Month: December 2024

The effectiveness of the selected bacterial carrier to protect encapsulated phytochemical compounds was assessed based on the thermal treatment of both the encapsulated phytochemicals and the phytochemicals in a control juice matrix. In summary, this study demonstrates the potential of using inactivated probiotic bacterial cell carrier for the binding and encapsulation of phytochemicals from a complex juice matrix and characterizes the binding and encapsulation efficiency of diverse phytochemicals and stability of encapsulated compounds in bacterial carriers using a combination of chemical analysis, spectral imaging, and antioxidant properties.Fruit juice contain a variety of phytochemicals, which in general can be classified into alkaloids, carotenoids, nitrogen-containing compounds, organosulfur compounds, and phenolics. Many in vitro and in vivo studies support that the antioxidant property of the phytochemicals plays a major role in their essential health benefits such as anti-inflammation and anti-carcinogen. To characterize the overall efficiency of encapsulating complex profiles of phytochemicals using the bacteria cell carriers, the relative encapsulation efficiency was measured based on the difference in antioxidant concentration of the juice sample before and after incubation with cells. To quantify this ratio, drainage pot antioxidant concentration in juice sample and juice residue after the encapsulation process was measured using the FRAP assay.

The method for FRAP assay is described in detail in Section 3.6. These differences in the FRAP values before and after the encapsulation process reflect the relative amount of antioxidant compounds, including phenolics that are infused or bound to a selected cell-based micro-carrier. Table 1 shows the total antioxidant capacity of MG juice sample measured using the FRAP assay. The encapsulation efficiency in the selected bacterial carrier was 72.67% for MG. This percentage indicates the total fraction of antioxidant compounds bound and encapsulated in a bacterial cell carrier compared to the total antioxidant content in the juice sample. This result suggests that a simple incubation method allows phytochemicals to passively diffuse from a juice matrix to inactivated L. casei cells and results in an efficient binding and encapsulation of the antioxidant compounds in the cell carrier.In addition to characterizing the encapsulated antioxidant content, the encapsulation efficiency of the anthocyanin pigments from the juice to the cells was also evaluated. Anthocyanin, being water soluble, is one of the major polyphenolic fractions in fruit juice and has a significant contribution to its antioxidant properties. To assess the anthocyanin content in the juice before and after encapsulation, the juice matrix was ex-tracted using methanol as described in the materials and methods section and the total anthocyanin content in the extract before and after incubation with cells was measured using a UV-Vis spectrophotometry.

The measured absorbance at 530 nm was converted to an equivalent keracyanin chloride concentration using a standard curve. Results show that MG juice had approximately 8.21 µM/mL of the equivalent keracyanin content. After incubation with inactivated bacterial cells, 66.97% of the total anthocyanin from the MG juice was encapsulated or bound to the cell carriers. In summary, these results highlight a significant potential of the selected bacterial strain for encapsulating antioxidants and anthocyanin family of compounds from a complex juice sample.To help visualize the encapsulated compounds and their intracellular distribution in the cell carriers, confocal multispectral fluorescence images were acquired based on the endogenous fluorescence signals of phytochemicals. The images were collected with a 405 nm excitation and an emission in the FITC channel from 500 to 550 nm. The fluorescence intensity of the cells in each image was quantified by randomly selecting 20 cells and measuring their mean pixel intensity using the ImageJ software. The mean background intensity was subtracted from the cell signals to remove the background signal. As shown in Figure 1, the signal intensity of L. casei carriers increased approximately 24-fold upon incubation of cells with an MG juice as compared to the auto- fluorescence signal from the control cells . Differences in the fluorescence signal intensity between the controls and the modified cells with juice phenolics was statistically significant with a p-value ≤ 0.05. The zoomed-in views in Figure 1b indicated that the cell carriers retained the cellular structure after the encapsulation process, and the encapsulated material was localized relatively uniformly across the intracellular compartment.

The broad emission range is usually associated with the presence of a diverse class of polyphenolic compounds. Based on the previous literature related to fluorescence properties of polyphenolics, the emission band between 533 nm and 595 nm mostly corresponds to anthocyanin content. MG spectra with the secondary emission around 590 nm indicates the presence of anthocyanin compounds in the cell carriers from juice matrix. In addition, the major peak in the MG spectra around 515 nm suggests a possible encapsulation of other phenolic compounds. Plant phenolics such as ferulic acid are known to have fluorescence emissions centered around 520 nm–530 nm. The broadening of the peaks observed in Figure 2 could be attributed to other photo active compounds present in the complex juice matrix. The shift in the emission range compared to the peaks observed from prior literature could also be caused by multiple factors. Anthocyanin polymerization during the juice processing and storage process could cause the emission to shift towards shorter wavelengths. In addition, fluorescence emission spectrums are known to be sensitive to environmental factors, including the excitation wavelength, medium pH and polarity, present macromolecules, etc.. Thus, it may contribute to the shifts observed in Figure 2. In this measurement, phenolic compounds that emit blue fluorescence were not captured in Figure 2 due to the limitation of the available wavelength range in this imaging system. To address these gaps in the compositional analysis of encapsulated compounds, analytical measurements using a HPLC method with known standards were conducted.Among the diverse groups of bio-active compounds present in the fruit and fruit juices, phenolic compounds constitute one of the largest and most diverse groups of phytochemicals. To characterize the phenolic compounds profile of the juice and the encapsulation efficiency, the MG juice before and after incubation with cells was analyzed using HPLC and, based on these measurements, the encapsulation efficiency of the selected polyphenolics was quantified. The protocols for the evaluation of phenolics in a grape juice matrix were already developed by Oberholster et al.. Target compound classes included in this study were flavanols , phenolic acid , flavonols , and polymeric polyphenols . Catechin, epicatechin, gallic acid, and polymeric phenols were quantified using chromatograms at signal 280 nm, caffeic acid at 320 nm, and quercitin and glycosylated myricetin at 360 nm. These polyphenolic compounds have been determined to be among the leading polyphenolic compounds in a grape juice. The chromatograms of 20% MG juice matrix at signals 280, 320, and 360 nmbefore and after encapsulation are shown in Figure 3. Peaks corresponding to each analyzed phenolic compounds were assigned in chromatograms collected at each signal.As observed from Table 2 and Figure 3, most of the investigated compounds were present in MG juice at different concentrations and had different levels of encapsulation efficiency. For flavanols, catechin and epicatechin were both present in the MG juice matrix. Despite these differences in absolute concentration levels, 17.40% of catechin and 18.77% of epicatechin were encapsulated upon incubation of cells with MG juice. For the phenolic acids, large pot with drainage the concentration of gallic acid in MG juice was 1.69 mg/mL and its encapsulation efficiency was 18.43% in L. casei cells. The content and encapsulation efficiency for gallic acid was significantly higher than the amount of coutaric acid and its encapsulation efficiency in L. casei cells. The amount of caffeic acid was below the detection limit in MG juice. Among flavonols, 20% MG juice contains 21.70 mg/mL of quercetin. The encapsulation efficiency of quercetin in L. casei was limited as compared to the quantified flavanols and phenolic acids. Glucoside derivatives are commonly found in grapes and wines, particularly delphinidin-3-glucoside, petunidin-3-glucoside, and malvidin-3-glucoside . MG juice contains 3.53 mg/mL myricetin 3-glycoside and its encapsulation efficiency was 69.85% upon incubation of cells with MG juice. Another common abundant polyphenolic compound in MG juice was polymeric phenols. The 20% juice contains 26.09 mg/mL of polymeric phenols.

The polymeric phenols identified using this protocol represent a mixture of polymeric pigments, which are formed based on reactions between grape anthocyanins and other components in the juice such as tannin, catechins, and proanthocyanidins. A total of 97.97% of the polymeric phenol was infused into the cell carriers upon incubation with MG juice. Taken together, the imaging and HPLC measurement results illustrate that cell carriers can simultaneously encapsulate diversity of bio-active compounds from a complex juice matrix. Compared to previous studies that have predominantly focused on yeast cells for the encapsulation of purified hydrophobic polyphenolic compounds, the results of this study suggest a potential of diverse cell carriers, including bacterial cell carriers, to simultaneously encapsulate multiple compounds from mixtures. Furthermore, since the encapsulation process was conducted using water soluble compounds in fruit juice, this study demonstrates that bacterial cell carriers can bind and encapsulate compounds from water extracts and juices. Together with prior studies, the results of this study illustrate the potential of cell carriers to encapsulate both hydrophobic and hydrophilic bio-actives. The encapsulation process of these compounds from cell carriers can be attributed to both composition and structure of cell carriers. Besides the structural integrity that withstood the encapsulation process as shown in Figure 1, bacterial and yeast cell carriers have a relatively high fraction of protein content on a dry basis. In the case of L. casei cells, the protein content can be as high as 80% or higher on a dry basis. Similarly, the protein content in yeast cells can range from 25 to 60% on a dry basis. In addition, cell carriers also express both soluble and structural proteins including membrane associated proteins. In previous studies, protein–polyphenolic interactions have been explored and the binding between protein isolates and polyphenolic compounds from juice or other plant extracts has been demonstrated. Thus, it is likely that a relatively high concentration and diversity of proteins in micro-scale cells carriers significantly promote the binding of diverse polyphenols from a juice matrix. In addition to proteins, bacteria and yeast cells also contain a diversity of carbohydrate bio-polymers mostly concentrated in cell walls and lipids that are integral parts of the cell membranes. Prior studies have shown interactions between polyphenols and cellular polysaccharides, and the binding mechanism could be attributed to a range of physical and chemical interactions. The complex and porous structures and surface properties of the cell wall has also been proposed to be important for the binding process. These compositions and cellular structures can provide a rich environment for the partitioning and compartmentalization of diverse compounds in cell-based carriers.The results illustrate that the encapsulated content and efficiencies varied by the chemical class, compounds, and juice matrix. Quercetin as a monomeric flavonol showed low incorporation rates from MG juice, while the glycosylated myricetin has a significantly higher encapsulation efficiency . In contrast, polymeric polyphenols yielded the highest encapsulation efficiency among all compounds tested from MG juice . This trend of differences in encapsulation efficiency of compounds of the same class was also observed in the case of flavonols and phenolic acids. Furthermore, based on these measurements, no clear correlation between encapsulation efficiency and relative hydrophilicity of the compounds was observed. These observations suggest that the partitioning of compounds in cells from a juice matrix significantly depends on the interactions among the polyphenolic compounds and the composition of cells. The characterization of these interactions is beyond the scope of this study, but these results suggest that it may be possible to select cellular compositions among the diverse class of microbes that may promote the binding of selective polyphenols from a given plant extract and juice.One of the important functionalities of encapsulation carriers is to protect the bio-active compounds from adverse environmental factors and food processing conditions. These adverse conditions may cause damage by oxidation, less favorable pH, and thermal induced reactions in bio-active compounds in food matrices. In order to produce a shelf-stable and microbially safe food, thermal processing methods such as pasteurization or sterilization are commonly used. Thus, in this study the effectiveness of the selected bacterial carrier in protecting and stabilizing the encapsulated juice polyphenols was evaluated.

Genetic map building with quantitative trait loci analysis is needed to identify possible candidate genes and to predict molecular markers associated with fruit quality characteristics. The most preferred molecular markers have been used as single nucleotide polymorphisms , polymorphic insertions or deletions , or microsatellites recently. Genome-wide association studies and linkage mapping need high-throughput molecular marker assays. Although genetic mapping studies are still limited in mandarin due to the insufficiency of phenotype data and the complexity of fruit quality traits, some studies have been conducted on fruit quality traits that are important for Citrus breeding . The QTL analysis would help select parents and improve hybrids of the parents with the target gene of a favorable trait by preventing the long juvenile period of Citrus and eliminating Citrus breeding expenses . Improvements in next generation sequencing and genotyping array technologies have helped to understand the genetic basis of quantitative trait variation. SNP genotyping became the most widely used genotyping method for GWAS and QTL mapping due to being inexpensive and producing many, greenhouse vertical farming codominant SNPs. In addition, SNP genotyping can be performed with SNP arrays or produced by genotyping-by-sequencing , or whole-genome sequencing.

