Month: June 2024

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Previous research from our group reported that consumption of hamburger meat with spice mix added prior to cooking resulted in a reduction in the concentration of malondialdehyde, a lipid peroxidation marker, in the meat and in plasma and urine of healthy volunteers, and improved postprandial endothelial dysfunction in men with Type 2 diabetes . Subsequent study reported that commercial spices in dry or fresh form exhibited significant antioxidant capacity that correlated with total phenolic content butnot with the concentration of chemical biomarker . There is limited amount of information regarding the activity of culinary spice extracts against clinical isolated intestinal bacteria, and a limited number of bacterial strains have been assessed for their susceptibility or antimicrobial activity against spices. Gunes and colleagues reported that minimum inhibitory concentration of curcumin against 7 standard bacterial strains is in the range of 129 to 293 µg/mL . Cinnamaldehyde, a bio-active component of cinnamon, was shown to exhibit more potent in vitro antibacterial properties against 5 common foodborne pathogenic bacteria with MIC being 125 to 500 µg/mL as compared to crude cinnamon stick extract , but cinnamaldehyde did not modulate the population of selected Lactobacillus and Bifidobacterium counts in mouse cecal content . Supplementation of rosemary extract was reported to increase Bacteroides/Prevotella groups and reduce the Lactobacillus/Leuconostoc/Pediococcus group in the caecum of both obese and lean rats . Based on potential health benefits demonstrated from our group, this study investigated major chemical constituents, antioxidant activity, and in vitro effect of 7 spice extracts on the growth of 33 beneficial Bifidobacterium spp. and Lactobacillus spp.,square plastic pot and established their antimicrobial activity against 88 intestinal, pathogenic, and toxigenic bacterial strains.

Eighty-eight strains of bacteria included in this study were clinical isolates from patients in the Greater Los Angeles VA Healthcare Center as published , or purchased from American Type Culture Collection . Altogether 17 strains of Bifidobacterium species consisting of 11 strains recovered from human intestinal contents and 6 ATCC Bifidobacterium type strains were studied. Similarly, 16 strains of Lactobacillus species consisting of 12 strains recovered from human intestinal contents and 4 ATCC Lactobacillus type strains were studied. Other bacterialstrains, including 1 Akkermansia, Bacteroides spp., Clostridium spp., including 12 toxigenic Clostridium difficile, E. coli, Finegoldia magna, Fusobacterium spp., Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Ruminococcus spp., Salmonella typhi, and S. aureus strains, were also tested. These bacteria have been identified by 16S rRNA sequence analysis as published . Brucella agar plates containing serial dilutions of each spice extract and control plates without the tested spice were prepared . Spice extracts were 1st diluted by 20 times to give 9.0 mg/mL concentration which was further diluted to give 4.50, 2.25, 1.13, 0.56, 0.28, 0.14, and 0.07 mg/mL of extracts. A suspension of colonies was used to prepare Brucella broth tubes with a density equal to that of a 0.5 McFarland standard corresponding to density of 108 bacterial colony forming units /mL. A volume of 10 µL of these suspensions was inoculated on the spice extract-containing plates, achieving a final inoculum of 105 CFU/spot. Brucella agar plates were used as a positive growth control. The plates were incubated in an anaerobic chamber for 48 h at 37 °C. Anaerobic conditions consisted of a gas mixture of 5% CO2, 5% H2, and 90% N2; the residual oxygen was removed by palladium catalysts . After incubation, the plates were examined. The MICs were determined. MIC is defined as the lowest concentration of each spice resulting in no growth or a marked change in the appearance of growth as compared to the control plate, as described in the Clinical and Laboratory Standards Institute approved protocol .

Further, the concentrations resulting in the change of the appearance of growth as compared to the control plate were recorded to establish for each test strain the concentration where the tested spice had no effect on the growth, where the spice stimulated the growth, and where the growth stimulation by the test spice reached a plateau .Results from our chemical investigations indicated that most spice extracts contain either moderate or high content of spice specific phytochemicals. The identification and quantitation of these phytochemicals provide insight into the potential influence of these chemicals on the gut microbial communities and activities. Among the chemicals characterized, majority belongs to polyphenols and phenolic acids. Convincing evidences suggest that the beneficial effects attributable to dietary phenols depend on their biotransformation by the gut microbiota. Most polyphenols present in the diet are in the form of esters, glycosides, or polymers that cannot be absorbed in their native form. It is estimated that about 5% to 10% of dietary polyphenols are absorbed and reach plasma. Over 95% of the intake passes to the colon and is fermented by gut microbiota to produce small aromatic or phenolic acids . For example, rosmarinic acid, found abundantly in ORE, ROS, and in many Lamiaceae plants, is an ester of caffeic acid and 3-lactic acid. A recent study reported a complete degradation of rosmarinic acid, and the generation of microbial product caffeic acid after in vitro fermentation using human feces . The caffeic acid undergoes subsequent microbial transformation to yield hydroxyphenylpropionic acids as major metabolites, a process mediated by microbial chlorogenate esterases or by Lactobacillus johnsonii . Results of human intervention study confirmed these in vitro findings . These and other microbial metabolites were found to selectively inhibit the growth of pathogenic bacteria and stimulate the growth of beneficial microorganisms .Of the 16 Lactobacillus strains tested, the growth of 1 strain was enhanced by BLP and another one by CAP at the concentration of 0.56 mg/mL. At 1.13 mg/mL, 4 strains were stimulated by BLP, 5 by CAP, 7 by GIN, and 10 by ORE. At 2.25 mg/mL, 2 strains were stimulated by BLP, 7 by CAP, 4 by GIN, and 2 by TUR. Overall, all spice extracts showed better growth stimulatory effect on the Lactobacillus spp. as compared to Bifidobacterium spp. Some foods can exert growth stimulatory effect because their ingredients can be used as substrates by these bacteria, or they can enhance nutrients consumption by affecting bacteria metabolism .

Prebiotics are defined as substances that induce the growth or activity of microorganisms that contribute to the well-being of their host . Lactobacillus and Bifidobacterium are widely established bacterial genera as prebiotic target organisms. These 2 genera produce acetic acid and lactic acid as the major end-metabolites and do not contain any known pathogens. Previous studies by our group and others have identified dietary sources functioning as prebiotics by stimulating the growth of Bifidobacterium spp. and Lactobacillus spp. in both in vitro and human studies . Evidence is growing in support of the prebiotic effect of foods high in polyphenols. Our results suggest that some of the tested spices exhibit prebiotic-like effect by stimulating the growth of a number of Bifidobacterium and Lactobacillus species. Bifidobacteria are normal inhabitants of the gastrointestinal tract. The composition, diversity, or relative abundance of Bifi- dobacterium species has been implicated in several intestinal disease conditions . Bifidobacteria have been shown to alleviate infectious diarrhea through their effects on the immune system . Lower levels of bi- fidobacteria were linked to higher prevalence of E. coli in obese children ; similarly,square pot lower levels of bifidobacteria have been demonstrated in overweight subjects and in patients with long-term asthma , suggesting a role of bifidobacteria in human health. Our study reported new findings in that among 7 spice extracts ORE was most active in promoting the growth of Bifi- dobacterium whereas GIN, BLP, and CAP produced more modest stimulatory activity. Studies of Lactobacillus rhamnosus GG have shown promising results in treating diarrhea caused by viruses and bacteria , atopic disease , and in prevention of gastrointestinal and respiratory tract infections . In our study, all spice extracts were found to promote the growth of LGG strain at concentration ranging from 0.56 to 4.5 mg/mL. L. reuteri and L. rhamnosus are often added to dairy products, or formulated as dietary supplements for controlling dysbiotic bacterial overgrowth during an active infection. BLP, CAP, GIN, and ORE enhanced the growth of these species at either 1.13 or 2.25 mg/mL. Our data are consistent with a study by Sutherland who reported that aqueous extracts of ginger and red chili enhanced the growth of L. reuteri and L. rhamnosus .Both medical students and faculty recognized the need for accessible health care among marginalized populations in the Zona Norte, and in 2011, a bi-national student-run free clinic was established in the heart of Tijuana’s red-light district . The clinic known as HFiT, or Health Frontiers in Tijuana, partners with Tijuana’s Autonomous University of Baja California School of Medicine and the University of California, San Diego School of Medicine. HFiT is equipped to provide medication, basic laboratory tests, and social services to patients living in poverty. Despite the clinic’s success in providing continuity care for more than 600 patients in 2012, the HFiT clinical faculty and student leadership recognized the need for a vulnerable women’s health clinic. An HFiT survey conducted between May and December 2012, found that of the 211 female patients, 12% stated they required obstetrical or gynecological care. Apart from HIV and syphilis diagnostic testing, the HFiT clinic currently lacks the supplies to perform other sexually transmitted infection testing, pap smears, endometrial biopsies, or provide contraception.

