in their levels of specific purines and that within an individual, such molecules are also non-randomly distributed, might well be an intriguing finding prompting more investigation. However, a spatial map with dense sampling of the same individuals makes it obvious that the molecule is something that is touched and consumed, and sometimes spilled, allowing one to guess that it is probably caffeine and that one person likely spends time in the ocean based on the distribution of Synechococcus spp. .However, the fact remains that for most microbes and for most molecules, we have no idea where they are in and on the human body, in natural environments, or in human-impacted environments including built environments. Just as John Snow’s map of cholera instantly led to the hypothesis that this disease was water-borne and stemmed from the Broad Street pump, reinforced by the map’s revelation that the block that drank alcohol had no incidence of disease . The power of maps is shown by the history that this visual display of disease incidences by street became the foundation for the science and practice of epidemiology. In an analogous manner, systematically collected maps of microbes and of molecules across different spatial scales will dramatically improve our ability to make useful inferences from this data. Integration of these maps with other data layers ranging from air pollution to food deserts and neighborhood walk ability, together with zoomable user interfaces , will fundamentally transform the types of questions that can be asked of microbiome and metabolomics data. The value of abstract maps, whether ordinations such as principal coordinates analysis , non-metric multidimensional scaling , t-distributed stochastic neighbor embedding , network diagrams obtained from object similarity , or from co-occurrence across samples, is also considerable. In particular, when the right data frame and metrics are chosen, the key result is often immediately obvious.