Fruit size is among the most crucial fruit quality traits for mandarin breeding. Generally, mandarin fruit size varies from small to medium, but tangelo and tangor hybrids have larger fruit sizes. Amparo is a small-fruited mandarin with about 40 mm diameter and 30 g weight. Moreover, Ugly has a much larger fruit size, it is a tangelo with a fruit diameter of 120 mm and a fruit weight of 580 g . Obtaining fruits in uniform fruit size is one of the essential elements for mandarin breeding. Therefore, it is important to construct a mandarin genetic map and identify markers related to fruit size. QTL identification associated with fruit size for mandarins has been conducted in several studies . In the study, Fortune , Murcott , and 116 F1 mandarin individuals derived from were analyzed for fruit quality traits by Yu et. al . It was carried out in Fortune , Murcott , and 116 F1 mandarin individuals derived from were analyzed for fruit quality traits by Yu et. al . It was carried out in January and February, with four samplings in 2012 and 2013. The map was performed by using a 1536-SNP Illumina GoldenGate assay. The constructed genetic linkage map of “FOR” consisted of 189 SNPs, while “MUR” consisted of 106 SNPs. A total of 48 QTLs related to fruit quality traits were defined in the study. 3 QTLs of them were associated with fruit weight and 3 QTLs of them were related to fruit diameter . The repeatable QTLs were determined as FW5.1 and FW8 and non-repeatable were FW4.2, FD4.2, FD5.1, and FD9.3.

FW4.2 was detected on MUR4.2 with 25.69 cM and explained % 24.60 of the phenotypic variance . QTL analysis study on fruit quality characteristics in the mandarins detected QTLs associated with fruit size. A SNP-based genetic linkage map and QTL mapping were conducted using an F1 segregating population derived from by Imai et. al . The map for “Harehime” consisted of 442 SNPs, and for “Yoshida” consisted of 332 SNPs. 4 QTLs were identified for fruit weight with 14.9-26.5 % of the phenotypic variance. FWq3 was identified spanning 15.3-31.0 cM on the Clementine genome scaffold 4. The most striking point in this study is the claim that these two QTLs can correspond since FWq3 and FW4.2 are located on the same Clementine reference genome scaffold 4 .The fruit flavor is one of the main determinants of fruit quality and consumer preference in mandarins. The ratio between sugar and acid content, which is defined as the total soluble solids: titratable acidity ratio , is significant for the taste of the fruit . Only the high sugar content and less acidity rate or less sugar and high acidity level do not affect the fruit taste positively. For this reason, fruit is required to contain a certain amount of acid content in terms of fruit taste. Fruits with a high TSS/TA ratio have a bland taste, while fruits with a low TSS/TA ratio have a sour taste. Goldenberg et. al stated that the TSS/TA ratio for mandarins that are highly desired is about 13. In addition, Citrus fruit development in relation to solids and acids. Solids gradually increase, and acids first increase and then decrease.

So meaningful comparison of varieties is only possible in relation to the date sampled. reported that tangerines, which is a popular variety in terms of their taste, are the variety of mandarin with mean of the highest sugar content , acid content , and sugar/acid ratios in Table 2. Some QTLs associated with the fruit sugar content in Citrus have been identified in some previous studies. For Soluble Sugar Content , Yu et. al detected five non-repeatable QTLs on scaffolds 2,3,4, and 8 of the Clementine reference genomes. In addition, three non-repeatable QTLs for Soluble solids content: titratable acidity were identified by Yu et. al . Moreover, they identified two non-repeatable QTLs and one repeatableQTL for TA were positioned at scaffolds 7, 8, and 9 of the Clementine reference genome. The QTLs identified for ST were positioned on the scaffold 1, 7, and 9 of the Clementine reference genome . In this study, some QTLs for TA overlapped the QTLs for ST and SSC. For example, TA9 and ST9, and TA8 and SSC8 overlapped. Imai et. al studied one of four mandarin fruit quality traits: sugar content . Only SCq1 was identified for SC on LG5 .Seedlessness is a desirable trait for Citrus breeding since the seed in the fruit may negatively affect the taste and aroma of the fruit due to effects of seeds on chemical composition . Navel orange, Satsuma mandarin, and Clementine mandarin are the most popular Citrus crops that are seedless . For Citrus fruit to be defined as seedless, it must contain no seeds, contain aborted seeds, or the number of seeds of a multi-seeded variety must be significantly reduced . Many factors play a role in mandarin seedlessness, including parthenocarpy, male and female sterility, self-incompatibility, abnormal ovules, embryo sac abortion, environmental conditions, and plant growth regulators . Seedlessness is one of the most important fruit quality characteristics in mandarin breeding and is important for obtaining seedless fruits. Thus, QTLs responsible for this trait are required to select for it more effectively. Five genomic regions were detected for seed number by using 201 full-sib population, which were crossed reciprocally between Fortune and Chandler . Four QTLs associated with seed numbers were detected in 2014 and 2015. The QTL SN11 was identified on the linkage group Gr9b on locus CTUCH7 with 8.8 %, moreover the other QTLs on the Clementine linkage groups with 8.4, 10.7, and 11.3 % of the explained phenotypic variance, respectively. Yu et. al detected non-repeatable QTLs associated with seed number. These QTLs on scaffolds 3 and 9 of the Clementine reference genome explained phenotypic variance with 21.32% and 19.59 %, respectively .Fruit color is one of the most influential fruit quality attributes of Citrus features because the first thing that affects consumer preference is the appearance and color of the fruit . Citrus peels consist of the pigmented peripheral epicarp or flavedo and albedo .The part responsible for the fruit peel color is the flavedo of the fruit . The color of mandarin peels is usually greenish-yellow, yellow, yellow-orange, orange, and reddish. Fruit pulp color is as important as a fruit quality feature as peel color. Color pigments are responsible for the color of fruit flesh and skin. These pigments are carotenoids and flavonoids. In general, mandarins have a yellow to orange hue. Carotenoids are the primary pigments for yellow to orange colors.There are three main cultivar groups in terms of including carotenoids in Citrus. The first group is the carotenoid-poor group. There are pomelos, lemons and limes, nft vertical farming and grapefruits in this group. The other group is the violaxanthin-abundant group, which includes oranges. The third is the cryptoxanthin-abundant group, primarily containing mandarins . Lycopene, which is responsible for the pink color, is also a carotene and is found in grapefruit such as ‘Star RUBY’ and navel orange such as ‘Cara Cara.’ Anthocyanins are a subgroup of flavonoids that give blue, purple, and red colors to the fruit. Anthocyanins are phenolic compounds responsible for the red color of Citrus fruits such as blood oranges . In addition, with the increase in the popularity of red-colored fruits, mandarin hybrids with red flesh have been introduced to the global market.

These red-fleshed mandarin hybrids are rich in anthocyanins, and examples include ‘Sun Red,’ ‘Early Sicily,’ and ‘Sweet Sicily’ varieties released in Italy . There are some significant genes in the carotenoid biosynthesis pathway in Citrus. They are PSY, PDS, LCYB1, LCYB2, CHY, CHYB, and CCD. The gene responsible for anthocyanin biosynthesis in Citrus is the RUBY gene which encodes an MYB transcription factor. MYB transcription factor regulates anthocyanin biosynthesis with a basic helix-loop-helix transcription factor and a WD40 repeat protein. The expression of the RUBY gene depends on environmental conditions. This gene is upregulated under cold temperatures, causing anthocyanin accumulation in the fruit .Previous QTL studies on fruit color were based on detecting QTLs associated with carotene content in Citrus fruits. Papers reporting QTLs associated with anthocyanin content in Citrus have not yet been published. created a population derived from the female parent ‘Okitsu-46’ and the male parent ‘Nou-5’ . For QTL mapping, EST-based CAPS markers were used to generate linkage maps from 51 progenies and their parents. Their extracts were prepared to measure by HPLC the carotenoid content. According to the results, the A255 map was generated with 345 markers and covered 660cM. In contrast, the G434 map was constructed with 254 markers covering 642 cM. It was cited that there was transgressive segregation for total and each carotenoid contents in progenies. Transgressive segregation arises from the distribution of alleles between parents. It leads to the form of extreme phenotypes in segregated populations compared to the parental phenotypes. QTLs for fruit pericarp and 35 QTLs for pulp were identified. Each QTL for flavonoids in fruit pericarp was explained by phenotypic variance from 30.2 to 71.8%. At the same time, each QTL for flavonoids in fruit pericarp was explained by phenotypic variance from 26.7 to 63.0%. The QTLs for flavonoids were on Chr3, Chr7, and Chr9.Since it is directly related to the external appearance, the rind or peel thickness is a vital fruit quality feature. The peel or rind of the fruit has flavedo and albedo sections and it is reported that mandarins have thinner albedo thickness among other Citrus varieties in general. For example, the albedo thickness of sweet oranges varies from 5 mm to 10 mm. Grapefruits have an albedo thickness of more than 10 mm. Lemons have ±5 mm, and mandarins have an albedo thickness of less than 3 mm on average. Asins et. al performed a QTL analysis associated with rind thickness. A total of QTLs for rind thickness was on the Clementine linkage group, with phenotypic variation explained by each QTL ranging from 9.0 to 21.3% . The juice volume is an essential trait for inner fruit quality. Pulp segments filled with many vehicles are called juice sacs in the Citrus flavedo part. Juice sacs include sugars, organic acids, vitamins, and polyphenolic plant compounds. Juice volume is considered an important criterion, especially for the fruit juice industry . 2 QTLs for juice volume were detected on Clementine linkage groups by Asins et. al . The QTL JV_11 on Cl3 explained 7.4% of phenotypic variance, and JV_11 on Cl4b explained 10.8% of phenotypic variance. In addition, 7 QTLs were identified for juice content . The paper reported that JC was calculated as a percentage of JV and fruit weight . All QTLs related to juice volume and content are defined only on the Clementine map . Yu et. al identified only one QTL associated with juice percentage . JP was calculated by using juice volume and fruit weight. The QTL JP7.2 was on MUR 7.2 with a 2.78 LOD score . In this study, the parents of the mandarin hybrids differ in several fruit traits related to size, seediness, sugar content, and acidity. We sought to achieve a better understanding of the genetic basis of variation in fruit characteristics, including fruit quality and rind color in an outcross F1 population of mandarin hybrids.

Grapevines were cane pruned with 12–14 nodes per cane. Vines were drip irrigated from May to September . A single irrigation pipeline was positioned on the soil with three drippers for each vine . Soil water potential was kept below −300 kPa, and leaf water potentials were maintained at values < −0.6 MPa. Fertilizer addition, pest control, and other vineyard operations were conducted according to local practices. A randomized block design was used with three blocks and three treatments, and each treatment in the block consisted of six grapevines selected with a uniform number of clusters. Each treatment consisted of: control, ethephon at 1445 mg/L, ethephon at 2890 mg/L. The concentrations used in this trial were established on results obtained in preliminary studies . Ethephon was dispersed in water with 0.1% of a surfactant and applied directly to the clusters of vines selected for abscission treatments. Clusters from control vines were treated with water containing the surfactant only. The ethephon or control solutions were applied with a handheld sprayer until run-off when the fruits reached sufficient soluble solids for harvest . After the berries dried, raspberry cultivation pot each cluster was enclosed in a mesh bag to collect any berries that may abscise.Berries were sampled before treatment, 2 h after treatment and in successive days, as reported in Table 1.

Measurements of FDF, berry skin color, and firmness were as described previously. In brief, FDF was determined as the force required to detach the berry from the rachis as measured with a mechanical gauge. A hand-held, temperature compensating digital refractometer and an automatic titrator were used for the following determinations: soluble solids content , pH, titratable acidity . For all these measurements, 10 clusters from each vine were selected and three berries from each cluster were sampled to measure the FDF and three berries for the other measurements. Pre-harvest abscission was determined by counting any abscised berries that had collected in the mesh bags on observation days . Abscised berries were placed in plastic bags and stored in a portable ice box for transport to the laboratory where the integrity of the berry, including the presence/absence of a pedicel, and a wet or dry stem scar was observed with the aid of a binocular microscope at 30× . Berries that abscised pre-harvest and those that fell during harvest, handling or after light shaking constituted the total percentage of dropped berries. The abscised berry percentage was calculated as [/ × 100].Ethephon residues were determined according to the method proposed by Takenaka . For each treatment 30 berries were randomly collected from 10 clusters, stored in a portable FIGURE 1 | Mesh bags to prevent pre-harvest berry loss of Thompson Seedless and Crimson Seedless table grapes. ice box, and carried to the laboratory for analysis. Cartridges SPE NH2 500 mg of Phenomenex activated as suggested by manufacturer were used in the purification step. The purified samples were evaporated to dryness with a rotavapor at 40◦C, taken up with 1 ml of methanol and subjected to derivatization.