Furthermore, increasing HIV prevalence among female sex workers in Tijuana emphasizes the need for interventions dedicated to the education and prevention of HIV transmission. In a study of 924 FSWs, nearly one third of all questions on HIV transmission and prevention mechanisms were answered incorrectly . Therefore educating and refuting common HIV misconceptions among FSWs demonstrates a potentially powerful intervention method for HIV prevention. The HFiT clinic has provided the physical space for a women’s clinic. This includes a private exam room and an exam table. Our plan of action is geared towards formally establishing a Women’s Clinic that will address the needs of our vulnerable patient population and will sustain long-term contribution to the clinic.Beginning the summer of 2013, after each Saturday clinic two clinic volunteers and I went around Tijuana’s Zona Roja in order to distribute flyers advertising our services and clinic location. We also distributed condoms and handouts on how to properly use a condom. Although we handed out maps/flyers to females all around the clinic, we tried to target the areas where most sex work took place in the Zona Roja. Here we asked female sex workers if they were willing to fill out a brief, anonymous survey asking where they currently go for medical attention, what medical services they need, and whether they were aware of our HFiT clinic. The surveys evolved over the course of the summer as we found some questions to be more effective than others and many FSWs did not have more than 5 minutes to devote to answering survey questions. Although responses varied for desired medical services, based on the results most sex workers did not know about our clinic prior to taking the survey. With a finalized version of the survey currently in place, we will continue to assess the population and evaluate how responses change over time. After researching the most cost-effective options for an ultrasound, including contacting the Professor of Clinical Emergency Medicine and Director of Instructional Ultrasound at UC Irvine School of Medicine, we found that our past contact for much of the clinic supplies was the best and most reliable option. We were able to secure an ultrasound complete with wide-angle probe, vaginal probe, and printer for a total of $2300. The ultrasound is refurbished, recalibrated and in great condition. Although the budget for the summer did not allow for purchase of both ultrasound and colposcope, we are very pleased that the ultrasound with endoprobe will allow us to perform transvaginal ultrasounds to assess gynecological anatomy.Following the clinic’s goal of empowering its patients, we have designed a Women’s Health curriculum that explores female anatomy, body perception, and general nutrition. The implementation of this program and its success can be attributed to the collaboration with Por La Vida . Por La Vida began as a pilot health education-advising program in San Diego in 1987. Evidence of its success and longevity 25 years later is seen through the over 200 “Consejeras” or advisors that currently lead small group sessions in the medically under served Latino Community of San Diego.

To estimate the extent of incorrectly-predicted pathways in the PMN databases, and to measure the overall accuracy of the computational predictions, both alone and in conjunction with manual curation, we evaluated the prediction of 120 randomlyselected pathways on both the released single-species databases in Pathway Tools and naïve prediction versions generated using only computational prediction . Biocurators evaluated the pathway assignments to the 126 organisms currently in PMN, and classified them as “Expected” , “Broader” , “Narrower” , or as NonPMN Pathways . In the naïve prediction databases, only 15% of selected pathways were predicted within the phylogenetic ranges expected from the literature, and 58% were NPPs. In the released PGDBs, however, 78% of evaluated pathways were predicted as expected . In addition to correcting the prediction for 94% of all NPPs of the surveyed pathways, incorporating curated information also reduced the percent of pathways predicted beyond their expected phylogenetic ranges from 13% to 4%. Thus, the applicationof phylogenetic information and manual curation drastically improves the quality of pathway prediction throughout PMN databases over the use of computational prediction alone. To determine whether different groups of plants can be differentiated solely by their metabolic capacity, we performed multiple correspondence analysis , a type of dimension reduction analysis that is similar to principal component analysis but can be used for categorical data . MCA was carried out using presence-absence matrices for pathways, reactions, and compounds . Reactions were considered present only if at least one enzyme in the species was annotated as catalyzing the reaction. Independently,25 liter pot the plants were categorized according to phylogenetic groups. Dimensions 1 and 3 of the pathway and compound MCA, and dimensions 1 and 2 of the reaction MCA, separated the species into several phylogenetic groups .

Phylogenetic groups that clearly cluster together and away from other groups include algae, non-flowering plants, Brassicaceae, and Poaceae . Dimension 1 separates the chlorophytes from land plants and dimension 3 separates certain angiosperm families such as the Brassicaceae and Poaceae well. No clear separation was observed among other eudicot groups. In addition, dimension 2 of the pathway and compound MCA mostly separated a small number of highly curated species from all the rest . Overall, the MCA clustering shows that some groups of plants can be readily differentiated based on their metabolic information in PMN, while other groups cannot, suggesting that further curation of species in these groups may be beneficial. We next asked which metabolic pathways drive the separation of the taxonomic groups on each dimension . Seventy percent of the variance in dimension 1 was described by 109 pathways, all of which were predicted to be either embryophyte-specific pathways or present in a larger proportion of embryophytes than chlorophytes. This mirrors the separation of the Chlorophyta cluster in dimension 1 of the MCA plot . Similarly, 70% of the variance along dimension 3 was captured by 150 pathways, of which 81 were associated more strongly with Poaceae and 69 were associated more strongly with Brassicaceae . The pathways that contributed 95% of the variance in dimension 1, which separates chlorophytes from embryophytes, were enriched for hormone metabolism . Hormone metabolism may have helped support the increased complexity of land plants compared to their algal ancestors . In contrast, pathways responsible for clustering along dimension 3 were enriched for specialized metabolism , which is more lineage-specific than other domains of metabolism and can help distinguish between clades of angiosperms . Thus, it appears that metabolic data in PMN can effectively differentiate groups of species not only by the presence or absence of specific pathways and reactions, but also by the types of metabolic processes which are related to their evolutionary divergence. One of the major advantages of PMN 15 is the ability to quantitatively and qualitatively analyze omics data in the context of global metabolism for the 126 species represented in the resource.

Here we demonstrate two applications of integrating omics data with PMN 15 to gain novel insights about plant metabolism. Pathway Tools’ Omics Dashboard allows users to visualize omics data across experimental timepoints and conditions across a broad range of cellular subsystems and at multiple scales of resolution . To demonstrate the utility of the Omics Dashboard within a metabolic context, we used a transcriptomic survey of two sorghum cultivars, RTx430 and BTx642, subjected to drought stress . RTx430 is tolerant to pre-flowering drought, whereas BTx642 is tolerant to post-flowering drought. To see if there was any difference in metabolic gene expression between the two cultivars in response to post-flowering drought, we examined differentially expressed genes in droughted plants compared to well-watered plants from the last week of watering to the first two weeks of post-flowering drought . We observed a consistent down-regulation of biosynthetic activity from root tissues in the post-flowering drought sensitive cultivar RTx430 compared to relatively stable expression in the post-flowering drought tolerant cultivar BTx642 . This observation is consistent with the authors’ findings that BTx642 demonstrated higher levels of redox balancing and likely experienced lower levels of reactive oxygen species stress, compared to RTx430, as a result of drought. By analyzing expression patterns of all metabolic genes, we observed a widespread metabolic down regulation in RTx430 root tissue, which was not reported previously . Drought-responsive DEGs were enriched in metabolic genes among both leaf and root tissues. However, contrary to the clear cultivar-specific trends shown in the root DEGs , there was no clear trend in expression patterns of metabolic genes in the leaves of either cultivar as a result of drought . To determine whether the consistent reduction of metabolic gene expression observed in RTx430 roots in response to drought was a global trend in the transcriptome or specific to metabolic genes, we compared relative expression levels of all non-metabolic root DEGs to all metabolic root DEGs in both cultivars during the same 3-week period.

While the average relative expression decreased each week among both metabolic and non-metabolic genes in RTx430, the down regulation was greater among metabolic genes at both time points . In contrast, BTx642 roots showed no difference in expression among both metabolic and non-metabolic genes in response to drought , suggesting a global metabolic homeostasis in sorghum drought tolerance. By comparing the patterns of expression among DEGs in root and leaf tissues, rather than solely the number of DEGs, analysis via the Omics Dashboards revealed that roots exhibited stronger genotype-specific responses to drought than leaves, which was not observed previously . In addition to offering a visual overview of metabolism via the Omics Dashboard, PMN’s analytical toolkit allows researchers to easily conduct enrichment analyses among a set of genes or compounds of interest. From within a SmartTable, users can view the pathways associated with a set of genes or compounds, and can then ask whether those genes or compounds are enriched for specific pathways. Broader metabolic classifications can also be added to the list of enriched pathways to better understand which area of metabolism are most enriched. For example, among the set of drought-responsive DEGs in RTx430 roots, we observed an enrichment in various domains of carbohydrate and amino acid biosynthesis and degradation, in addition to chitin degradation, consistent with the authors’ observation of drought-induced responsiveness of biotic defense genes . Thus, by combining PMN’s analytical capabilities with its broad set of metabolic data, users can find additional means of supporting existing hypotheses, uncovering novel insights, and finding new avenues for exploration in their own research. PMN 15 data can also be integrated with other cutting-edge datasets to investigate novel biological questions. As an example, we integrated A. thaliana root single-cell RNA-seq datasets from five independent studies to examine cell type specificity of metabolic domains and pathways . We define cell type-specific metabolic domains as those whose constituent genes show significantly higher expression levels in certain cell types compared to their average expression level in total cells. Different metabolic domains showed overlapping as well as distinct cell type specificity . First,25 liter pot epidermal and cortex cells were most metabolically active throughout the various domains of metabolism . This is consistent with previous observations that the major groups of metabolites detected in Arabidopsis roots, including glucosinolates, phenylpropanoids, and dipeptides, were highly abundant in the cortex . In contrast, maturing xylem showed relatively low metabolic activity as the major roles of these cells are structural support and water/soluble transport . Viewed from the level of metabolic domains, this analysis demonstrates a diverse range of metabolic activity across unique cell types in Arabidopsis roots. We next probed cell-type specificity of individual pathways. Among the 198 pathways associated with at least 10 genes, 40 pathways showed specificity in at least one cell type compared to their background gene expression levels represented by the average expression level of the pathway across all cell types . For example, in actively dividing cells, such as meristematic xylem cells, pathways involved in pyrimidine, histidine, arginine, and lysine biosynthesis showed high activity . These pathways are involved in essential metabolism, which are critical for maintaining cell division and growth. On the other hand, hormone biosynthesis pathways, such as cytokinin glucoside and gibberellin, showed high activity in the cortex. This is consistent with current understanding that the cortex is one of the predominant cell types that synthesizes these two hormones in the Arabidopsis root .