One hundred microliters of reconstituted samples were transferred to 1.5 mL eppendorf, diluted with 500 µL of acetone and derivatized by adding 10 µL of trimethylsilyldiazomethane . The reaction vials were maintained at 50◦C for 30 min, then 10 µL of 1 M acetic acid in methanol were added in order to stop the reaction. After centrifugation, 2 µL of the clear upper phase were injected in the GC-MS system.Ethephon application did not affect berry color of Thompson Seedless until 14 days after treatment . At that time, ethephon-treated fruit was darker in color , and had lower C ∗ and a greater h◦ , indicating the fruit were somewhat more yellow colored than non-treated fruit and generally had a more mature appearance. These findings are consistent with other reports that ethephon affects berry skin color by stimulating the accumulation of phenolic compounds . Ethephon treatments clearly reduced FDF because most of the berries on treated clusters were so loosely attached that they abscised before harvest or during handling . However, the few remaining berries on treated clusters were just as tightly held as the berries on non-treated clusters, so no treatment effects on FDF could be measured . A similar result was reported for Thompson Seedless treated with methyl jasmonate, another abscission agent . FDF may decline within a few days of treatment with abscission agents , so timely harvest may be needed when reductions in FDF are large. Abscission agents did not reduce fruit firmness, but FDF and berry firmness decreased from the time of ethephon application whether the clusters were treated or not . As suggested earlier, ethephon at either concentration tested stimulated an almost complete berry abscission from the rachis .

The effects of the two concentrations were similar, with only a few berries still attached to the rachis by harvest time , and the abscised berries generally had dry stem scars . Dry stem scars could be desirable for fresh-cut fruit since the scars help prevent juice leakage and minimize the exposure of interior berry tissues to the atmosphere and to pathogens that might reduce shelf-life or berry quality. However, pre-harvest berry abscission could lead to significant yield losses , though yield loss might be minimized byearlier harvest or the use of catch systems, i.e., nets under the canopy. Ethephon did not affect SSC, pH, or TA . Few studies have examined the effect of abscission agents on grape berry composition, but our results generally agree with Uzquiza et al. who reported few and minor treatment effects on winegrapes. Even though a registered use of ethephon on grape is the promotion of fruit maturity, effects on grape composition are often variable, and ethephon applications to promote fruit maturity are made at veraison, a much earlier stage of fruit development . Abscission agents are applied to mature fruit, so there is less opportunity to affect fruit composition. Moreover, abscission agents quickly initiate the development of an abscission layer between the pedicel and berry . The rapid action of abscission agents necessitates a short time period between application and harvest, further limiting the potential for differences in composition to develop.Ethephon reduced the lightness and purity of the skin color as previously observed for Thompson Seedless, and similarly to that observed by others . The FDF was significantly reduced , whereas SSC and acidity were not affected as in a previous work . A short post-harvest interval limits the possible compositional effects , as discussed above. However, in a previous trial on Crimson Seedless, an increase of tartaric acid, procyanidin P2, terpenoid derivatives, and peonidin-3-glucoside as well as a decrease of catechin and epicatechin was observed after treatments with ethephon a few days before harvest .Treatment with either concentration of ethephon stimulated significant pre-harvest abscission , both >40% and almost 55% at the dose of 2890 mg/L . A similar effect on Crimson Seedless has been recently reported . The treatments tested were less effective at inducing abscission of Crimson Seedless than they were at inducing abscission of Thompson Seedless. Differences among varieties in responsiveness to abscission agents has been previously reported in grape , low round pots and it has also been observed that some table grape varieties are more susceptible than others to “shatter,” or “dry drop,” a post-harvest disorder characterized by the development of an abscission layer between the pedicel and berry . The physiological basis for varietal differences in responsiveness to abscission agents is uncertain, but the application of very high rates of ethephon can induce abscission in varieties that are otherwise non-responsive , suggesting that the less responsive varieties may be less sensitive to ethylene. As observed with Thompson Seedless, SSC, pH, and TA of Crimson Seedless were not affected by abscission agents .The lack of compositional effects are probably due to similar reasons identified and discussed earlier for Thompson Seedless.No present model of solids can predict all microscopic electrical properties of a solid. Two approaches are used in the quantum mechanical study of the electrical properties of solids: free electron models, and bound electron models. Models such as the nearly free electron model, suppose that electrons are free of atoms, and then impose restrictions on how electrons may move. Models such as the tight binding model suppose that electrons are bound to atoms, and then provide ways in which electrons may move.

Meanwhile, bound electron models are necessary for explaining the subtle microscopic properties of solids. A cornerstone the microscopic theory of solids is the Quantum Hall Effect. Within real solids, there is disorder: wave functions and crystal lattices are not uniform or periodic throughout space. With disorder, electrons tend to localize, and materials become insulators. However, within two dimensional materials such as graphene, for certain magnetic fields, even in the presence of disorder, a delocalization of electrons is observed. This conduction is the Quantum Hall Effect, which experimentally exhibits conduction values of exact integer numbers of electrons, or, surprisingly, fractional numbers of electrons known as quasiparticles. Each phase of conduction, or topological phase, is described by a quantum number called the Chern Number, which is an integer that uniquely specifies that topological phase. It is particularly interesting that the transitions between topological phases, known as Plateau Phase Transitions, may described using exactly the same model as for phase transitions between states of matter. This model is the Landau Theory of Phase Transitions, in which a phase transition is quantified through a set of numbers known as critical exponents. Within quantum mechanics, the two basic states of particles are standing waves and traveling waves. Particles in the absence of a global potential take the form of traveling waves and are delocalized. Particles in a global potential take the form of standing waves and are localized. In the global potential created by a magnetic field, electrons behave as standing waves that are circles perpendicular to the magnetic field, also known as cyclotron orbits. In the local, periodic potential created by uniformly spaced atoms, electrons behave as traveling waves of wavelengths such that they will miss the atoms. The Hofstadter Model combines a global magnetic field with a linear combination of atomic orbitals. It is a tight binding model that first assumes that electrons are bound to atoms, and then provides ways to conduct electrons to nearby atoms. Within the Hofstadter Model, the degree of locality is quantified by a distance known as the “localization length.” This length is strongly dependent on the magnetic field and electron concentration, and at Plateau Transitions, this length becomes infinite. In this paper, we proceed by way of an introduction with background given on the theories of electronic conduction, the Classical Hall Effect, Landau Levels, and the Quantum Hall Effect. These sections give a semi-classical and experimental motivation for the notions of localization and topological phase. Next, we introduce the tight-binding model for electronic motion in one dimension, solve it analytically and numerically using the modules, similar results are then obtained for the two-dimensional tight-binding model. The Hofstadter Model is covered, presented as a result of a sum over translation operators, and is then solved numerically using the modules at a variety of fluxes to find the Hofstadter Butterfly. Chern numbers are introduced with the experimental motivation of the Quantum Hall Effect. A method to calculate Chern numbers through the Berry Curvature is presented, as is an efficient algorithm by Fukui, Hatsugai, and Suzuki. This algorithm forms the basis of the Chern number module. This is then applied to a simple model of a Topological Insulator as well as the Hofstadter Model. After that, the concept of localization is introduced and presented in the form of the Anderson Model. This model is then numerically analyzed using modules for transfer matrices and results are described. Finally, the Chalker-Coddington Model is analyzed using the modules and the results are consistent with the literature.Free electron models are a class of models of electron conduction that work well on metals or near-metals. Free electron models assume that there is either no, or very little interaction between an electron and the lattice If there is no interaction with the lattice, then the electron behavior is that of a Fermi gas, where momentum is pF which is given by the Fermi energy _x000F_ F = p 2 F /2m. This is a smooth quadratic function in momentum, and it cannot explain the behavior of insulators which have discontinuities in their energy dispersion.

When 3.9 µmol/kg per day of L or Z for 24 to 101 weeks were supplemented, the rhesus monkeys showed significant increases in their corresponding serum, retinal, and adipose tissue concentrations. In their retina samples, L and meso-Z, but not Z, appeared in the L-supplemented group, while only Z was found in the group supplemented with Z. In humans, a study investigating the serum and macular responses of L, Z, and meso-Z from dietary supplements found that 13.13 mg/d for 12 weeks provided maximum MPOD improvement, whereas 7.44 mg/d was the amount that increased serum levels at the highest efficacy. These and other studies support the need for dietary recommendations for L and Z, particularly as conditionally essential nutrients due to their protective effects on eye health. Lutein and Z fulfill many criteria as essential nutrients, including high concentrations in select tissues, biological plausibility for eye health, depletion outcomes such as vision impairment in primates, and inverse associations with certain diseases. In addition to their role in eye health, L and Z are involved in cognitive function at all stages of life. A randomized controlled trial reported that L and Z supplementation improved neural efficiency and learning performance by increasing the interaction of numerous brain regions in older adults.

Other reports have demonstrated an association between L intake or circulating levels and preserving age-related cognitive decline, dutch bucket for tomatoes reducing the risks of certain cancers, coronary heart disease, stroke, metabolic syndrome, and achieving higher levels of physical activity. A systematic review of in vivo, ex vivo, and in vitro studies concluded that L may benefit vascular health by improving endothelial function, reducing inflammation, regulating favorable lipid profiles, and maintaining glucose homeostasis. Systematic reviews summarizing the amount of L needed for cognitive functions and enhancement of gray matter volume estimated that at least 10 mg/d for 12 months could be beneficial. Age must be considered when creating DRI values, since MPOD values are lower in older compared to younger individuals. Whether the proposed intake of L and Z should be based on the amount that can reduce AMD risk, benefit visual maturation in newborns, protect cognitive health, or reduce the risk of other diseases requires further consideration. Sex differences in AMD prevalence must also be considered, especially in relation to pregnancy and lactation, as discussed above. Nevertheless, L and Z are not included for DRI consideration due to inadequate details from food databases, limited large-scale dietary intake studies, and insufficient knowledge regarding their metabolism and biological functions. 

Many continue to advocate for a DRI for L, since it satisfies all nine criteria for bio-active compounds. A rich dietary source of L and Z is goji berry, , also called wolfberry or Gou Qi Zi. The bright orange-red colored oval fruit, has been used for millennia in traditional Chinese medicine for its role in visual health, to provide immunoregulatory, neuro-protective, and anti-inflammatory benefits, and to help regulate liver and kidney meridians . Commercially-available goji berries and their products come primarily from the Ningxia and Xinjiang autonomous regions in western China. Goji berry is known for its high amount of carotenoids, with the Z content higher than any other known food.140,141 In addition to carotenoids, other bioactive compounds found in goji berries include Lycium barbarum polysaccharides , flavonoids, vitamins, minerals, betaine,cerebrosides, phenolic acids, and certain amino acids which may also support the overall health of the eye, particularly when working synergistically. Although the TCM use of goji berry also includes the leaves and bark of the plant, this review will discuss the potential benefits of the fruit on eye health. In addition to a robust amount of Z, goji berries contain modest amounts of β-cryptoxanthin, β- carotene, neoxanthin and L. 

The Z and L content among different varieties of dried goji berries cultivated in Ningxia province ranged from 25 to 152 mg/100g, and 0.3 to 1.9 mg/100g, respectively.143 According to the United States Department of Agriculture food database, one serving of goji berries is 28 g, which would provide up to 42.6 mg of L + Z, depending on the cultivar. Moreover, the predominant form of Z in goji berries is a dipalmitate, found with a diester linkage. The ratio of Z dipalmitate to total carotenoids was up to 55% and 88%, in fresh and dried goji berry fruit, respectively. This esterified form of Z showed a significantly higher intestinal absorption than monoester and free Z due to the high efficacy of hydrolysis, mainly by carboxyl ester lipase. Plasma Z was significantly increased in individuals consuming 15g goji berries daily for 28 days in comparison to those on a habitual diet.  Participants consuming 5 mg of Z dipalmitate extracted from goji berries showed a higher plasma Z concentration than when they consumed the same amount as unesterified Z over a nine to 24 hour period. The high Z content of goji berries has been proposed as a dietary source to reduce the risk of AMD, although studies are limited. In one study, circulating Z levels were significantly higher in healthy older individuals who consumed 10 mg of Z extract from goji berries daily for 90 days. No change in macular pigmentation or soft drusen was observed, but MPOD was not measured. In an uncontrolled trial, individuals with early stage AMD who consume a beverage containing 12 mg of L and 2 mg of Z derived from marigold flower and goji berry, respectively, daily for five months, showed higher circulating levels of L and Z, lower intraocular pressures, and better best-corrected visual acuity scores. Unfortunately, the study lacked a control group, did not test the effect of Z separately, and did not clarify whether the form of Z extracted from goji berry was the dipalmitate. Another study investigating the effects of an herbal formula among healthy adults with dry eyes noted that those chewing tablets containing L , Z , extracts from blackcurrant, chrysanthemum, and goji berry showed dose-dependent reductions in eye fatigue symptoms, improved tear secretion as well as MPOD, compared to placebo. The basis of this formula was derived from TCM, so the multi component formulation could not directly inform the role of any single ingredient. A study in patients with early AMD reported that the MPOD was significantly higher in those consuming 25 g/day of goji berries for 90 days, compared to their baseline levels and to a habitual diet control group. The BCVA was also significantly improved in the goji berry group compared to their baseline values.We recently reported that MPOD and skin carotenoid scores were increased in healthy middle-aged individuals consuming 28 g/day of goji berries five times a day for 90 days compared to a group taking a supplement with 6 mg of L and 4 mg of Z.These results illustrate that MPOD levels can increase in healthy individuals even without early signs of AMD. While these results are encouraging, longer intervention periods with a larger number of participants are necessary. In addition to AMD, goji berries have been studied as a therapy for retinitis pigmentosa, an inherited retinal disease. Patients who consume 0.35 g/d of LBP for 12 months showed a significant improvement in visual acuity and macular thickness, compared to control subjects who did not consume L or Z.156 Examples of human studies that evaluated the effects of supplements containing goji berries on retinal health are shown in Table 1. Based on preclinical evidence, potential benefits of goji berry intake on glaucoma and diabetic retinopathy may also exist. Goji berry extract ameliorated the high glucose-induced blood-retinal barrier disruption in human retinal pigment epithelial cells.Studies reported that LBP showed significant neuroprotective effects over retinal ganglion cells in male C57BL/6N mice and Sprague-Dawley rats with ocular hypertension.In db/db mice, goji berry extract restored the thickness of the retina, the ganglion cell number, blueberry grow pot and the integrity of RPE after daily intake over eight weeks.Although research on the upper limit of goji berry intake is scarce, goji berry allergy risk has been associated with the existence of cross-reactivity to nonspecific lipid transfer proteins from peaches, tomatoes, tobacco, tree nuts, and select pollens.In addition, bleeding symptoms after consuming goji berry juice, tea, or wine have been described in case reports among patients taking warfarin, an anticoagulant medicine.

Although the potential value of foods high in L and Z during pregnancy and lactation has been discussed above, the utilization of goji berry products during these unique periods in a woman’s life needs special caution. Clinical studies of goji berries on eye health have been conducted primarily in Asia, with emerging research reported from Italy, Lithuania, and Switzerland.Potential gene-nutrient interactions must be considered when comparing results from Asian with Caucasian populations.Macular xanthophylls cannot be synthesized de novo in primates. The oxidative defense and blue light filtering characters of L, Z, and meso-Z are important for visual function and potentially reducing the risk of AMD. Because L and Z storage in the maternal body during pregnancy and lactation may become depleted due to active transfer to the offspring, more attention to safe and robust intake of foods and dietary supplements containing these xanthophylls is warranted. Since many infant formula products are not fortified with L or Z, addition of these two xanthophylls should also be considered. The impact of foods and dietary supplements rich in L and Z on MPOD and visual function among AMD patients and in healthy individuals deserves further attention. Since Z may play a different role than L in terms of macular pigment development or protection, better analytical techniques are needed to reassess food composition databases to distinguish these compounds, with greater attention paid to Z as a stand-alone food component, rather than grouping it with other xanthophylls. Goji berries have the highest known content of Z of any commonly consumed food, which also comes in a unique dipalmitate form. Given the increasing rates of AMD worldwide, goji berries, along with L and Z supplements, may help reduce this escalation. As of 2010, safe and accessible drinking water has been explicitly recognized as a human right by the United Nations General Assembly and deemed essential for public health and economic prosperity. Despite this designation, 785 million people lack essential drinking water services globally, and at least two billion people use drinking water sources contaminated with feces. Diseases like cholera, diarrhea, dysentery, hepatitis, typhoid, and polio are linked to such sources of contamination . Despite the risks associated with a lack of essential water services, many drinking water utilities face challenges meeting regulations due to limited water supply, strict budgets, high demands due to population growth, aging infrastructure, and increasingly strict regulations for water quality. Analytical testing and water quality monitoring must be carried out regularly to ensure water is free from priority biological and chemical contamination. Unfortunately, these methods often take multiple days to conduct, in which case contaminated water may already have had negative impacts on public health. These challenges can contribute to public water utilities violating regulations established by the EPA, increasing interest in solutions such as point-of-use filtration, and real-time water quality monitoring. Even if violations are not observed from water utilities there is still potential for contamination between treatment and the tap. This contamination can source back to community water system’s distributional networks and property owner’s premise plumbing . Before reaching taps, consumers can install POU drinking water treatment systems in their water supply lines to provide on-site treatment to the water they consume. These systems encompass many treatment technologies such as membranes, filtration, UV disinfection, activated carbon, etc. The utility of implementing POU filtration shows in its ability to reduce contamination’s acute and chronic health effects. Still, there is little evidence of commercially available technologies to improve the “smartness” of point-of-use water systems in terms of their ability to perform tasks such as monitoring and reporting on water quality Possible ways to enhance the smartness of point-of-use filters include integrating Internet of Things enabled sensor technologies. Commercially available sensors measure parameters including pH, oxidation-reduction potential , and electrical conductivity , which can all be affected by chemical and biological contaminants—making these parameters effective indicators for the overall water quality as well as capable of detecting changes in water quality. When combined with the integrated circuit microcontrollers and micro processing technologies that have emerged in the last decade, there has become a platform for developing robust and customizable data loggers for relatively low costs.

Scientists hypothesized that cocoa may be an influencing factor in the low prevalence of CVD in this population, due to its high concentration of flavonoids. The primary flavonoids in cocoa are flavanols, including monomeric catechins, epicatechin, and polymeric procyanidins. Cocoa also contains methylxanthines, i.e., theobromine and caffeine, which remain in flavanol-poor cocoa butter and cocoa solids after pressing. The effect of cocoa flavanols on vascular function was first reported more than two decades ago. Extending the observations about cocoa flavanols to broader dietary patters, studies exploring the association between the intake of flavonoids and the risk of CVD have yielded inconsistent results, suggesting that certain sub-classes may be more effective than others in terms of cardio-protection. Some observational studies have shown that the dietary intake of flavanols was associated with a decreased risk of CVD and ischemic heart diseases. Epidemiological studies have reported that a moderate amount of chocolate consumption was associated with a decreased risk of CVD. A Cochrane meta-analysis concluded that the consumption of flavanol-rich chocolate and cocoa products had a small but statistically significant effect on reducing systolic and diastolic blood pressure, both by 1.76 mmHg. 

Similarly, another systematic review and meta-analysis included acute and short-term randomized controlled trials concluded that chocolate, cocoa, vertical hydroponic nft system and flavanol intake significantly improved vasodilation function as measured by flow-mediated dilatation , reduced diastolic blood pressure by 1.6 mmHg, and marginally improved the serum cholesterol profile. However, these results were strongest among individuals with moderately elevated blood pressure and untreated hypertension. The vasodilation function in healthy males, measured by FMD, was significantly increased one to four hours after taking a cocoa drink containing 917 mg of cocoa flavanols, compared to baseline values and to those consuming a control beverage with 37 mg of flavanols. The pattern of improvement in FMD from the flavanol-rich cocoa beverage was closely mirrored when participants also consumed 1 or 2 mg/kg of body weight of -epicatechin dissolved in water, suggesting -epicatechin and its metabolites were the main contributors of the vascular effects. Additional research from the same study found that while -epicatechin is a primary contributor to the vasodilation function, dimeric and oligomeric procyanidins that are metabolized by the gut microbiome may also contribute to the vasculo-protection.

In healthy young men, daily intake of a cocoa extract containing 130 mg of -epicatechin and 560 mg of procyanidins for 30 days significantly improved FMD and reduced blood pressure and arterial stiffness as measured by pulse wave velocity , while 20 mg of -epicatechin and 540 mg procyanidins, or a control capsule, did not. However, total cholesterol was decreased after in both groups consuming cocoa, suggesting synergistic effects of -epicatechin and procyanidins, possibly through gut microbiome-mediated catabolism. Cocoa also contains methylxanthines, which are biologically active. While the intake of theobromine and caffeine alone did not result in a significant change in vascular endothelial function measures,interestingly, the combination of methylxanthines plus a high cocoa flavanol drink induced a significant improvement in FMD response than the beverage only. In addition, plasma metabolites of -epicatechin were higher after consuming the flavanol-rich cocoa drink with methylxanthines than when the flavanols were consumed alone, indicating a likely interaction between theobromine, caffeine, and cocoa flavanols on vascular function. Several molecular mechanisms regarding the effects of flavanols on blood pressure and vasodilation have been proposed.

Flavanols such as -epicatechin can increase nitric oxide production directly by increasing endothelial nitric oxide synthase expression. The release of NO consequently increases intracellular cGMP which then induces a relaxation of vascular smooth muscle cells. The increased eNOS activation may also be modulated by flavanols through a calcium/calmodulin pathway by increasing the intracellular calcium concentration, or by phosphatidylinositol 3-kinase/protein kinase B -dependent eNOS phosphorylation. In addition, -epicatechin has been shown to down-regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase by inhibiting the synthesis of vasoconstrictors such as endothelin-1, and therefore increase the utilization of NO. Flavanols may also directly inhibit angiotensin-converting enzyme activity, which increases NO production. Apart from modulating NO production, flavanols may also induce the release of endothelium-derived relaxing factors such as hydrogen peroxide and prostacyclin. Cocoa flavanols also benefit cardiovascular health by inhibiting platelet activation and adhesion. In healthy individuals, platelet aggregation induced by collagen and adenosine diphosphate, and the expression of P-selectin, was significantly decreased compared to a placebo group after the daily intake of 234 mg of cocoa flavanols and procyanidins for 28 days. Some studies have reported that the reduction in platelet aggregation was not different between flavanol-rich darkchocolate or low-flavanol dark chocolate mixed with white chocolate, suggesting potential antiadhesive effects from methylxanthines. While the exact mechanisms to explain the interaction between flavanols and platelets are still under investigation, proposed mechanisms from in vitro and ex vivo models include an inhibition in the expression of endothelial adhesion molecules , and the down-regulation of pro-inflammatory factors such as interleukin -6 and tumor necrosis factor-α, which also decrease the recruitment of other proinflammatory compounds. Mango originated from the Indian subcontinent and has been cultivated for thousands of years. The bark, leaves, roots, and flowers of the tree, and the peel, kernel, and pulp of the fruit, have been used in traditional medicine in tropical and sub-tropical regions throughout the world. The bark and leaves have been used for treating diarrhea and diabetes in Bangladesh, and Ghana, with the pulp and kernel used for hemorrhaging in the lungs and intestines in India. Mango fruit is a rich source of fiber, vitamins C and E, folate, potassium, β-carotene, and phenolic compounds. Dietary intake of vitamins C and E, and β-carotene, are associated with reduced risks for CVD.  Major phenolic compounds reported in mango pulp include mangiferin, quercetin, kaempferol, myricetin, catechin, gallic acid, ferulic acid, protocatechuic acid, and chlorogenic acid.23,57 One study observed that a higher mango intake was associated with improved nutrient intakes, diet quality, and body mass index , factors known to reduce the risk of CVD.58 Clinical trials also suggest that mango fruit may have protective effects against the development of CVD. Daily intake of 200g of fresh-cut Ataulfo mango for 30 daysdecreased blood lipids and increased the plasma antioxidant capacity in healthy adults. In obese men and women aged 20-50 years, supplementation of 10 g/d freeze-dried mango pulp for 12 weeks decreased blood glucose levels but not inflammatory or any cholesterol markers. Another study reported that the daily intake of 400 g of fresh frozen mango pulp significantly decreased systolic blood pressure only in individuals with a BMI of 18-26.2 kg/m2 . In contrast, plasminogen activator inhibitor 1, IL-8, and mitochondrial pyruvate carrier-1 were significantly reduced in individuals when the BMI was partitioned as > 28.9 kg/m2 . In addition, in participants with impaired glucose sensitivity, the supplementation of 100 or 300 mg/d of mango fruit powder with 250 ml water daily for four weeks significantly increased the vasodilation of arteries as measured by the reactive hyperemia index compared to a placebo group. Mangiferin is a unique compound in mango that has been studied for its vasculo protective effects .