By elucidating cell type-level activity of metabolic pathways, we can begin to map metabolism at cellular and tissue levels, which will be instrumental in understanding how metabolism affects plant development and responses to the environment as well as enabling effective engineering strategies. Similar to cell-type specificity, the concept of pathway divergence at the individual cell level can also be explored using single-cell transcriptomics data. To probe this question, we asked whether isozymes catalyzing the same reaction are more likely to be expressed in different cells compared to enzymes catalyzing different reactions in the same pathway. Isozymes are defined as enzymes encoded by different genes catalyzing the same reaction, which are usually the result of gene duplication events. We computed Spearman’s correlation coefficient to measure gene expression pattern similarity between a pair of enzymes across Arabidopsis root cells. The coefficients computed based on single cell data were generally lower than that generated by bulk RNA-seq, which may be due to the sparseness of single cell transcriptomic profiles or high heterogeneity of gene expression across cells. Nonetheless, metabolic genes in the same pathway showed higher correlation than randomly sampled metabolic genes , which suggests functional coordination between genes involved in the same pathway at the cellular level. Isozymes were much less correlated than enzyme pairs catalyzing different reactions in the same pathway. This indicates that isozymes may have evolved divergent expression patterns in root cells . Since isozymes are often the results of gene duplication events, this diversified expression between isozymes may contribute to retaining duplicated genes through sub-functionalization or neofunctionalization and fine-tuning metabolic pathways at the cellular level . Recently we introduced the Pathway Co-Expression Viewer, which integrates information from PMN 15 and ATTED-II , a database of gene co-expression, to visualize co-expression of the genes in a pathway for species represented in ATTED-II , Solanumlycopersicum , Oryza sativa , Zea mays , Brassica rapa, Vitis vinifera , Populus trichocarpa , and Medicago truncatula. An example is shown in Supplemental Figure S4A-B; Lysine biosynthesis is currently known to occur via two distinct routes, utilizing either diaminopimelate or α- aminoadipate as an intermediate. Its biosynthetic pathway in plants, cyanobacteria, and certain archaebacteria converts tetrahydrodipicolinate to L,L-diaminopimelate via L,L-diaminopimelate aminotransferase and is distinct from that of other prokaryotes and of fungi . Lysine biosynthesis is of particular importance as it is both an essential amino acid not biosynthesized by mammals and it is the least abundant essential amino acid in cereals and legumes . The co-expression levels of six pairs of genes are in the top 1% of coexpressed gene pairs within ATTED-II, while an additional 10 gene pairs are in the top 5% . This tool provides a convenient way of visualizing the co-expression of genes in a pathway and thus provides clues as to how the pathway may be regulated. PMN 15 introduces an additional feature which provides a new way of visualizing pathways that span intracellular compartments and include transport reactions. For example, the glutamate-glutamine shuttle from AraCyc is a pathway in which glutamate and glutamine are exchanged between the mitochondria and chloroplast as a means of ridding the mitochondria of ammonium produced during photorespiration .

The bear’s habitat may also have included honeybees, but this is speculative. The genus appears to have originated in Europe dispersing into Asia, Africa as well as North America. In North America the fossil record of this lineage is represented by a single species from the Miocene of Nevada. The most likely route that the Apis lineage took to arrive in North America would have been via the Bering Isthmus, which was present throughout the Neogene until ~5–7Ma. This land connection would have allowed for the existence of expansive high-latitude terrestrial continuity, spanning the northern reaches of the Eurasian and North American continents. Thus Apis in North America may have originally inhabited this Arctic biome before dispersing southward into the mid-latitudes of North America. In which case, the polar P. abstrusus may have had opportunity to supplement its diet with honey. Aside from the Beaver Pond site fossil bear, all other basal ursines are known from the northern mid-latitudes of Eurasia and North America . The lack of fossil bears in the intervening latitudes reflects the scarcity of northern Neogene vertebrate fossil sites in these regions. Thus, the discovery of the Beaver Pond site P. abstrusus at 78°N fills a substantial geographical gap. The finding also shows that early ursines were adapted to northern forests with snowy winters. Moreover, the Beaver Pond site bear is a small-bodied bear with dental caries and associated with a polar forest, rich in seasonal fruits ,large pots with drainage suggesting that the northern populations of P. abstrusus likely consumed large amounts of sugar-rich foods in the fall, a pattern consistent with preparation for hibernation seen in modern bears.

If so, the Beaver Pond site bear represents the earliest known, and most primitive bear, to have hibernated. Modern ursid hibernators include high latitude/ altitude Asian black bears , northern American black bears , all brown bears , and female polar bears. Also, the fossil species of cave bears are inferred to have hibernated. All living bears also employ a reproductive strategy of embryonic diapause with implicit adaptive value of reducing the cost of reproduction by truncating embryonic development and of optimizing birth season at the most appropriate time. Furthermore, these reproductive cycles may regulate metabolism by facilitating earlier entry of pregnant females into winter-dormancy state. In the context of the phylogeny of modern bears the northern americanus-arctos-spelaeus-maritimus clade appears to have acquired hibernation from a single ancestor. The case for Asian black bear is ambiguous because its nearest relatives are not known to hibernate; namely the sloth bear of India, the sun bear of Southeast Asia. The early diverging spectacled bear of South America is also a non-hibernator . If the northern adapted Beaver Pond bear was a hibernator, then hibernation can be traced to the ancestor of all modern bears. This would imply that the Asian black bear retains the primitive condition, and the Eurasian ancestor of the spectacled bear, which would have passed through cold Beringian habitat when it first immigrated to North America, also employed hibernation as part of its repertoire for winter survival. In this evolutionary scenario modern non-hibernating bears, are interpreted to have secondarily lost this trait, in association with adaptation to warmer habitats.Fire-induced turbulence has considerable impact on scalar dispersion and momentum transport both during and after the passage of the fire-front across the fuel in wild land fire environments.

The dispersion of scalars such as smoke in turn affects visibility and human health, especially for residents in the wild land-urban interface, while the transport of firebrands poses a severe risk of spotting up to several kilometers away from the fire-front . Moreover, fire behavior is closely linked with the ambient turbulence . Depending on the environment, turbulence in the canopy or the atmospheric boundary layer demonstrably affects fire spread-rates , occasionally in unexpected ways. With a deep necessity to understand fire-induced turbulence both in grassland and forested environments, fire simulations as well as in-situ measurements from burns on small as well as management scales have gained importance. However, the excessive computational overhead involved in simulating fires in forested and grassland environments make them less amenable for extended use. While a fast-running simulation platform like QUIC-fire is able to circumvent this issue, its wind solver outputs the time-averaged flow field, which makes it difficult to analyze the turbulence-driving fire behavior. Given these constraints on computational models, careful examination of observed data from burn experiments can be useful in investigating the coherent motions that characterize fire-induced turbulence. Such examination can potentially also allow for the simplification of the governing equations describing fire behavior based on the relative importance of the different terms involved. In the past, empirically derived models based on the widely used model by Rothermel have been successful in simplifying the complex set of governing equations for successful operational use via the parameterization of several physical quantities. However, these models are based on laboratory experiments so that their application on management scales is limited.

Another limitation is their inability to provide insights into the complex turbulent environment arising from fire-atmosphere interaction. Despite their usefulness in understanding the physics underlying fire-induced turbulence, studies relying on experimental data have only recently gathered momentum because of the high risk of damage posed to equipment during data collection in the presence of a flame . Here, we review the key findings of some of the few leading empirical studies that attempted to untangle the complex dynamics underlying fire-atmosphere interaction. Recent studies by Heilman et al. explored turbulent kinetic energy levels at different heights of a 20 m measurement tower, the energy spectrum of the velocity components, and patterns in the turbulent heat and momentum fluxes for two different backing surface fires in the New Jersey Pinelands National Reserve , one of high intensity in 2011 and the other of lower intensity in 2012. For the higher intensity fire, fire-induced TKE near the canopy top was found to be much more substantial compared to the lower heights, making turbulent diffusion of smoke near the canopy top more important than mixing at lower heights within the canopy. Analysis of 1 minute averaged turbulent momentum fluxes showed that vertical turbulent fluxes contributed 40%–80% to the net momentum flux before, during, and after fire-front-passage . The relative contribution of the vertical turbulent heat and momentum fluxes to the net heat and momentum fluxes was found to be highest at the mid-canopy height when compared to near the surface or the canopy top. Estimates of the turbulence anisotropy obtained by Heilman et al. demonstrated the significance of horizontal velocity perturbations over vertical velocity perturbations pre- and post-FFP as well as during FFP periods at all measurement heights. Analysis of the relative contribution of the terms in the TKE budget equations showed that shear production was higher than the buoyancy production at all measurement heights within the over story vegetation layer pre- and post-FFP. Furthermore,square pot the turbulent transport term resulted in a loss of TKE at all measurement heights during FFP in 2011, while its effect was found to be greatly diminished in the lower-intensity 2012 burn. In a separate study, Heilman et al. studied the contribution to turbulent fluxes of sweep-ejection events before, during, and after the passage of a heading surface fire-front through a network of 20 m measurement towers during an operational burn in the New Jersey Pine Barren. It was found that pre-FFP and post-FFP periods were characterized by sweeps and ejections, while FFP periods were characterized by sweeps and outward interactions . These studies encapsulate some of the most recent turbulence analyses on experimental surface fires in forested regions. The FireFlux experiment was a pioneering study on fire–atmosphere interactions in a grassland fire and set a benchmark for similar studies in the future . It comprised a heading experimental grassland fire conducted at the Houston Coastal Center in Texas on 23 February, 2006. Temperature and velocity measurements taken during the experiment by a 43 m instrumented tower were utilized by Clements et al. for a first-order analysis. Derived quantities such as the velocity spectra, turbulent momentum fluxes, and TKE were analyzed to comment on the turbulence levels induced by the fire-front passage. The turbulence intensity generated by the fire was found to be four to five times greater than the ambient atmospheric turbulence. Furthermore, turbulent fluxes demonstrated a downward transfer of high momentum downstream of the fire-front. Heilman et al. took the analysis further with their study of sweep-ejection-like events, which also included results from the three prescribed fires in the New Jersey Pinelands described above.