A mango bark extract with a high concentration of mangiferin decreased cholesterol in plasma and liver, and reduced oxidative stress in mice. A subsequent human study showed that the daily intake of 900 mg of Vimang® for 90 days reduced a measure of serum oxidative stress compared to a control group among older individuals. In both the animal and human studies, the marker of oxidative stress, nft hydroponic system while considered valid at the time of the study, is now viewed with limitations. In overweight hyperlipidemic individuals, the daily intake of 150 mg of mangiferin for 12 weeks significantly improved lipid profiles and glucose homeostasis. In hyperuricemic rats, mangiferin intake significantly reduced SBP, serum uric acid and inflammatory markers, and increased the expression of eNOS. One potential mechanism to help explain the vasodilatory effect of mangiferin may be due to the increased expression of eNOS, and therefore enhanced the production of NO. Reports have shown that the composition of phenolic compounds in mango varied significantly among different varieties. Mango varieties with high polyphenol content, such as Ataulfo, may play a more prominent role in cardiovascular health, but the interaction between the polyphenols, carotenoids and other bioactive compounds in mango must be considered. Further studies may also focus on the potential effects of mango by-products on metabolic health, since the total concentration of phenolic compounds is higher in the kernel, peel, leaves, and bark compared to the edible fruit. Such explorations may be useful in processing what is considered as agricultural waste into useful extracts.Low-bush blueberry and high-bush blueberry are two common species originally grown in North America. Native Americans have a long folklore history of using both types of blueberry plants to treat rheumatism and infection.  Anthocyanins are responsible for the red, blue, and purple color in ripe berries. Blueberries are one of the most abundant sources of anthocyanins in commonly consumed fruits. The total anthocyanin level in fresh blueberries is significant, reaching up to 487 mg/100g. Blueberries also contain appreciable amounts of proanthocyanidins and hydroxycinnamic acids , along with vitamins and minerals, fiber, and small quantities of flavonols and flavanols. Epidemiological studies suggest that a higher dietary anthocyanin intake is associated with a lower risk of hypertension, and reduced arterial stiffness in women, though these studies do not specify blueberries as the sole source of these bioactives. A meta-analysis of 19 cohort studies reported that the dietary intake of anthocyanins was associated with a decreased risk of coronary heart disease and CVD mortality, but not myocardial infarction, stroke, or total CVD risk. The effects of blueberries on markers of CVD risk have been studied. In obese postmenopausal women with pre- and stage I-hypertension, daily consumption of 22 g of freeze-dried blueberry powder containing 469 mg of anthocyanins for eight weeks significantly reduced systolic and diastolic blood pressure by 7 mmHg and 5 mmHg, respectively, and arterial stiffness measured by brachial-ankle PWV, compared to their baseline values or to a placebo group. In healthy males, FMD was significantly increased one, two, and six hours after the intake of 34, 57, and 80 g of blueberry powder mixed in water , compared to a control drink. However, no changes were seen in arterial stiffness measures. In addition, the increase in polyphenol metabolites and decrease in neutrophil NADPH oxidase in plasma were correlated to FMD, suggesting that the phenolic metabolites after blueberry powder consumption effectively improved vasodilation functions by elevating the bio-availability of NO through inhibition of NADPH oxidase. Later the research group identified that the FMD improvements were mainly due to anthocyanin metabolites. The blood pressures of overweight and obese smokers who consumed 250 g of blueberries for three weeks showed no significant changes from baseline values . Among mid-aged women who were at risk for type II diabetes, daily consumption of 240 ml of wild blueberry juice with 314 mg of anthocyanins forseven days significantly improved serum nitrates and nitrites, but no change were noted for in glucose metabolism parameters, cholesterols, inflammatory markers, platelet adhesion molecules, vasodilation, or blood pressure, compared to baseline and the placebo group. Taken together, the above results suggest that clinical trials with blueberries may need require a few weeks or longer of regular intake in order to observe clinically significant changes. Similar to blueberries, the American-cranberry is also a plant that is native to North America and has a long history of botanical uses by indigenous people, such as for urinary tract disorders and diarrhea. Cranberries are rich in numerous phenolic compounds, including A-type procyanidins, anthocyanins, flavanols, benzoic acid, and ursolic acid. Due to the extremely low sugar and high tart and astringency nature, cranberry products often contain added sugar or are blended with other fruits to improve palatability. To date, no systematic reviews or meta-analysis has noted an efficacy of cranberries in reducing CVD risks. However, potential cardio-protective effects may exist due to mechanisms discussed above.Goji berry , also termed wolfberry or Gou Chi Zi, has been used in TCM for its “eye-brightening” effects for millennia. In Chinese culture, goji berries can be consumed as a snack, an ingredient in soup, or as a tea alternative. Goji berries have the highest known content of Z of any commonly consumed food. In addition, goji berries also contain modest amounts of β-cryptoxanthin, β-carotene, neoxanthin and L.

Looking more closely at pest management, soil fumigation is the highest cost category for conventional production at $3,302 per acre, with weed control, another labor-intensive practice, the highest cost in second year and organic strawberries at $1,212 and $2,506 per acre, respectively . However, for organic strawberries the cost to control insects ran a close second at $2,488 per acre, which was dominated by control for lygus bug with a bug vacuum, and two-spotted spider mite with the release of predatory mites. By comparison, estimated costs for insect control in conventional strawberries were lower at $702 per acre and still lower at $579 in second year conventional berries.Raspberry and blackberry production were not routinely studied in years prior to 2003. Since then, several primocane-bearing raspberry and floricane-bearing blackberry cost and return analyses have been performed, with the most recent studies conducted in 2012 and 2013, respectively. Both studies detail establishment and first year production and harvest costs for not-yet-fully-mature crops. For raspberries, first year of production includes a $12,460 per acre construction, stackable planters management and investment cost for protective tunnels. Costs for a mature raspberry crop are analyzed in the second production year and total $48,210 per acre .

For blackberries, costs for a mature crop are shown for the second through fifth production years, and total $43,406 per acre per year. Harvest costs again represent the vast majority of total costs, at 81% and 71% of total costs for raspberries and blackberries, respectively. For raspberries, cultural costs represented a much smaller share of total costs at $4,656 per acre, roughly half of which was for trellis and tunnel management. Blackberry cultural costs totaled $5,709 per acre, of which over half was for pruning and training canes.Each study also includes an analysis of potential net returns to growers above operating, cash and total costs for a range of yields and prices. When evaluating net returns above total costs, gains are shown for higher yield and price points; losses are also documented at many lower yields and prices . Farms with productive soils, experienced managers, optimal production conditions and robust market plans generally realize higher net returns. In contrast, farms with less-than-optimal production conditions, reduced yields, poor fruit quality or inexperienced managers may contribute to lower net returns. Results from the strawberry analyses show that on a per acre basis, organic strawberries tend to be more profitable than conventional berries, even with lower yields.

Organic price premiums explain the result; in this example price per tray for organic strawberries ranged from $12 to $18, while price per tray for conventional berries ranged from $7.30 to $11.30. Prices for second year conventional strawberries were slightly lower still to account for a portion of the crop that was diverted to the freezer market. Net returns for both caneberries were mostly positive. Other noteworthy entries in all recent berry studies include per acre costs for pest control advisers , management of invasive pests and food safety and regulatory programs for water and air quality. Though each alone represents a relatively small portion of total costs, they provide readers with insights into the changing nature of berry production activities and costs over time.Cultural practices in the berry industry have evolved to address changes in soil, water and pest management needs. Based on historical trends, and to meet both industry needs and consumer demands, we expect to see new varieties continually developed over time. Businesses have responded to consumer and market demands for fresh, safe and organic products by implementing food safety programs and/or transitioning more lands to organic production.

Water and air quality programs have been developed to comply with state regulatory requirements. In the past, growers customarily hired those with expertise in financial and market management; they now also enlist the support of experts in food safety, organic agriculture and environmental quality to assist with farm management. But challenges remain, and management of key agricultural risks — including those for production, finances, marketing, legal and human resources — have become increasingly important. Invasive pests pose significant management and regulatory constraints and increase production, financial and market risks. Two recent examples are light brown apple moth and spotted wing drosophila . LBAM infestations can lead to loss of part or all of the crop because of field closure from regulatory actions, increasing production and financial risk. SWD presents substantial market risk to growers in that its larvae can infest fruit and render the crop unsaleable. Growers minimize the risk of loss from these two organisms with the routine use of PCAs. PCAs monitor fields more frequently than growers alone would be able to do, identify pests and recommend actions, for example, the use of pheromone mating disruption for LBAM and field sanitation for SWD. Since their introduction, the soil fumigants CP and MB have unquestionably contributed to the expansion of the berry industry. However, the full phaseout of MB as a pest management tool — it will no longer be available for use in berry production after 2016 — presents both production and financial risks. While a substantial research commitment has been made to finding alternatives to MB, nothing has yet come close to offering the same level of protection from the large-scale loss to soil pathogens or the gains in productivity associated with the application of CP and MB as synergistic preplant fumigants. We anticipate that the berry industry will adapt to the MB phaseout by using alternative fumigants and preplant soil treatments, but these are likely to carry a higher level of risk for berry production in the short term and may lead to a decrease in planted acreage and production. However, this may also stimulate an even more robust research agenda directed towards soilborne diseases and plant health to minimize disruption to the industry. Reliance on fumigants as the primary strategy for pest management is almost certainly a thing of the past. Instead, adoption of integrated approaches, including alternatives to fumigants, to manage diseases, weeds and other pests will be key to sustaining berry production over the longer term . Labor is also a current and significant challenge for growers of berry crops. Social and demographic changes in Mexico — the source of a majority of the area’s agricultural labor — have resulted in markedly lower immigration rates into the United States, a shrinking labor pool and upward competition and wage pressures for the agricultural workers who remain . In recent years, growers have reported difficulty in securing and retaining sufficient numbers of workers to ensure timely and effective farm operations. The lower production figures seen in strawberries in 2014 may in part have been the result of an insufficient labor pool from which to draw . However, no known regional employment or wage data are available to specifically document this. Some growers minimize labor risk by paying higher wages and providing year-round employment when possible. However, these strategies can be difficult for some businesses to justify economically. Arguably, the area’s berry industry, and agriculture more generally, stacking pots increasingly face political risk. Immigration legislation that may assist with the current labor challenge languishes at the federal level, with major policy changes unlikely before 2017 . Farming practices are under ever more scrutiny by consumers, local municipalities and state and federal agencies. Soil fumigants and pesticide use have been the focus of many intense debates and discussions, especially in Santa Cruz and Monterey counties. At the time of this writing, several new regulations related to pesticide application notifications, pesticide and fumigant application buffer zones and worker safety have been proposed by the California Department of Pesticide Regulation or the U.S.

Environmental Protection Agency but have not yet been finalized. It is anticipated that implementation will begin in 2017, with full compliance required in 2018. And, as California struggles through a fifth year of drought, water use, quality and cost has become a more robust part of the local, state and federal discourse, with directives issued and new legislation proposed. Compliance with each new directive or regulation presents production and logistical challenges for growers and can be costly to manage. Although it is unlikely that regulatory pressures will lessen in the future, there is every expectation that growers will continue to adjust business practices to meet or exceed any new requirements or standards. The economic sustainability of individual farming operations and the area’s berry industry in total will ultimately be impacted by and continue to evolve with the ever changing business environment, and by an array of risks and challenges.Bunch grapes , notably European , are considered among the major fruit crops worldwide, producing roughly 70–80 million tons each year . Cultivars of V. vinifera L. are used for wine, juice, and table grape production. Grape berries are classified as non-climacteric fruits, exhibiting a double-sigmoid developmental pattern with two rapid growth phases: the berry formation and the ripening phase , separated by an intermediate lag phase called the green plateau . The exponential increase in berry size characterizes both growth stages , but not the lag one . During phases and , also known as immature stages, organic acids, mainly tartrate and malate, accumulate leading to induction of acidity levels . At the end of the lag phase, a step-change point takes place known as veraison, where acidity starts to decline while sugars, mostly glucose and fructose, as well as anthocyanins in colored varieties, increase. Of particular interest are phenolic compounds, which are major and ubiquitous plant secondary metabolites derived from the shikimate/phenylpropanoid and polyketide pathways, with three utmost categories: proanthocyanidins , also known as condensed tannins, the gallo- and ellagitannins , and the phlorotannins . Such diversity of polyphenols, with more than 8000 structural variants, bestows them a wide range of biological functions ranging from growth, development, and protection inside the plant to, to some extent, human-related issues . In grapevines, the accumulation pattern of phenolic compounds, along with the aforementioned berry attributes, distinguishes each of the berry phases throughout berry development . Indeed, berry quality and sensory characteristics are notably defined by its polyphenol content . Remarkably, astringency is among the hardest sensory traits to depict and interpret as many intricate processes underpinning its perception . For instance, a sensory characterisation of the astringency of 11 varietals of Italian red wine revealed that neither total phenols nor PAs can predict how all astringency subtleties will be perceived . It is worth noting that the amounts, compositions, and proportions of polyphenols in a given species may vary widely depending on several factors, such as genotypic variations, developmental stages, and environmental circumstances . Scarlet Royal is a mid-season ripening table grape variety, producing seedless, red-skinned, oval-shaped, firm, and moderate to large berries with a sweet to neutral flavor . In the San Joaquin Valley, California, it typically ripens in mid to late August, filling the harvest window between Flame Seedless and Crimson Seedless, and has thus become a very popular red table grape variety in California. However, an undesirable astringent taste has been observed occasionally in some cases. In fact, the economic value of grapevines depends substantially on the environmental conditions, including climate, soil, cultural practices, cultivar, and rootstock. Hence, the term “terroir” is used in viticulture to describe the effect of such an interactive ecosystem on grapevine and wine quality . The current study aimed to understand the underlying mechanism of astringency development in Scarlet Royal berries at two contrasting vineyards . The first location produces well-colored, non-astringent berries; however, the second site yields astringent taste, poorly colored berries . The data showed a large variation in berry astringency within the same vineyard and from year to year. The data illustrated that the divergence in berry astringency stemmed from alterations in its polyphenol composition , most notably tannins. Additionally, the ripening stage was the most distinguishing platform for such variation between both vineyards. We were able to determine the tannins’ threshold level that causes the Scarlet Royal astringency taste to be ~ 400 mg/L. Given the changes in the levels of polyphenols during berry ripening, the question was raised: what is the mechanism governing the distinctive tannins accumulation pattern between V7-berries and V9-berries, and hence astringency diversity?