Their focus was on examining how the frequencies of occurrence of sweep-ejection-like events and their contributions to the mean vertical turbulent fluxes of heat and momentum were modulated by the vicinity or presence of the fire. Contrary to conditions in the absence of surface fires, it was found that ejections were completely dominant in their contribution to heat-flux events and outward interaction events substantially overshadowed the contribution of sweeps and ejections to momentum-flux events in both environments. The most frequent events during FFP were heat-flux inward interaction events and momentum-flux outward interaction events in grassland and forested environments, respectively. As seen above, previous empirical and semi-empirical studies have taken a keen interest in exploring turbulent fluxes and terms of TKE budget equation pre-, during, and post-FFP for insights into the dominant physical processes characterizing fire-induced turbulence. However, the interpretation of coherent structures inferred from the turbulent fluxes and the TKE budget terms is sensitive to the averaging scheme employed to decompose turbulent signals into the mean and turbulent fluctuations from the mean. Additionally, the effect of the fire on the mean kinetic energy budget terms remains to be explored using the existing data. Furthermore, several differences in the fire-turbulence behavior between surface fires in forested and grassland environments remain to be articulated. For instance, we expect the nature of the turbulent transport term to be different, considering that it assumes different levels of importance within the canopy and in the open atmospheric boundary layer in no-fire conditions . Heilman et al. also reflected on the need to understand differences in the behavior of turbulent fluxes for backing and heading fires across all the measurement heights in the canopy. Evidently, there is a need to unify and synthesize observations from burn experiments that have already been conducted via an inter-comparison to develop an over-arching description of the process involved. The analyses presented by Clements et al. and Heilman et al. for four experimental management scale burns conducted in Texas in 2006 , in the New Jersey Pinelands National Reserve in 2011 and 2012, and in the New Jersey Pinelands National Reserve in 2019 are synthesized and re-investigated in this study. Here, we aim to fill the gaps in the previous analyses on these data regarding the coherent motions arising out of fire-atmosphere interaction when juxtaposed with the background canopy turbulence or open atmospheric turbulence, depending on the surface-fire environment. High-frequency 10 Hz measurements in the case of the sub-canopy fires and 20 Hz measurements in the case of the grassland fire for velocity and temperature are utilized. We first introduce the data by exploring the slowly-varying parts of the velocity components and temperature for changes occurring in the flow over relatively longer time scales. We then investigate pre-, during, and post- FFP wind-rose statistics for a visual representation of the combined changes induced by the presence of the fire and ambient wind shifts. Finally, we compute the turbulent fluxes and terms of the TKE budget equation; we comment on the effect of the former on the latter and on the physical significance of both. From these quantities, computed at different heights above ground level , we seek insights into the more persistent coherent structures, the key fire-spread mechanisms, and the dominant turbulence generation and TKE redistribution processes, along with the relevant vertical length scales. Another important and relatively unexplored question of interest is the effect of FFP on the terms of the MKE budget equation, which is investigated here for the sub-canopy surface fires. We aim to draw a comparison of insights among all the three scenarios considered here: heading surface fire beneath the canopy, backing surface fire beneath the canopy, and heading surface fire in a grassland.

The Food Rating Log included a 9-Point Hedonic Scale for each food item consumed, on which participants rated each food from 1 = “dislike extremely” to 9 = “like extremely.” This Hedonic Scale was originally developed by the U.S. Armed Forces to measure soldiers’ food preferences , but is now used by NASA to assess astronauts’ food preferences, as well. For each participant, I averaged their daily food ratings so that each participant had 8 average daily food rating scores, with 1 being the lowest possible rating and 9 being the highest possible rating. I also conducted exploratory analyses in which I evaluated each food item’s average rating. These exploratory analyses provided insight into which individual food items should be replaced, but these analyses were not included in the study’s main analyses. Every evening , participants completed the Food Monotony Scale , adapted from questions previously used by Redden in NASA-funded studies. The scale included three questions, with two questions asking participants to rate their satisfaction with food variety from 1 to 9 . The remaining question asked participants to rate their boredom with food from 1 to 9 . However, this question was sometimes negatively correlated with the other two questions, suggesting that participants misread the wording of the response options and interpreted 9 as indicating high boredom. Consequently, only the first two items were used in analyses. This resulted in consistent positive correlation between the two items every day. These two items were averaged to calculate 8 average daily satisfaction with variety scores, with 1 indicating lowest satisfaction with variety and 9 indicating highest satisfaction with variety.

If a participant ate half of a food item on Monday and finished that food item on Wednesday,nft vertical farming only their Monday rating was included when calculating that participant’s daily average food rating. On two instances, one participant ate half of a food item in the morning and the other half of that food item in the afternoon. In these instances, I averaged the food ratings and counted them as a single rating. If a participant smelled or tasted but did not eat a food item, the ratings were included in analyses, just as if the food had been eaten. Three participants requested and ate additional food items during days 3 and 4 of the experimental condition. Providing these additional food items was considered acceptable because the trading and sharing of food would likely be common during spaceflight. One participant was provided with another Asian Noodle and Indian Potato Entrée, one participant was provided with two more BBQ Chicken Sandwiches, and one participant was provided with two more applesauce pouches and two more yogurt pouches. Participants’ ratings of these additional food items were included when calculating participants’ daily food ratings. When computing the average rating a particular food item, I averaged ratings if one participant rated the food twice in one day. However, I used only the first rating if a participant rated the same food item on two separate days. This is the same way that I assessed ratings when computing participants’ daily food ratings. However, when computing average ratings of particular food items, I did not include the ratings of any additional items that were requested by participants, so that each participant’s evaluations were weighted the same . Two items were provided twice in the experimental condition. All ratings for the Italian-Style Sandwiches were averaged together to compute its average food rating.

The same approach was used for Turkey Jerky. Eight individuals participated in the study. One participant had a medical emergency on Thursday of the Control week, leading to extreme changes in diet , the inability to weigh the participant in-person, and the delayed completion of surveys. Additionally, it was unknown if the medical event led to altered eating on the other days of the study. Consequently, this participant was excluded from all analyses, leaving a sample size of N = 7.The remaining seven participants were comprised of four females and three males . Participants were not asked to report their precise age, but they all appeared to be in their late 20’s to early 30’s – largely mirroring NASA selected astronaut candidates, whose average age is 34 . The seven participants were of Caucasian descent. A sample that more accuracy reflected the U.S. population would have been preferred, but the sample nevertheless reflected NASA’s former astronaut population . Prior to participating in the study, participants reported they ate the breakfast that was served at work a mean of 2.43 times a week , the lunch a mean of 3.86 times a week , and the dinner a mean of 3.00 times a week . Prior to participating in the study, participants reported most commonly eating their meals at the cafeteria/Mezz at work , at their desks , and at home . No participant reported that they ate foods such as juice or meal replacement bars most days. However, participants did report having food patterns, such as eating protein shakes or oatmeal most days. The anonymous personality survey that participants completed on the last Friday of the study revealed that participants did not have extremely unusual personalities, as compared to norms based on 1,813 respondents . Compared to these norms, participants in this study scored 1.42 points higher on Extroversion, 0.60 points higher on Emotional Stability, and within a half point of every other measured personality trait. These comparisons were computed using the worksheet designed by DeNeui to compare TIPI scores to TIPI norms . These comparisons are shown in Table 5, with each participant depicted as a number.The primary aims of this study were to investigate the impact of a possible commercial space food diet on nutrient intake, food satisfaction, psychological health, and physical health. These aims were investigated by having a sample of aerospace employees eat a diet of commercial, ready-to-eat food for four days one week, and comparing their outcomes to when they ate essentially as normal for four days another week. The seven employees that participated in this study were an ideal sample, given that they had a high workload and represented the type of space enthusiast that will likely seek out commercial space flights, once they become more readily available.

These employees had relatively ordinary eating habits and average personalities, being just slightly more extraverted and emotionally stable than a normedsample. Their adherence to the study was extremely high, largely driven by their desire to contribute to the future of space food.The experimental diet was designed to meet nearly all nutrient recommendations set forth by NASA and to be highly satisfying. Consequently, I had hypothesized that while in the experimental condition, participants would consume the same amount of calories as they did while in the control condition. I had also hypothesized that there would be no significant decline in caloric intake across the four days of each condition. The results of this study coincided with these hypotheses. When caloric intake was computed based on participants’ self-reported food intake, there was no significant difference in caloric intake between conditions and no significant change in caloric intake over time. Although the experimental diet was designed to be highly satisfying, it was nevertheless made entirely of commercial, ready-to-eat food. Consequently, I had hypothesized that while in the experimental condition, participants would rate the food as being slightly less hedonically rewarding and slightly more monotonous, with “slightly” being defined as less than 2-point decreases on the 9-point scales assessing hedonic food ratings and satisfaction with variety. In line with these hypotheses,vertical tower for strawberries the experimental food, as compared to the control food, was rated 1.345 points lower on the hedonic food rating scale and 1.750 points lower on the scale assessing satisfaction with variety. In both instances, these differences between conditions were statistically significant . Consequently, as predicted, the experimental diet was rated as being slightly less hedonically rewarding and slightly more monotonous.I had also hypothesized there would be no significant decline in food satisfaction over the four days. In line with this hypothesis, there was no significant interaction between condition or time and no significant main effect of time on either hedonic food ratings or on satisfaction with variety. Thus, although participants rated the experimental diet as being slightly less hedonically rewarding and slightly more monotonous, the diet did not become less rewarding or more monotonous over time. Because the diet was comprised of long shelf-life, ready-to-eat food, it contained few vegetables and was a relatively sweet, salty diet. Consequently, I had hypothesized that during the experimental condition, as compared to during the control condition, participants would show higher cravings for vegetables, lower cravings for salty food, and lower cravings for sweets. The results showed that, in line with the hypothesis regarding sweets, participants reported lower cravings for sweets while in the experimental condition. However, contrary to my hypotheses regarding cravings for vegetables and salt, there were no significant differences between conditions on cravings for vegetables or salt. In post study interviews, some participants reported they would have liked to have seen more vegetables and more salty food. Unexpectedly, there was a significant interaction between condition and time on cravings for meat, with participants in the experimental condition craving meat significantly more than participants in the control condition on day 2.

Although cravings for meat were not significantly different on the other days of the week, this significant result coincides with participants’ qualitative feedback that they would have liked to have seen more meat in the experimental condition. The experimental condition was designed to be nutritious and highly satisfying, and was therefore expected to meet participants’ psychological needs. Consequently, I had hypothesized that while in the experimental condition, participants would show psychological health that was equivalent to their psychological health while in the control condition, as indicated by no significant differences in mood, perceptions of social disconnection, and perceived stress. I also hypothesized there would be no significant decline in psychological health over the four days, as indicated by non-significant time by interaction terms. In line with these hypotheses, participants showed no significant differences in perceptions of social disconnection, perceived stress, or mood between conditions. There were also no significant differences in perceptions of social disconnection or perceived stress over time. The only mood that changed significantly over time was fatigue; participants reported being less fatigued on the fourth day of each week than they were on the first day of each week. The time term was also significant for the mood of vigor, however pairwise comparisons showed no significant differences on any particular day. Thus, there was no significant decline in psychological health over the four days. The experimental diet was designed to be nutritious, and I had anticipated participants would consume equivalent calories in both the experimental and control conditions. Consequently, I had hypothesized that while in the experimental condition, participants would have physical health that was equivalent to their health while in the control condition, as indicated by no significant differences in sleep or self-reported health and no significant weight loss. I also hypothesized there would be no significant change in self-reported health or weight over time. In line with my hypotheses for sleep and self-reported health, participants showed no significant differences in global sleep scores or self-reported health between conditions, and no significant decline in self-reported health over time. However, contrary to my hypothesis for weight, participants in the experimental condition lost significantly more weight than participants in the control condition. In the control condition, participants lost just 0.071 pounds from Monday to Friday, a change that was not significant. Conversely, in the experimental condition, participants lost 2.157 pounds from Monday to Friday, a change that was statistically significant. Why did participants lose more weight in the experimental condition if they didn’t eat fewer calories? There are many possible explanations. It is possible that participants lost fat mass or lean mass due to eating less in the experimental condition. However, given that there were no significant differences in caloric intake between the two conditions, and that my visual checks of the leftovers showed that participants accurately reported their food intake, this is unlikely. Participants were not asked to record their exercise, and consequently, it is possible that participants exercised more during the experimental week, resulting in weight loss. However, there were no significant differences in vigor or fatigue between the two conditions, and consequently this is also considered unlikely.

Small molecules have a long history of acting as drugs for the treatment of hypertension, infections, heart failure, diabetes, asthma, rhinitis, and tumors and comprise approximately 75% of the anticancer drugs approved by the FDA from 1981 to 2014.Recently, many small molecules target EMT and exhibit good therapeutic effects on retarding progressive tissue fibrosis and cancer in experimental conditions.In addition, it is worth noting that many small molecules, such as tivantinib, trametinib, linsitinib, nintedanib, and binimetinib, are in ongoing clinical trials for the treatment of tumors.These cases show promising prospects for treating fibrosis and cancer, which encourages researchers to find effective small molecule drugs for treating fibrosis and cancer. In this review, we describe some important transcription factors, signaling pathways, RNA‐binding proteins and microRNAs and several novel regulators that contribute to EMT and further present some small molecules that exhibit therapeutic effects against EMT. Targeting these mediators with these compounds may be a promising therapeutic strategy to treat fibrosis and cancer.Briefly, histological and pathological analyses show that epithelial cells are present in single cell layers or multilayers and show apical‐basal polarity. Epithelial cells not only interact with the basement membrane with integrins but also communicate with each other via specialized intercellular junctions,round plastic pots including desmosomes, subapical tight junctions, adherens junctions and scattered gap junctions.

These interactions help maintain epithelium integrity and function. However, some stimuli drive EMT in pathological conditions. During the process of EMT, epithelial cell‐cell junctions are dissolved, and the epithelial cells lose their apical‐basal polarity and acquire front‐rear polarity. In addition, the cytoskeletal architecture is reorganized, and E‐cadherin expression is replaced by N‐cadherin expression, which enhances cell motility and invasiveness. In fibrosis, mesenchymal‐like cells transform into myofibroblasts, which can be activated to produce excessive collagen, and in tumors, these mesenchymal‐like cells migrate along with the circulatory system to a secondary location where they form a secondary tumor via mesenchymal‐epithelial transition .It is worth noting that tissue fibrosis and tumors share the common process of EMT, which suggests that targeting EMT may be an effective strategy to treat both fibrosis and tumors . EMT is regulated by various mediators such as transcription factors, signaling pathways, RNA‐binding proteins, and miRNAs. In addition, there are many small molecules that exhibit effective therapeutic effects on tissue fibrosis and tumors by suppressing EMT via targeting these mediators.Snail is a zinc‐finger transcriptional repressor that consists of three isoforms, Snail1, Snail2, and Snail3. Among them, Snail1 and Snail2 are activated in the EMT during development, fibrosis, and cancer. The upregulation of Snail1 is found in many different types of cancer such as gastric, colorectal, and prostate cancer.In addition, the over expression of Snail1 enhanced the motility and invasion of prostate cancer cells, and the silencing of Snail remarkedly suppressed the adhesion, migration, and invasion of Hep‐2 cells.Further study revealed that the knockdown of Snail blocked the EMT, which was accompanied by the down regulation of the expression of MMP‐2, MMP‐9, vimentin, N‐cadherin, and fibronectin.

Moreover, Snail expression was demonstrated at different stages of kidney fibrosis, and the reactivation of Snail‐induced renal fibrosis20 . Furthermore, hypoxia‐inducible factor 1α mediated EMT by regulating Snail and the β‐catenin signaling pathway in early pulmonary fibrosis induced by paraquat.Based on these findings, it was suggested that Snail played a key role in the EMT during fibrosis and cancer, and the targeted inhibition of Snail expression might be an effective therapy to treat fibrosis and tumors.Metformin is a first‐line drug for treating type II diabetes mellitus that has shown a therapeutic effect on many cancers. Further study revealed that metformin increased the expression of E‐cadherin, miR‐200a, miR‐200c, and miR‐429 and decreased the expression of miR‐34a, vimentin, Snail1 and ZEB1 in transforming growth factor‐β ‐induced EMT in the colorectal cancer cell lines SW480 and HCT‐116 .In addition, some compounds derived from natural products have also shown significant intervention in fibrosis and tumors by targeting Snail . Toosendanin, a triterpenoid isolated from Melia toosendan Sieb. et Zucc , isused to prevent and control agricultural pests.Recently, it was reported that toosendanin significantly inhibited TGF‐β1‐induced EMT in lung cancer cells via the extracellular signal‐regulated kinase /Snail pathway and suppressed EMT and tumor growth in pancreatic cancer by deactivating the RAC‐α serine/threonine‐protein kinase /mechanistic target of rapamycin pathway , which suggests that toosendanin is a promising pharmacological agent for the treatment of cancer.A recent study revealed that neferine enhanced oxaliplatin sensitivity by inhibiting EMT via the Snail pathway.In addition, ponicidin is a major diterpenoid compound extracted from Rabdosia rubescens Hara that exhibited a therapeutic effect on TNF‐α‐induced EMT and the metastasis of colorectal cancer by targeting the Akt/glycogen synthase kinase‐3 β /Snail pathway. Moreover, ferulic acid is a bioactive component derived from Ligusticum chuanxiong Hort that suppressed EMT induced by TGF‐β1 via inhibiting the Smad/integrin‐linked kinase/Snail signaling pathway in a renal proximal tubular epithelial cell line , suggesting that ferulic acid might be potent fibrosis antagonist.The two ZEB family members, ZEB1 and ZEB2, are transcriptional inhibitors that are involved in cell proliferation, migration, invasion, and apoptosis.