Future studies which directly compare climatic variables and mortality of A. glauca – of various ages – across ecoregions during extreme drought would therefore be of great value, and may elucidate a better understanding of the driving factors and relative vulnerabilities of plants in high-risk ecosystems.Consistent with our hypothesis, plants located at lower elevations generally experienced more physiological stress and greater dieback severity than those at higher elevations. However, there was also considerable variability within sites and along the elevational gradient. We hypothesize this is due to the extreme heterogeneity of the Santa Ynez mountain landscape, and that certain landscape features outside the scope of this study may act as refugia for A. glauca resilience. Since A. glauca is a relatively shallowly-rooted species, variables such as slope, aspect, slope angle, concavity, and rockiness – all of which vary greatly across the region and influence temperature, water availability, or both – likely impact shrub functioning and vulnerability to drought on a very local scale. In considering long-term predictions, it is possible that while some stands of A. glauca may suffer high levels of dieback and even mortality during extreme drought, populations as a whole may be resilient because of microsite and landscape heterogeneity.Although higher elevations in our study area historically record greater rainfall, we found this trend to be slightly disrupted during our study period: rainfall at the highest elevation site was lower than at the intermediate site from 2015-16 to 2018-19 . Furthermore, nft channel while elevation is known to influence temperature such that lower elevations are generally hotter than higher ones within a region, human-induced climate warming has been shown in some cases to occur more rapidly at higher than at lower elevations.

For instance, Giambelluca et al. found more rapid warming at elevations above 800m in Hawaii, and a review by Pepin et al. found enhanced warming with elevation in high mountain regions globally . Therefore, higher elevation sites in our study may also be experiencing more warming. We do not have temperature records for our six sites so we cannot separate temperature trends or anomalies from other elevational impacts. Nonetheless, the lack of a consistent elevation effect could be due not only to the confounding effects of other topographic features , but also to lower rainfall and increased warming in some higher elevation sites. From the early studies of Whittaker ecologists have long recognized the influence of aspect on plant composition and vegetation dynamics in montane environments. Yet rarely have investigators tied performance or mortality of individuals within a species population to subtle differences in aspect that occur within a single population . Here we found that aspect was the strongest predictor of dieback over time, with plants in SW aspects showing greater dieback than plants on more north-facing aspects. This influence of aspect may be the result of direct negative effects of sun exposure and higher temperatures ina semi-arid environment or maybe indirect whereby influences of aspect are mediated through aspect-effects on soil and microbes. For example, Gilliam et al. 2015 demonstrated greater soil organic matter accumulation and different microbial community composition in N facing compared to S facing slopes. Regardless of mechanism, the finding that SW aspect is detrimental to these plants could be important for restoration practitioners seeking to identify promising sites on the landscape where restoration of individual species may be more successful .

Another source of variability both within the landscape region we studied, and across Southern California chaparral, which was not directly measured in this study, is the occurrence of fog. The Santa Barbara region experiences predictable fog in May and June, and late summer cloud shading and fog events have been demonstrated to reduce vapor pressure deficit , slow plant seasonal water loss and provide direct foliar uptake of water . We observed significant variation in fog intensity between sites, particularly at night during the early summer months , even between sites on a given night. Sites C and D in particular often experienced heavy fog drip, while other sites remained dry. We found plants saturated by fog to exhibit higher water potentials on such nights, similar to findings in Mahall et al. , suggesting that fog drip may be sufficient enough to percolate to shallow roots. As mentioned previously, A. glauca is considered to be a relatively shallowly rooted chaparral shrub species and has been shown to be sensitive to even small changes in rainfall . Additionally, some coastal California shrub species have been shown to exhibit foliar water uptake from fog , though this has not yet been demonstrated for A. glauca. Regardless, variations in summer fog patterns have previously been shown to influence late-season water potentials in other Arctostaphylos spp. , possibly causing populations with greater exposure to fog to have higher “safety margins” with which to avoid cavitation . Similarly, Emery et al. have shown that fog can reduce the rate of water loss in coastal shrublands in California. Mortality of A. glauca during drought documented in other studies occurred in more interior sites, with less fog influence.

Thus, we believe the role of local and regional variation in fog occurrence in A. glauca drought resistance warrants further research.While dieback severity increased over time, the fact that no new plant-level mortality was observed during the study supports previous findings that A. glauca are resilient to prolonged drought. The relatively more mesic climate of Santa Barbara may have contributed to resiliency in our study, compared to studies in more xeric shrubland regions that reported high mortality of A. glauca during the same drought . Landscape heterogeneity, while likely confounding some of our results, may also be advantageous in creating differential water availability across the landscape, thus providing refuges that can ultimately allow resilience of A. glauca populations in a region. However, severe and widespread dieback, like that which we observed, still represents a threat to healthy ecosystem functioning, and has implications for fuel management in regions that are already fire-prone. Lower elevations and exposed, southwesterly slopes may continue to seethe highest levels of dieback during future drought events, and thus should be identified as high-risk and potential focus areas for management.Supported metal catalysts consisting of finely dispersed metal nanoparticles on high-surface-area supports are key in realizing cleaner and more efficient chemical conversions by lowering the energy cost and increasing the selectivity to the desired product. Efficient use of precious metals is facilitated by dispersing the catalytically active metal in sub-10 nm parti-cles. Preventing the growth of these small NPs into larger agglomerates, for example, at elevated temperatures and pressures, is challenging and remains a major cause of catalyst deactivation. Traditionally, high surface-area supports are used to retard NP sintering by increasing the interparticle spacing. Most prominent approaches are to distribute the particles in well-defined mesoporous supports, to encapsulate the individual NPs in porous oxides, to induce a thin metal oxide coating via strong metal-support interactions, or by incorporating the NPs in a microporous framework, such as, for example, zeolites. These designs drastically increase the thermal stability of the NPs and successfully protect the particles from sintering and Oswald ripening during catalysis. However, the encapsulation can lead to a partial loss of catalytic activity due to blockage of the NP surface by the surrounding metal oxide. A key challenge is to mitigate this effect, hydroponic nft and to develop methodologies to probe the chemical accessibility of the NPs in these novel catalyst designs. An emerging class of materials developed by our group that addresses this challenge is raspberry-colloid-templated catalysts . The RCT materials exhibit exceptional thermal and catalytic stability, while maintaining high catalytic activity and selectivity. The material design consists of an ordered macroporous metal oxide framework with sub 10-nm metal NPs distributed at the pore walls. The colloidal preparation approach that involves infiltration of the metal oxide precursor into the assembled sacrificial colloids decorated with catalytic NPs offers high versatility of the structural design, that is, particle size, metal composition, metal oxide composition, and micro- and macro-porosity can be tuned independently. The robust, macroporous matrix allows facile mass transport throughout the catalyst. So far, the potential of the RCTs for thermal catalysis has been demonstrated in selective alkyne hydrogenation, HD exchange, CO oxidation, oxidative alcohol coupling, and the oxidation of volatile organic compounds. A unique feature of the RCT catalysts is their excellent stability during catalysis and thermal treatment, allowing long catalyst lifetimes and facile reactivation via thermal treatment. A particularly noteworthy finding by Shirman et al. is the observation that not only the stability but also the activity of the RCT catalysts can be strongly enhanced compared to commercial catalysts, allowing drastic reductions in precious metal use and in energy cost in oxidation catalysis.

The origin of the superior thermal stability of the RCT catalyst has remained an open question thus far. Furthermore, the crystal structure and accessibility of the catalytic NPs, in particular at their interface with the support, are still unknown despite the relevance of these properties to understanding the origin of sinter-resistance of these materials and their activity in catalysis. Here, by using theoretical modeling, 3D electron microscopy, and epitaxial overgrowth, we assess the NP embedding, the crystal structure, and the chemical accessibility of the metal NPs in RCT catalysts and discuss the implications of these structural characteristics for the catalysts’ functional properties, and in particular for NP stability.The RCT preparation relies on multi-step synthetic route . The first step comprised the preparation of the metal NPs and amidine-functionalized polystyrene colloids . Here, the metal particles consisted of 96 atom % Au and 4 atom % of Pd, abbreviated as Au96Pd4, and were capped with polyvinylpyrrolidone . However, the desired composition, size, and shape of both the NPs as well as the size and functional surface groups of the PS colloids can be readily altered, independent of the subsequent synthesis steps, allowing a tailored design of catalysts for specific chemical reactions. Next, the metal NPs were attached to the PS colloids, yielding so-called raspberry colloids . An ordered colloidal crystal of raspberry colloids assembled by solvent evaporation was infiltrated with a pre-hydrolyzed silica sol-gel solution . In the final step, the PS template and stabilizing ligands were removed via calcination , generating an ordered macroporous silica support with metal NPs decorating the pore walls. Additionally, the high-temperature calcination ensured the mixing of Pd and Au into a homogeneous alloy. The resulting RCT catalyst contained ≈5 wt.% bimetallic Au96Pd4 NPs as determined with inductively coupled plasma mass spectrometry . The weight loading closely matches the theoretical weight loading of 4.8 wt.% calculated based on the synthesis parameters, indicating that no metal leaching occurred during the RCT preparation. The sinter-resistance of the NPs in the RCT catalyst is evident from the overlapping size distributions before and after thermal treatment at 800 °C in static air . The average particle sizes before and after treatment closely match and are 7.5 ± 2.5 and 7.4 ± 2.4 nm, respectively. Additional long-term stability experiments show that thermally treating the catalyst for 10 h in either oxidizing or reducing gas atmospheres does not lead to particle growth . These findings are in line with previous reports in which particle growth was not pronounced in RCT catalysts at temperatures up to 950 °C, and in strong contrast to significant agglomeration and growth of NP in silica-supported catalysts in which the NPs were introduced after the macroporous silica framework was prepared. Thermal treatment of the latter already led to particle growth to > 20 nm after thermal treatment at 500 °C in static air. In addition to the thermal stability of the NPs during synthesis and in oxidizing and reducing atmospheres, the long-term stability and sinter resistance of the PdAu and Pd NP RCT catalysts have been demonstrated for a range of reactions under catalytic conditions: CO oxidation, methanol oxidation, 1-hexyne hydrogenation, and oxidation of simulated exhaust mixtures. It has been shown that the sinter resistance is maintained in RCT catalysts even at high metal loadings and small NP sizes. We postulated that the infiltration step in the synthesis procedure is crucial in determining the NP–silica interface and the degree of NP embedding into the matrix after calcination. More specifically, the geometry of the NP pocket within the silica support is defined by the wetting of the surfaces of the gold NPs and PS colloids by the infiltrating silica sol-gel solution.