Extensive studies have confirmed that ZEB1 is upregulated in the EMT and plays a key role in the development of tumors and fibrosis.It was reported that the Np63‐miR‐205 axis increased epithelial marker gene expression and decreased mesenchymal marker gene expression in oral squamous cell carcinoma by down regulating ZEB1 and ZEB2.Moreover, the heterogeneous expression of ZEB1 induced by EMT played an important role in metastasis through the regulation of miR‐200c.In addition, miR‐302a‐3p exerted a protective role via inhibiting ZEB1 and EMT in diabetic kidney disease.These results suggest thatvarious mediators contribute to EMT via the ZEB signaling pathway, suggesting that ZEB is a promising target for the treatment of fibrosis and tumors. Decitabine is a drug used to treat myelodysplastic syndromes that inhibited EMT by regulating the miR‐200/ ZEB signaling pathway in non–small‐cell lung cancer PC9 cells.In addition, a phase Ib/II clinical trial revealed that low‐dose decitabine enhanced the efficacy of immunotherapy in patients with drug‐resistant relapsed/refractory alimentary tract cancer, making it an attractive therapy for cancers.104 Silibinin, a bio-active component from Silybum marianum Gaertn., exhibited anticancer properties in multiple types of cancer, such as bladder and lung cancer . Recent studies revealed that silibinin reversed EMT by inhibiting the expression of ZEB1, vimentin, and MMP‐2 as well as the transactivation of β‐ catenin in bladder cancer metastasis.Furthermore, the combined treatment of silibinin with trichostatin A or decitabine suppressed EMT in non–small‐cell lung cancer.These studies suggest that silibinin is an important candidate anticancer drug. Honokiol was extracted from the seed cones of Magnolia grandiflora L. and showed a therapeutic effect on various cancers. Honokiol effectively inhibited EMT in breast cancer through the STAT3/ZEB1/E‐cadherin axis and reversed EMT in renal cell carcinoma via miR‐141/ZEB2 .STAT3, a member of the STAT protein family, can be phosphorylated as a transcription factor and plays a key role in various cellular processes, including cell growth, apoptosis, and differentiation. Hypoxia is a key mediator that induces EMT during tumor initiation and metastasis, and STAT3 promotes HIF‐1α expression to induce EMT by binding to the promoter of HIF‐1α. In addition, STAT3 was identified as a positive regulator that aggravated TGF‐β1‐induced EMT.In particular, the JAK2/STAT3 signaling pathway had a significant effect on the EMT in multiple cancers. In addition, STAT3 cooperated with other transcription factors promoting EMT, such as Twist and ZEB1.For example, the expression of p‐STAT3 and ZEB1 was positively associated with metastasis, and they cooperatively enhanced EMT in colorectal carcinoma Moreover, STAT3 was upregulated in TGF‐β1‐induced EMT during renal fibrosis, implying that STAT3 is an innovative target for the prevention of fibrosi.Furthermore, there are many mediators that induce EMT through the activation of STAT3, such as Pin1, HOXB8, miR‐30d, and IL‐6.Sepantronium bromide is an inhibitor of survivin that exhibits anticancer properties in multiple cancer types. A recent study revealed that sepantronium bromide reduced the invasion of glioblastoma induced by radiation and reversed EMT by targeting STAT3.Tanshinone IIA is a major bioactive component from the famous medical herb Salvia miltiorrhiza that was reported to attenuate the proliferation of bladder cancer cells . Further study revealed that tanshinone IIA suppressed EMT in bladder cancer cells via the modulation of the STAT3‐CCL2 signaling pathway.In addition,hydroponic bucket tanshinone IIA blocked EMT by regulating the TGF‐β/Smad pathway in peritoneal fibrosis.From these data, it was suggested that compounds that target EMT might have both anti-fibrotic and anticancer properties. In addition, polyphyllin I, a steroidal saponin derived from Paris polyphylla, has shown anti inflammatory and anticancer properties. It was suggested that polyphyllin I, which reversed EMT by modulating the IL‐6/STAT3 pathway, served as a novel solution to conquer EGFR‐TKI resistance in non–small‐cell lung cancer. Therefore, combined treatment with polyphyllin I and erlotinib is a promising therapy for lung cancer patients to strengthen drug efficacy and reduce drug resistance.The other compound, quercetin, is a flavonoid widely distributed in fruits, vegetables, and beverages that shows antioxidative, anti inflammatory, and anticancer properties.

Quercetin reversed IL‐6‐induced EMT in pancreatic cancer cells through STAT3.Collectively, the data indicate that the use of compounds targeting STAT3 might be an effective approach for curing fibrosis and cancer.TGF‐β1 plays a crucial role in various cellular functions, including cell growth, proliferation, differentiation, and apoptosis, and is considered a key mediator in EMT during the processes of tumor formation and fibrosis.Many components contribute to EMT through the TGF‐β1 signaling pathway, which indicates that the inhibition of the TGF‐β1 signaling pathway may be effective in the treatment of cancer and fibrosis. Several excellent reviews have discussed the role of the TGF‐β1 signaling pathway in tumors and fibrosis well. Here, we present several important small molecules that suppress EMT in tumors and fibrosis by targeting the TGF‐β1 signaling pathway . Galunisertib, also known as LY2109761, is a TGF‐β receptor kinase inhibitor that specifically down regulated the phosphorylation of Smad2 and upregulated E‐cadherin expression in cultured human hepatocellular carcinoma cell lines, implying that it could suppress the EMT.Galunisertib is now in ongoing clinical trials in patients with glioblastoma, pancreatic cancer, and hepatocellular carcinoma.Nobiletin is a flavonoid compound isolated from Citrus depressa that suppressed EMT by antagonizing the TGF‐β1/Smad signaling pathway in human non–small‐cell lung cancer cells.In addition, oridonin is a diterpenoid from Rabdosia rubescens Hara that inhibited EMT via blocking TGF‐β1/ Smad2/3 in osteosarcoma.Moreover, GW788388 is a novel inhibitor of TGF‐β type I receptor that inhibited TGF‐β‐ induced EMT and fibrogenesis in db/db mice that showed significant diabetic nephropathy.Furthermore, oxymatrine, a bioactive alkaloid from Sophora japonica L., alleviated EMT induced by high glucose through inhibiting the TGF‐β1/ Smad signaling pathway in NRK‐52E cells, indicating that oxymatrine might be a therapeutic agent for diabetic nephropathy.Isoviolanthin, a flavonoid isolated from Dendrobium officinale Kimura et Migo, suppressed TGF‐β1‐ induced EMT through inhibiting the TGF‐β1/Smad and phosphatidylinositol 3‐kinase /Akt/mTOR signaling pathways in hepatocellular carcinoma cells.In addition, vitamin D, a well‐known inhibitor of EMT, inhibited EMT by negatively regulating the TGF‐β1 signaling pathway in human bronchial epithelial cells, and MART‐10 suppressed metastasis via down regulating EMT in pancreatic cancer cells.The NF‐κB signaling pathway is widely involved in multiple cellular activities, such as cell proliferation, apoptosis, invasion, and inflammation. NF‐κB plays a vital role in the inflammation of tissue fibrosis, and inhibiting NF‐κB activity reduced inflammation and enhanced recovery from CCl4‐induced liver fibrosis.In addition, NF‐κB contributed to inflammation, apoptosis, growth, migration, and invasion of cancer cells.Recently, it was found that the mediator of RNA polymerase II transcription, subunit 28 modulated EMT through NF‐κB in human breast cancer cells, which suggests that the NF‐κB signaling pathway also plays a key role in the process of EMT.Therefore, targeting the NF‐κB signaling pathway may be a good choice to treat fibrosis and tumors. Osthole, a dominant component in Cnidium monnieri Cuss., exhibited various biological activities including neuroprotective, osteogenic, cardiovascular protective, immunomodulatory, hepatoprotective, and antimicrobial effects. Osthole was found to block TGF‐β1‐induced EMT, adhesion, migration, and invasion by the inactivation of the NF‐κB/Snail pathways in A549 cells .In addition, osthole also attenuated insulin‐like growth factor‐ 1‐induced EMT by the PI3K/Akt pathway and inhibited hepatocyte growth factor‐induced EMT via the c‐Met/Akt/ mTOR pathway in human brain cancer cells.Pterostilbene is a stilbene containing in blueberries hat effectivelyblocked the EMT in breast cancer stem cells by the NF‐κB/miR‐488 circuit.Moreover, pterostilbene negatively regulated EMT and inhibited triple‐negative breast cancer metastasis via inducing the expression of miR‐205.

A far less common practice is to determine the effects of pesticides via oral exposure, in which the study organism is provided with a pesticide-treated food source. This approach has more frequently been used for testing pesticide toxicity among pollinators. Pesticides are added to a diet of sugar water and fed to pollinators, such as honey bees and bumbles bees . This type of bio-assay may be used to determine acute toxicity and/or effects on foraging behavior. With respect to natural enemies, a treated diet may be used to assess oral exposure. For instance, treated honey has been used to assess oral toxicity of several insecticides on parasitoids . Cryptolaemus montrouzieriis a mealybug predator used in integrated pest management programs for citrus. To determine non-target effects of acaricides, C. montrouzieri was provided treated prey, the citrus mealy bug Planococcus citri. Another key natural enemy in IPM programs is Coccinella undecimpunctata , a generalist predator of aphids. In order to evaluate how the neuroactive pesticides pirimicarb and pymetrozine may affect C. undecimpunctata consumption of aphids, the predator was provided a diet of treated Myzus persicae. As some pesticides are specifically designed for ingestion, oral exposure of natural enemies is an important factor to consider when assessing pesticide impacts. Historically, one of the most common toxicological measurements of pesticide exposure is the median lethal dose or the lethal concentration . Though acute toxicity assays are a good method for beginning to understand the effects of pesticides,stacking pots they are limited to the measurement of survivor ship only. Pesticides can induce a much wider range of sublethal effects that impair physiology or behavior .

Studies which consider sublethal effects are viewed as more accurately measuring toxic effects than those which only measure acute effects . For instance, when the parasitoid Aphidius erviwas exposed to lambda-cyhalothrin, the LD20 was found to be 50 times lower than the recommended field rate . Additionally, the study found, that even at such low concentrations, exposure to lambdacyhalothrin decreased the response of A. ervi females to its host aphid, M. persicae. This same parasitoid species also exhibited a change in response to plant-host odor when exposed to triazamate . Although it is worthwhile to determine lethal doses of pesticides for natural enemies relative to applied field rates, this measurement alone could not have predicted the disruptive behavioral effects on A. ervi females. Studies on sublethal effects can be used determine changes in all aspects of the life history performance of natural enemies that result from pesticide exposure. For instance, pesticides have been shown to significantly affect reproduction in the predatory mirid bugs, Nesidiocoris tenuis and M. pygmaeus . For N. tenuis, fresh residues of azadirachtin reduced fertility but those of spinosad did not. Conversely, for M. pygmaeus, spinosad reducedfertility, but azadirachtin did not. Another study found that methoxyfenozide significantly reduced fecundity of Aphidius colemani , while exposure to pure azadirachtin had no significant effect . Interestingly, a pesticide may display chronic toxicity, yet have no significant effects on reproduction. For example, exposure to any one of three insect growth regulators caused an 80% reduction in adult emergence of the lacewing C. externa, but there was no significant impact on the number of eggs laid per female or hatch rates . In this case, solely examining sublethal reproductive effects would have been misleading as a measure of potential demographic effects because the low adult emergence indicates otherwise. Therefore, it is best practice to link several effects of pesticides, lethal and sublethal, to estimate changes in the demography of natural enemy populations.

Demographic toxicology was initially used in 1962 to study the effects of gamma radiation on the intrinsic rate of increase Daphnia pulex . Since then, demographic toxicology has been increasingly employed, as it provides a more complete determination of the effects of a toxicant on the population growth rate of an organism rather than simply the acute effects on individuals . For example, using life table data , critical extinction thresholds were estimated for four economically important parasitoids: Diachasmimorpha longicaudata, Psyttalia fletcheri, Fopius arisanus, and Diaeretiella rapae . Though all species are braconid parasitoids, D. rapae was far less vulnerable to pesticide exposure, suggesting that risk assessments cannot be generalized for an entire guild. Another demographic study found that imidacloprid exposure reduced adult emergence of the egg parasitoid Trichogramma cacoeciae , yet mean longevity of emerged females or the mean number of female offspring per female was not significantly different from controls . In order to more fully understand the effects of pesticides, it is useful to measure both lethal and sublethal effects on natural enemies. Acute toxicity studies can be a useful starting point for determining lethal doses relative to recommended field rates. This is especially necessary when maximum field rates cause 100% mortality, making it difficult to study sublethal effects. Moreover, by determining both lethal and sublethal effects, we can begin to measure how these effects translate to changes in population dynamics. The accuracy of these tests can be increased even further by incorporating multiple routes of exposure, thereby predicting what actually may occur to natural enemies in a field setting. Taken together, the aforementioned bio-assays yield a more complete assessment of pesticide impacts on natural enemies, ultimately informing more effective integrated pest management strategies. The purpose of this dissertation was to assess the effects of reduced-risk pesticides on Hippodamia convergens. This coccinellid is an important predator in high-value tree crops in western North America, namely apples, pears, and walnuts. Hence, the pesticides included in this study are those that are commonly used in these cropping systems. In order to more fully understand pesticide impacts, several different assessment methods were used and are described below.

There were three primary objectives, each one addressed in a different chapter. For more than a decade, regulatory limitations have caused a reduction in the use of organophosphates in the U.S. as successive products have been restricted or can celled according to the Food Quality Protection Act . Additionally, the U.S. EPA’s Reduced-Risk Initiative has created a shift away from OPs toward other classes of insecticides . The goal of this initiative was to encourage the development and registration of insecticides that, compared to OPs, pose lower human and environmental health risks. This included lower health risks to mammals, aquatic organisms, birds, and other non-target organisms, such as beneficial insects . In principle, this initiative would be expected to enhance the potential for biological control of arthropod pests by the natural enemies that had previously been limited by the use of OPs. However, while reduced-risk insecticides may lower human and environmental health risks, they do not necessarily have lower toxicity to natural enemies . It is important to determine the selectivity of insecticide alternatives to OPs, particularly to natural enemies that contribute effectively to pest management . For instance, several reduced-risk insecticides showed acute toxicity to important natural enemies in high-value tree crops in western North America. Spinetoram and spirotetremat were toxic at recommended field concentrations to the predatory mite Galendromus occidentalis. Acetamiprid was toxic to Deraeocoris brevis, a natural enemy of pear psylla, Cacopsylla pyricola. Furthermore,strawberry gutter system novaluron and lambda-cyhalothrin were highly toxic to larvae of two different green lacewing species, Chrysoperla carneaand Chrysoperla johnsoni Henry, Wells and Pupedis . On the other hand, some reduced-risk insecticides appear to be compatible with certain natural enemy species, demonstrating low toxicity. Chlorantraniliprole had no significant effects on survival, parasitism rates, or emergence of seven different species of parasitoid wasps, including two sensitive indicator species, Aphidius rhopalosiphiand Trichogramma dendrolimi. In addition, it had no significant impact on the mortality of the predatory ground beetle Harpalus pennsylvanicus DeGeer when fed treated food or on parasitism rates of the ectoparasitoid, Tiphia vernalis Rohwer when treated directly. Another diamide insecticide, cyantraniliprole, had no significant impact on the mortality of Tamarixia triozaeexposed to residue on either a glass or leaf surface , and caused only 23% mortality of nymphs and no significant mortality of adults of the predatory mirid, D. brevis . For many natural enemies, the selectivity of insecticide chemistries is not yet clear. The primary objective of this study was to determine the acute toxicity of a range of pesticides for Hippodamia convergens, a well-known generalist aphid predator in a wide variety of agricultural systems . H. convergens is especially prevalent in western North America , where it serves as an indigenous biological control agent for aphid pests in high-value tree crops .

As for other natural enemy species, reduced-risk pesticides have also shown acute toxicity to H. convergens. In laboratory and greenhouse bio-assays, the two neonicotinoid insecticides, acetamiprid and imidacloprid, were toxic to H. convergens , and indoxacarb residue was highly toxic even when aged for 14 days . In this study, seven pesticides were tested that are commonly used in high-value tree crops in western North America . These included five insecticides used for management of key pests such as codling moth, Cydia pomonella, plus two pesticides used for management of bacterial or fungal pathogens in these crops. Each pesticide was tested for acute toxicity using laboratory bio-assays for each of three different exposure routes. Natural enemies, such as H. convergens, can experience topical exposure from direct applications of pesticides, residual exposure from a treated plant surface, and/or oral exposure from ingestion of a treated food source . The objectives of this study were 1) to assess the acute toxicity of selected pesticides on two life stages of H. convergensand 2) to determine the difference in acute toxicity through three different exposure routes: oral, residual, and topical. Adult H. convergens were obtained from a commercial source and stored for up to two months in a screened wooden cage at 6 °C in order to maintain them in a state of overwintering hibernation. To prevent desiccation, several 8.5 cm petri dishes lined with cotton wool were included in the cage and sprayed with distilled water on a weekly basis. Prior to use in the laboratory bio-assays, adults were removed from hibernation storage and placed into an incubator maintained at 22 °C, 60-70% RH, and a 16:8 h photoperiod. Males and females were identified and approximately 20 adults were placed into 96.1 ml plastic cups . Cups were lined with cotton wool soaked in a 1:1 honey-water solution and covered with a perforated plastic lid to allow for ventilation. After a period of 24 h under these conditions, adults were then used for bio-assays. To obtain larvae of H. convergens for the laboratory bio-assays, adults were removed from cold storage, sexed, and individual mating pairs were placed into 96.1 ml cups, capped with perforated plastic lids for ventilation, and assigned to the same incubator. These adults were reared on a diet of pea aphids, Acrythosiphum pisum, which are known to provide a high quality diet for H. convergens . 20 adult A. pisum were provided to each mating pair every day. After 3-4 days, males were removed from the cups and the remaining females were fed approximately 20 adult A. pisum per day. When a female oviposited, it was moved to a new plastic cup, and when eggs hatched, the first instar larvae were fed a diet of A. pisum, ad libitum, for a period of 48 h before being used in bio-assays. For each of the seven pesticides tested in comparison to a control, three variables were manipulated 1) life stage , 2) pesticide concentration , and 3) exposure route . For each bio-assay, a minimum of 28 replicates was used. In bio-assays using adults, the gender ratio was 1:1 . For all bio-assays, test insects were placed individually in 15 x 45 mm glass vial arenas, with cotton wool placed over vial openings to allow for ventilation. Insects were kept at 22 ⁰C, 60-70% RH, and 16:8 h photoperiod. Bioassays lasted for a period of 48 h, after which acute effects were recorded. Acute effects were estimated as the number of live and dead or moribund insects , where moribund insects were those that were unable to right themselves. For adult bio-assays, acute effects were recorded separately for each gender.