Infection and dieback severity varied considerably across the landscape, however, there is some evidence to suggest that populations at lower elevations may be at highest risk for severe dieback, either due to increased water stress, close proximity to fungal inoculum sources, or both. Additionally, shrubs located on southwest-facing slopes may also be more vulnerable due to increased sun exposure and thus environmental stress. Management efforts may want to focus on these areas when this region experiences future drought. Finally, although extreme dieback was recorded throughout the study, none of the observed shrubs succumbed to mortality. This may be the result of overall physiological resiliency, and the ability of adult shrubs to allocate resources to keep portions of the canopy alive. It could also be that the region’s slightly more mesic climate offers a climatic buffer that prevented shrubs from reaching their mortality thresholds. More research is needed to identify these exact mechanisms and thresholds in A. glauca. Collectively, the results of this dissertation work provide valuable knowledge on the severe dieback of an important chaparral shrub during an historic drought, with the potential for ecologically and economically costly consequences. Additionally, the data I present provide insight into the scale and progression of A. glauca dieback in a chaparral system, and potential patterns of future dieback in the face of predicted climate change. Future research that seeks to further resolve landscape and environmental variables contributing to plant stress would help in identifying these patterns.

Heterogeneity and rugged topography across the landscape, round garden pot while likely beneficial for the resilience of regional A. glauca populations during drought, presents significant challenges for on-the-ground monitoring. Out of necessity for safe access , many of the plants surveyed were located on the outer boundaries of stands, where edge effects may have been a factor. Monitoring intact, undisturbed stands using drones would yield valuable additional insight into the extent of disease deeper into stands and in stands on steep terrain or that are outside of normal visual range. The challenges of working in rugged landscapes covered in impenetrable vegetation highlight the need for using and refining remote sensing technologies, such as drone imaging, Light Detection and Ranging , and hyperspectral imaging as monitoring tools. Large-scale, long-term monitoring using these tools would allow researchers to retrieve data in areas that have previously been inaccessible, while also gaining a larger scale understanding of drought impacts. They ultimately will enable future studies to reveal more nuanced patterns across the landscape and between years of varying climatic conditions.Plant pest and disease outbreaks play a major role in shaping ecosystems around the world. Outbreaks can alter ecosystem structure and function, often with substantial consequences . Over the past 200 years, pest/disease outbreaks have increased due to mass exchange of biological materials from global trade and a rise in unusual climate events resulting from global climate change .

Prolonged climate irregularities can subject plants to environmental stress outside of their normal resistance thresholds and make them susceptible to pests and pathogens . For example, the increase in extreme droughts, defined here as greater in intensity and duration than historical drought regimes, has been directly linked to enhanced mortality in woody plant systems worldwide, often in association with pest/pathogen outbreaks . Plant disease outbreaks are often economically costly , and can result in loss of ecosystem services in natural ecosystems. With global trade continuing to spread pests and pathogens, and global change-type drought events predicted to increase , incidences of plant disease outbreaks are expected to increase. Understanding the role of drought and pathogens in plant dieback and mortality is therefore of critical importance. Latent fungal pathogens are of particular concern for natural ecosystems yet their ecological roles remain poorly understood. These pathogens can live as asymptomatic endophytes within their hosts and remain undetected for long periods of time . The Botryosphaeriaceae fungi, a groupthat causes considerable damage to hundreds of agricultural, ornamental, and naturally occurring host species around the world , includes many latent fungal pathogens that are difficult to detect in wild plant populations. Members of this diverse family can occur as endophytes, pathogens, and saprophytes on diverse woody hosts . They are best known as pathogens that cause leaf spots, cankers, severe branch dieback, and death in economically important hosts such as grapevines , avocado , and eucalyptus . While Bot. fungi are rapidly becoming one of the most important agents of disease in agricultural plant hosts , relatively few studies have been conducted on these pathogens in natural systems .

The Bot. fungi have a long history of taxonomic confusion, in part due to indistinctive morphological characteristics among species and from other fungal taxa, as well as historically poor and inconsistent descriptions early on in their discovery . Furthermore, Bot. host specificity and pathogenicity can vary widely among species and across geographical regions, complicating our understanding of their influence in various host species and across systems . While advances in molecular sequencing and data basing have added clarity in this area , challenges remain in understanding the diversity and pathogenicity of Bot. species among hosts and across regions. As a result, there is a dearth of knowledge on their ecological roles, particularly in native ecosystems.One consistent finding is that disease outbreaks from Bot. fungi in agriculture are often associated with environmental stress, such as extreme heat fluctuations and drought . Furthermore, studies have shown latent pathogens like Bots cause more damage to water-stressed hosts , and some Bot. species have been shown to grow well in water potentials much lower than what their plant hosts can tolerate , suggesting drought conditions increase virulence by these pathogens. Therefore, regions that have historically dry climates or experience periodic extreme drought may be especially vulnerable to disease outbreaks from latent pathogens as they are predicted to experience an increase in drought events due to climate change . Mediterranean-type climate areas are projected to be global change “hot spots” , and dry shrublands are predicted to experience some of the most rapid increases in mean temperatures . Indeed, recent drought-related morality in California’s semi-arid Mediterranean climate shrublands has provided support for these predictions . Furthermore, the combination of dense human settlement and agricultural lands in close proximity to many natural shrubland habitats in southern California creates a likely pathway for exotic pathogen introductions and movement of pathogens from agricultural settings into wildland species. Not surprisingly, Bot. species have been retrieved on a variety of native chaparral shrub species in California, including Ceanothus spp. , Malosma laurina , and other species of Arctostaphylos . Understanding the response of native species and these pathogens to extreme weather conditions will help to predict future vegetation change and potential species losses . From 2011-2018, southern California experienced one of the most severe droughts in recorded history, with 2014 being the driest in the past 1,200 years . Field observations in winter 2014 identified high levels of branch dieback, and in some cases mortality, large round plant pots in a common ecologically important shrub, Arctostaphylos glauca in coastal California. Two well-known Bot. species were isolated from the symptomatic shrubs . Like other members of the Bot. family, both N. australe and B. dothidea infect a broad range of hosts, and are known to be responsible for disease outbreaks associated environmental stress in agricultural species . While B. dothidea is well established in California, with over 35 different host species having been identified , phylogenic evidence suggests N. australe may be more recently introduced . Its impact on shrublands of California has not been quantified.

Preliminary observations suggested high levels of branch dieback, and in some cases mortality, at lower elevation sites and along exposed ridges compared to higher elevations in coastal montane settings. We hypothesized that identifiable patterns would exist in the distribution of B. dothidea and N. australe across these landscapes that correlate with branch dieback and environmental variables associated with drought stress. Manzanita dieback has previously been causally associated with Bot. infection . A greenhouse experiment by Drake-Schultheis et al. , revealed that drought enhances onset of stress symptoms and mortality in young A. glauca inoculated with N. australe compared to shrubs subjected to drought or inoculation alone. However, to the authors’ knowledge no previous quantitative studies exist on the distribution of Bot. species in California shrubland environments with Mediterranean climates. To better understand the occurrence, distribution, and severity of Bot. infections in chaparral shrublands, we surveyed infection in A. glauca between April and September 2019. We also collected data on site elevation, aspect, and average percent canopy dieback at each site sampled for infection. While a variety of landscape variables are likely to influence plant stress at any given site , we focused on elevation because A. glauca already tends to occur mostly on xeric and rocky soils of exposed slopes, and therefore elevation was presumed to be the most significant factor influencing precipitation and water availability in this setting. Also, other studies have used elevation as a proxy for climate variation . We also recorded aspect of each sampled shrub since it influences sun exposure, temperature, and water stress. To test our hypothesis that Bot. fungi and level of stress each played a role in extensive canopy dieback in A. glauca, the following questions were addressed: What is the distribution of Bot. infection in A. glauca stands across the chaparral landscape in coastal Santa Barbara County? How do levels of infection by the two Bot. fungi, N. australe and B. dothidea, compare across elevation? and How do stand-level infection and elevation correlate with dieback severity? We predicted N. australe and B. dothidea to be presentacross all sites and elevations, but also that N. australe, having been previously isolated with high frequency in the area , would likewise have the greatest incidence in this study. Furthermore, we expected levels of Bot. infection and dieback severity to be greater at lower elevations compared to higher elevations, because lower sites typically receive less annual rainfall, thus exacerbating drought stress. This study presents the first quantitative survey summarizing the severity and distribution of Bot. fungi in natural shrublands, and seeks to identify important patterns of infection and dieback in A. glauca to predict future vulnerabilities across the landscape.The study sites were located on the generally south-facing coastal slopes of the Santa Ynez Mountains in Santa Barbara, California, USA . The sites range from a lower elevation of ~550m to an upper elevation of ~1145m, and cover an area of ~47km2 . This region is characterized by a Mediterranean climate, with wet winters and hot, dry summers. Mean annual precipitation ranges from 68.4cm at lower elevations to 90.6cm at upper elevations . During the 2013-2014 wet season, which was two years into a multiyear drought and one of the driest years on record in California , these areas received only 24.8cm and 31.6cm precipitation, respectively .Sites were initially randomly generated from polygons drawn in the field around relatively pure stands of A. glauca , and Drake-Schultheis, unpublished data. Polygons were then categorized according to elevation , and numbered within their respective elevation categories. Ten sites per elevation zone were randomly selected using random number generator for a total of 30 sites. When necessary, some randomly generated sites were substituted with nearby stands that were more accessible. Furthermore, any randomly selected sites that were discovered to be in recent fire scars were exchanged for nearby stands that contained intact, mature A. glauca.Elevation data were collected in situ using Altimeter GPS Pro and corroborated using Google Earth . Aspect was recorded in situ in degrees, then converted to radians and transformed to linear data for analysis of “southwestness” using cos according to Beers et al. . This yielded aspect values ranging from -1 to 1 , which were then used for modeling the effects of aspect on shrub dieback and Bot. infection. Sites were demarcated by >50% A. glauca cover within a stand, as determined by visual on the-ground assessments where the tops of the canopies could be viewed. Stand dieback was then visually estimated by two-to-three people as the percent of “non-green” vegetation compared to live, green vegetation within the defined boundaries of a site . Categories of NGV included yellow, brown, and black leaves, and bare/defoliated canopy, and percentages were summed to reflect total NGV within a site.

As wind turbines account for a major proportion of the wind farm initial investment, previous researches mainly focused on optimizing the number and locations of wind turbines. In [6] and [7], the genetic algorithm was proposed to iteratively optimize gridded turbine layouts on flat terrains. In [8] and [9], the greedy algorithm was introduced to search for the optimal gridded turbine layouts on hilly wind farms. The modified particle swarm optimization algorithm was applied to solve the turbine layout optimization problem in a continuous solution space. The civil works include the construction of access roads, turbine foundations, crane hard-standings, cable-trenching, and substation buildings. Their cost can be substantial. Access roads are greatly influenced by topographic characteristics, and their cost is much higher for hilly or mountainous wind farms than plain ones. A comparative strategy and the minimum spanning tree were proposed to search for the shortest road network for wind farms in flat areas. In [11], the Euclidean Steiner tree was introduced to further reduce the length of the road network. However, these methods are not directly applicable to hilly or mountainous wind farms where the road gradient must be considered to build winding roads capable of accommodating significant weight. Wind farms are generally constructed in rural areas with challenging topography. With the rapid expansion of wind farms, black plastic planting pots new farms have to be built in hills or mountains, where designing suitable access roads for construction, turbine erection and maintenance is not straight forward.

It is necessary to know roughly which route should be chosen to connect turbines and how much they will cost, while placing turbines at the peaks for high energy production. The selection of appropriate access roads is essential for the overall planning of the wind farm construction. A well-chosen road network can also reduce the wind farm construction period and lower the environmental impact. In this paper, the constraints on the maximal gradient of access roads are guaranteed when developing an automatic contour-based road-network design model. The design of access roads and the evaluation of their cost are simultaneously considered in the process of optimizing turbine layouts, which results in a more technically feasible and economically beneficial micrositing of wind farms. The remainder of this paper is organized as follows. Section II proposes an automatic model for road network design. The problem of wind farm micrositing is formulated in Section III. Section IV gives the simulation results. Finally, Section V makes some concluding remarks.In civil engineering, a feasible route between the starting and end points is selected by establishing a series of control points on the terrain. Using these control points as an initial alignment, the horizontal and vertical curves are then located, subject to the road design constraints including geometric specifications and environmental requirements. Heuristic optimization algorithms such as the genetic algorithm, the Tabu search, and the simulated annealing algorithm have been applied to optimize horizontal and vertical alignments of roads. These algorithms depend on a high-resolution digital elevation model to support the analysis of road design features such as ground slopes and other landform characteristics.