Targeted application of insecticide has the potential to reduce pesticide usage in conventional farming systems yet maintain an effective control over agricultural pests. Focusing spray along field borders can also potentially address the issue of spray coverage, one of the main problems faced in field-wide treatment of pesticides that severely reduce the efficacy of applied chemicals . An example of successful targeted application of insecticides would be the recent efforts to control brown marmorated stink bug, Halyomorpha halys Stål , in Northeastern USA by focus spraying along edges in peach orchards instead of orchard-wide application . The results are promising as pesticide usage was reduced by 25–61% using this method and the damage on peach caused by brown mamorated stink bugs was also reduced when compared with the common standard practices. In addition, better understanding of edge-biased distributions may be used to optimize deployments of anti-insect nets in the orchard industry . Finally, there is a growing body of literature on the potential use of banker plant systems . In these agricultural systems, the agroecological landscape is manipulated through integration of special plants along crop banks. These banker plants serve several purposes, including nectar and pollen for natural enemies, host plants for alternative prey of natural enemies, and shelter and oviposition sites for natural enemies. Thus,hydroponic nft banker plant systems represent ways to enhance the performance of biological control of crop pest. A very important aspect of banker plant systems is that the landscape manipulation is conducted along field edges, so edge-biased distributions of many insect pests are a contributing factor to the growing interest in this type of agricultural systems.

There are multiple ways in which removal of infected host plant tissue can be employed as an element of disease management. These include removal of reservoir hosts to limit pathogen spillover onto a focal host , roguing of infected focal hosts to limit secondary spread , and removal of localized infections within hosts to limit further infection or to retrain an unproductive plant . Studies of bacterial pathogens in perennial crops have evaluated the utility of pruning as a disease management tool, with mixed results . The removal of infected plant tissues is analogous to measures used for management of trunk diseases, often referred to as “remedial surgery,” as an alternative to replacing infected plants . In this study, we investigated whether severe pruning of Xylella fastidiosa-infected grapevines in commercial vineyards could clear vines of existing infections. Pierce’s disease is a lethal vector-borne disease of grapevines caused by the bacterium X. fastidiosa . After susceptible plants are inoculated by X. fastidiosa, pathogen populations multiply and move through the xylem network, leading to symptoms of reduced water flow , including leaf scorch, cluster desiccation, vine die back, and eventually death. There is no cure for grapevines infected with this bacterium; current strategies for management of PD in California vineyards involve limiting pathogen spread to uninfected vines by controlling vector populations, disrupting transmission opportunities, and eliminating pathogen sources in the surrounding landscape . PD is notable for the numerous sources of variability in infection levels and symptom severity in plants. X. fastidiosa infection levels vary among plant species , grapevine cultivars , seasons , and as a function of temperature .

Like other bacterial plant pathogens , X. fastidiosa is often irregularly distributed within individual hosts. For example, X. fastidiosa infection levels in grapevines may vary by more than 10-fold between grapevine petioles and stems ; in other hosts, infection levels may vary by more than 100-fold between basal and apical sections of shoots . This within-host heterogeneity may be epidemiologically significant if it affects pathogen acquisition efficiency . Moreover, if such variation is associated with protracted localized infection near inoculation points, such heterogeneity may facilitate other disease management tactics. In addition to grapevines, other plant species that are susceptible to X. fastidiosa infection include citrus in South America . Management of the resulting disease in C. sinensis relies on clean nursery stock, vector control, and pruning infected plant tissue from established trees or roguing young plants . The concept of pruning of infected plant material is based on the fact that, in established trees , tissue with early symptoms of infection can be pruned ~1 m proximal to the most symptomatic basal leaf, effectively eliminating infections, as the remaining tissue is free of X. fastidiosa . However, pruning is not adequate for young trees or for removing bacterial infections if any symptoms are present in fruit . X. fastidiosa multiplies and spreads through the xylem vessels, reaching the roots of perennial hosts such as citrus , peach , alfalfa , and blueberry . Nonetheless, under field conditions, chronic infection of grapevines is temperature and season dependent. In regions with freezing winter temperatures, infected plants can recover in winter, curing previously infected and symptomatic grapevines . Infections that occur during spring lead to chronic disease ; however, infections that occur during late summer and fall may cause disease symptoms in the current year, but a high proportion of vines lack symptoms of X. fastidiosa infection in the following year . The biological mechanism behind this winter recovery has been studied but is not fully resolved.

Nonetheless, models that incorporate low temperatures have substantial explanatory power in predicting rates of winter curing of X. fastidiosa infections in grapevine . Infections that occur early in the season may have a longer period during which X. fastidiosa can colonize and reach high infection levels, which may increase the likelihood of the disease surviving over the winter. Following this rationale, if most late-season infections remain in the distal ends of shoots and have lower infection levels, removing the symptomatic portion of the vine might eliminate X. fastidiosa. In other words, the efficacy of pruning infected grapevine tissue could depend on both the time of year in which the plant was infected and on winter temperature. A potential benefit of severe pruning versus replanting is that pruning leaves a mature root stock in place, which is likely to support more vigorous regrowth compared to the developing root stock of a young transplant . Recent attempts to increase vine productivity by planting vines with more well-developed root systems are based on this presumption. However, even if severe pruning can clear vines of infection,hydroponic channel it removes a substantial portion of the above ground biomass of the vine. Thus, a method for encouraging rapid regrowth of the scion after aggressive pruning is needed. We studied the efficacy of pruning infected vines immediately above the root stock graft union—the most aggressive pruning method—for clearing grapevines of infection by X. fastidiosa. We reasoned that if such severe pruning was ineffective at clearing vines of infection, less severe pruning would not be warranted; if severe pruning showed promise, less severe pruning could then be tested. We use the term “severe pruning” to refer to a special case of strategic pruning for disease management, analogous to the use of “remedial surgery” for trunk diseases . To test the efficacy of clearing vines of X. fastidiosa infection, we followed the disease status of severely pruned versus conventionally pruned vines over multiple years, characterized the reliability of using visual symptoms of PD to diagnose infection, and compared two methods of restoring growth of severely pruned vines.Pruning trials were established in Napa Valley, CA in commercial vineyards where symptoms of PD were evident in autumn of 1998. The vineyards used for these trials varied in vine age, cultivar, and initial disease prevalence . All study vines were cordon-trained and spur-pruned. We mapped the portions of the six vineyards selected for study according to evaluation of vines for disease symptoms. The overall severity of PD symptoms for each vine was recorded as follows: 0 = no symptoms, apparently healthy; 1 = marginal leaf scorch on up to four scattered leaves total; 2 = foliar symptoms on one shoot or on fewer than half of the leaves on two shoots on one cordon, no extensive shoot die back, and minimal shriveling of fruit clusters; and 3 = foliar symptoms on two or more shoots occurring in the canopy on both cordons; dead spurs possibly evident along with shriveled clusters. To test the reliability of the visual diagnosis of PD, petiole samples were collected from the six vineyard plots when symptom severity was evaluated for vines in each symptom category; these samples were assayed using polymerase chain reaction . Petioles were collected from symptomatic leaves on 25, 56, and 30 vines in categories 1, 2, and 3, respectively.Next, severe pruning was performed between October 1998 and February 1999 in the six vineyard plots by removing trunks of symptomatic vines ~10 cm above the graft union.

During a vineyard survey, severe pruning was conducted on 50% of vines in each symptom category; the other 50% of vines served as conventionally pruned controls. Sample sizes for control and severely pruned vines in each disease category ranged between six and 62 vines depending on the plot, with at least 38 total vines per plot in each control or pruned treatment. In spring 1999, multiple shoots emerged from the remaining section of scion wood above the graft union on severely pruned vines. When one or more shoots were ~15 to 25 cm long, a single shoot was selected and tied to the stake to retrain a new trunk and cordons, and all other shoots were removed at this time. We evaluated the potential of severe pruning to clear vines of infection, by reinspecting both control and severely pruned vines in all six plots for the presence or absence of PD symptoms in autumn 1999 and 2000. In all plots, category 3 vines were inspected in a third year ; in plot 6, vines were inspected an additional two years . Finally, in plot 6 we investigated chip-bud grafting as an alternate means of ensuring the development of a strong replacement shoot for retraining. To do this, 78 category 3 vines were selected for severe pruning, 39 of which were subsequently chip-bud grafted in May 1999. An experienced field grafter chip budded a dormant bud of Vitis vinifera cv. Merlot onto the root stock below the original graft union, and the trunk and graft union were removed. The single shoot that emerged from this bud was trained up the stake and used to establish the new vine. The other 39 vines were severely pruned above the graft union and retrained in the same manner as vines in plots 1 to 5. Development of vines in plot 6, with and without chip-bud grafting, was evaluated in August 1999 using the following rating scale: 1) “no growth”: bud failed to grow, no new shoot growth; 2) “weak”: multiple weak shoots emerging with no strong leader; 3) “developing”: selected shoot extending up the stake, not yet topped; and 4) “strong”: new trunk established, topped, and laterals developing.All analyses were conducted using R version 3.4.1 . We used a generalized linear model with binomial error to compare the relative frequency of X. fastidiosa-positive samples from vines in the different initial disease severity categories . Next, we analyzed the effectiveness of chip budding versus training of existing shoots as a means for restoring vines after severe pruning. This analysis used multi-nomial logistic regression that compared the frequency of four vine growth outcomes the following season: strong, developing, weak, or no growth. This main test was followed by pairwise Fisher exact tests of the frequency of each of the individual outcomes between chip budded-trained and trained vines . We analyzed the effect of severe pruning on subsequent development of PD symptoms using two complementary analyses. First, we compared symptom return between severely pruned and control vines in the three symptom severity categories for two years after pruning. To appropriately account for repeated measurements made over time, our analysis consisted of a linear mixed-effects model with binomial error, a random effect of block, and fixed effects of treatment , year , and symptom severity category . Next, we analyzed the rate at which PD reappeared in only severely pruned vines from category 3 in subsequent years using a survival analysis. Specifically, we used a Cox proportional hazards model with a fixed effect of plot .