The calculation of an optimal route between two given locations is time consuming and could even take several hours. The micrositing optimization process usually involves hundreds or thousands of iterations to evaluate the wake effects between turbines and to update the turbine layout. The process without road design is already intensive. If a complex road network for the whole farm needs to be designed in each iteration by the aforementioned heuristic optimization algorithms, the computation burden is prohibitive and even impossible. In this paper, only the control points of access roads are selected during the micrositing optimization process. Such a relatively simple and functional road design is suitable for low-volume access roads, which are mainly for wind farm construction and maintenance. It is also adequate for the design at the strategic and tactical level. Based on the above assumption, a fast automatic method for road-network design is developed to estimate the cost of access roads during the optimization of wind farm micrositing.Forest engineers usually use large-scale contour maps to select preliminary routes with dividers to connect two locations, a process known as route projection. Control points of a road can be selected during the route projection process. In this paper, contour lines as a group of nested closed curves are chosen to store topographic information of wind farms. A distinct advantage of the contour-line data over a digital elevation model is that the vector representation lends itself to the object-oriented modeling of terrains, which provides a natural mechanism for sorting terrains and facilitates the search through a contour map.

Given a technically feasible gradient, a route is projected from the starting point toward the end across adjacent contour lines. This process can be efficiently automated with a mathematical model. The basic idea of the route projection process is to determine the projected route segment between two neighboring contour lines, as the elevation of the terrain rises continuously. Every projected segment of the route must begin from a point on a contour line and end on another one.Two comparative cases are studied to investigate the effectiveness of the proposed road network design model and the significance of road network optimization for farms in hills or mountains. Due to the randomness of genetic algorithms, ten independent runs are performed for each case. Table IV summarizes the results, including the best, average, and worst values of the best solutions achieved in each independent run. In Case 1, the micrositing problem is solved in a two-stage manner, i.e., we first search for an optimized turbine layout without considering access roads and then design the road network for the given layout. The best configuration of the wind farm is shown in Fig. 7, where the blue triangle represents the entrance of the wind farm, red points mean the locations of wind turbines, and black polylines are the designed road network. In Case 2, the turbine layout and its corresponding road network are optimized simultaneously, i.e., in each optimization iteration, the road network design model is performed for every individual turbine layout generated by the genetic algorithm. The best configuration of the wind farm is shown in Fig. 8. The simulation results of both cases are summarized in Table IV. Note that, the simulations for both cases are time consuming. The simulations were carried out in an IBM Blade Center HS22 with 12 Intel Xeon X5650 CPUs at a frequency 2.66 GHz. Parallel computing was employed to speed up the simulation progress. In each run of the 10 independent simulations, 12 CPUs were utilized simultaneously and each CPU was responsible for each wind direction. Even with the parallel computing Fig. 7. Best wind farm configuration of Case 1. technique, plant plastic pots the running time for each simulation is still around 7–10 h. For both turbine layouts, reasonable road networks with feasible winding roads are obtained, which demonstrates the effectiveness of the proposed road network design model. Comparing Figs. 7 and 8, it is clear that the configuration of the road network depends on the turbine layout and the farm topology considerably. Therefore, to create a cost-effective road network, it is necessary to consider these two parts simultaneously during the microsting optimization process. Indeed, since the road network is simultaneously considered in Case 2, while the cost of the road network is ignored in Case 1, Case 2 consistently has a higher net present value than Case 1 in all independent runs. In the simulations, the net present value is determined by the annual energy production and the length of the road network.Methane released into the atmosphere as a result of agricultural activity, such as enteric fermentation and anaerobic digestion, significantly contributes to overall greenhouse gas emissions in the United States . The California Air Resources Board attributes approximately 60 % of recent anthropogenic CH4 emissions in California to agriculture, with 45 % of CH4 emissions directly related to dairy farm activity for 2013 . Reduction strategies proposed by CARB seek to lower California’s CH4 emissions to 40 % below 2013 rates by 2030 , thereby emphasizing the need for accurate methods to directly quantify the contribution of different CH4 sources within agricultural operations. Estimates of CH4 emissions due to dairy livestock can be calculated using inventory emission factors combined with activity data on animal populations, animal types, and details about feed intake in a particular country . Other methods to estimate CH4 emissions from ruminants involve direct atmospheric measurements.

Emissions from dairy farms have been estimated in the Los Angeles Basin, California, using downwind airborne flux measurements . Farmscale measurements of CH4 have been made using a variety of techniques and instruments, such as open-path infrared spectrometers , tunable-infrared direct absorption spectroscopy , and column measurements employing solar absorption spectrometers with comparisons to cavity ring-down spectrometers . Several studies of various CH4 sources assert that inventory-based calculations tend to underestimate emissions compared to atmospheric observations and modeling . Atmospheric studies have often used specific gases as tracers to distinguish a sample of interest from background conditions or interferences. Tracer gases released at known rates have been employed in experiments looking at chemical transport , dispersion , source allocation , and model verification using mobile laboratories , radiosondes, sampling towers, and ground-based equipment. Application of tracer gases in agricultural studies have involved insertion of a sulfur hexafluoride permeation tube into the rumen of a cow with subsequent collection of time-integrated breath samples . Inverse-dispersion techniques have employed line-source releases of SF6 within a dairy farm combined with open-path measurements to understand whole-site emissions . Release of a tracer gas directly into the atmosphere, 2–3 m above ground level, can be used to determine and distinguish CH4 emissions from various sources within a site . This study quantifies CH4 emissions using the well-established tracer flux ratio method at two dairy farms over the course of 8 summer days . Controlled releases of tracer gas from various areas on each farm mixed with site-derived emissions were observed by an instrumented aircraft and mobile laboratory . Using this technique provided the flexibility to estimate entire dairy farm emissions and apportion emissions among sources on multiple scales. Uncertainty in measurements from low-flying airborne studies has been attributed to the need to extrapolate results below the minimum safe flight heights as regulated by the Federal Aviation Administration . Prior to this study, Aerodyne Research, Inc. performed controlled ground releases of ethane in Colorado and Arkansas, while Scientific Aviation made measurements in a similar aircraft to the one used in this study . The original release rate of C2H6 was estimated via a refined mass balance technique, with a +2 % difference observed during tests in Colorado and +24 % difference in Arkansas as described in Conley et al. . These releases did not correspond to any CH4 source but demonstrated the feasibility of using a low-flying aircraft to successfully quantify flow rates from controlled tracer gas releases. Using tracer flux ratio in this study, we again utilized the aircraft to detect emitted tracer gas and then compared with dairy farm emissions to evaluate CH4 emission rates. This field study was originally focused on estimating CH4 emissions from dairy farms and distinguishing on-site sources using established techniques . An intentional effort was made to align measurement time windows of the mobile laboratory and aircraft for the purpose of inter-comparison between the tracer flux ratio and mass balance methods. As a result, the aircraft was exposed to several hours of ground-released tracer gas. Due to this overlap in time, we were able to further assess the viability of observing enhanced concentrations of a ground-released tracer gas from an aircraft at low flow rates, compare CH4 and C2H6 enhancements emitted from within dairy farms via tracer flux ratio to determine emission rates, and directly compare the application of tracer flux ratio methodology to simultaneous ground and airborne measurements of the same air mass.In a collaborative effort, SA and ARI attempted a flight-based tracer release experiment to quantify CH4 emissions from two dairy farms in central California. This study reanalyzes data collected as part of an Environmental Defense Fund coordinated project that occurred in June 2016 . ARI employed tracer flux ratio methodology with two tracer gases and a mobile laboratory, while SA conducted a mass balance experiment from a light aircraft.

While surveys often provide a way to overcome time and budget constraints to learn about farmer knowledge, this study suggests that to achieve specificity and depth in analysis of farmer knowledge requires an interactive approach that includes – at a minimum – relationship building, multiple field visits, and in-depth, multi-hour interviews. Accessing farmer knowledge necessitates locally interactive research; this knowledge may or may not be immediately generalizable or scalable without further locally interactive assessment in other farming regions.The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, large plastic gardening pots most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .

In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database. The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARSCoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future.

Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard bio-safety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available. With product accumulation in the range of 0.1–4.0 g kg−1 biomass , larger-scale quantities can be supplied after 4–8 weeks , making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number of candidates. Also, leaf-based expression can result in a coefficient of variation >20% in terms of recombinant protein accumulation, which reduces the reliability of expression data . The variability issue has been addressed to some extent by a parallelized leaf-disc assay at the cost of a further reduction in sample throughput .

The reproducibility of screening was improved in 2018 by the development of plant cell pack technology, in which plant cell suspension cultures deprived of medium are used to form a plant tissue surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner. The costs can be as low as €0.50 per 60-mg sample with a product accumulation of ~100 mg kg−1 and can typically result in a CV of <5% . These costs include the fermenter-based upstream production of plant cells as well as all materials and labor. The system can be integrated with the cloning of large candidate libraries, allowing a throughput of >1,000 samples per week, and protein is produced 3 days after infiltration. The translatability of cell pack data to intact plants was successfully demonstrated for three mAbs and several other proteins, including a toxin . Therefore, cell packs allow the rapid and automated screening of product candidates such as vaccines and diagnostic reagents. In addition to recombinant proteins, the technology can, in principle, also be used to produce virus-like particles based on plant viruses, which further broadens its applicability for screening and product evaluation but, to our knowledge, according results had not been published as of September 2020. In the future, plant cell packs could be combined with a recently developed method for rapid gene transfer to plant cells using carbon nanotubes . Such a combination would not be dependent on bacteria for cloning or gene transfer to plant cells , thereby reducing the overall duration of the process by an additional 2–3 days . For the rapid screening of even larger numbers of candidates, cost-efficient cell-free lysates based on plant cells have been developed and are commercially available in a ready-to-use kit format. Proteins can be synthesized in ~24 h, potentially in 384-well plates, and the yields expressed as recombinant protein mass per volume of cell lysate can reach 3 mg ml−1 . Given costs of ~€1,160  ml−1 according to the manufacturer LenioBio , this translates to ~€400 mg−1 protein, an order of magnitude less expensive than the SP6 system , which achieves 0.1 mg ml−1 at a cost of ~€360  ml−1 based on the company’s claims. Protocol duration and necessary labor are comparable between the two systems and so are the proteins used to demonstrate high expression, e.g., luciferase. However, the scalability of the plantcell lysates is currently limited to several hundred milliliters, and transferability to intact plants has yet to be demonstrated, i.e., information about how well product accumulation in lysates correlates with that in plant tissues. Such correlations can then form the basis to scale-up lysate-based production to good manufacturing practice -compliant manufacturing in plants using existing facilities. Therefore, the cell packs are currently the most appealing screening system due to their favorable balance of speed, throughput, large plastic growing pots and translatability to whole plants for large-scale production. In any pandemic, the pathogen genome has to be sequenced, made publically available, and freely disseminated in the global scientific community to accelerate therapeutic and vaccine development. Once sequence information is available, a high priority is the rapid development, synthesis, and distribution of DNA sequences coding for individual viral open reading frames. These reagents are not only important for screening subunit vaccine targets but also as enabling tools for research into the structure, function, stability, and detection of the virus .

Because many viral pathogens mutate over time, the sequencing of clinical virus samples is equally important to enable the development of countermeasures to keep pace with virus evolution . To ensure the broadest impact, the gene constructs must be codon optimized for expression in a variety of hosts ; cloned into plasmids with appropriate promoters, purification tags, and watermark sequences to identify them as synthetic and so that their origin can be verified ; and made widely available at minimal cost to researchers around the world. Not-for-profit plasmid repositories, such as Addgene and DNASU, in cooperation with global academic and industry contributors, play an important role in providing and sharing these reagents. However, the availability of codon-optimized genes for plants and the corresponding expression systems is often limited . For example, there were 41,247 mammalian, 16,560 bacterial, and 4,721 yeast expression vectors in the Addgene collection as of August 2020, but only 1,821 for plants, none of which contained SARS-CoV-2 proteins. Sharing plant-optimized SARS-CoV-2 synthetic biology resources among the academic and industry research community working on PMPs would further accelerate the response to this pandemic disease. Screening and process development can also be expedited by using modeling tools to identify relevant parameter combinations for experimental testing. For example, initial attempts have been made to establish correlations between genetic elements or protein structures and product accumulation in plants . Similarly, heuristic and model-based predictions can be used to optimize downstream processing unit operations including chromatography . Because protein accumulation often depends on multiple parameters, it is typically more challenging to model than chromatography and probably needs to rely on data-driven rather than mechanistic models. Based on results obtained for antibody production, a combination of descriptive and mechanistic models can reduce the number of experiments and thus the development time by 75% , which is a substantial gain when trying to counteract a global pandemic such as COVID-19. These models are particularly useful if combined with the high-throughput experiments described above. Techno-economic assessment computer aided design tools, based on engineering process models, can be used to design and size process equipment, solve material and energy balances, generate process flow sheets, establish scheduling, and identify process bottlenecks. TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into. The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high . Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for bio-facilities of the future .