Horticulture

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

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

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

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

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

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

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

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

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

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

The key to PLV’s success is a curriculum that has evolved to meet the needs of its female population in order to effectively provide guidance and support to group members. Topics covered by weekly PLV sessions include cardiovascular risk reduction , tobacco use prevention , Cancer Screening and Prevention , Breast Cancer Education , Nutrition and Cancer , and Family Communication, Parenting Skills and Community Resources . I had the pleasure of contacting PLV and recruiting a Consejera to come to the Tijuana clinic to teach a session . This was the first class of our “Mujeres Protegidas” series that covered topics such as female anatomy, menstrual cycles, body perception, and nutrition. A total of 5 women were in attendance, 4 of which were sex workers. The session took place in one of the upstairs rooms of the HFiT clinic that comfortably fit the participants, our PLV Consejera Gloria,square plastic planter and two observers from Flying Samaritans who were hoping to bring a similar program to their clinic in Ensenada. We began the class with a consent form and pre-survey to assess general knowledge on the topics we would be discussing during the class. We then began the discussion on female anatomy utilizing teaching aides provided by Por La Vida.

Our group instructor Gloria went above and beyond to make class enjoyable and very informative for the women. Gloria’s presence made the women feel comfortable and many were willing to share personal stories and experiences. The women were given diagrams of female anatomy and pocket calendars so they could chart their menstrual cycle as well as any reflections for the day. We concluded the class with a healthy snack with recipes to take home to their friends and family. We ended the class with questionnaires assessing how the women felt the day went. 5/5 of the questionnaires expressed high interest level in the topics discussed and that they learned a lot of new information from the day’s session. As part of our women’s health curriculum we also realized the importance of training medical students and maintaining participation in the student-run clinic. We will expand clinic participation through both the HFiT elective and the newly established HFiT student interest group. The HFiT elective includes a didactic course with a curriculum developed by clinical faculty from both UCSD and UABC. The course thread incorporates clinical shadowing. Lectures occur before clinic starts on Saturday and include topics such as health disparities and policy in Mexico, substance abuse in Zona Norte, and medical history taking. At clinic, medical students receive hands on training from attending physicians on how to perform a PAP smear and other gynecological exams. This is a very exciting opportunity since most medical students don’t receive training in this area until 3rd year clerkships. In addition, with our partnership with the Scripps Family Medicine program, we successfully completed an IUD placement workshop on 10/30/13.

Under the instruction of Dr. McKennett, medical students learned more about the different types of contraception and their application. All events were organized by the HFiT student interest group, which will strive to promote awareness and interest in the Tijuana clinic. Below is a picture from UCSD School of Medicine Activity Fair where incoming and current medical students can sign up for the HFiT interest group. Since the initiation of this project, numerous changes have challenged our progress. First of which, the clinic site was move to the Desayunador Salesiano “Padre Chava,” soup kitchen. The new site offers increased space and a large pool of potential patients. Coinciding with the clinic site change, an influx of Haitian migrants arrived in Tijuana. They are seeking refuge in the United States from political unrest in Haiti and the aftermath of Hurricane Matthew. Unable to keep up with the growing number of Haitians arriving at the border, the USA has recently reinstated deportations, a change from a 2010 policy, which accepted all Haitians. Fearing deportation, many Haitians are forced to wait in Tijuana to regroup with family and loved ones. Mexico has done its part in aiding these migrants who have often traveled far distances and put everything at stake to arrive at the border. Padre Chava has been transformed to house Haitians overnight. At one point, the space provided shelter for over 500 people with mattresses overflowing to the street in order to accommodate all the migrants. The women of the HFiT clinic needed an independent space where they could congregate and continue weekly sessions. As a result, a support group of 20 women meets every Saturday at a new clinic space close to the Zona Norte. The women take turns leading discussion in this dynamic group. Many of the women belong to marginalized populations in Tijuana. Similar to the design of the PLV sessions, the premise of this group is empowerment through knowledge. In support of the HFiT women’s group, during the week of February 6th, 2017, the UCSD SOM coordinated a drive to assist in items of basic hygiene and care. Feminine products were donated throughout the week culminating into the final collection at the Winter AC cup. The SOM donated over 100 items through the initiative of the medical students.Despite the changing global health environment that challenged this project, establishing the support services for women in Tijuana allowed me to work directly with the HFiT patient population in the exam room, in the classroom, and on the streets where they worked.

It gave me the opportunity to hear personal experiences and understand their concerns and health needs. Overall the expansion of the HFiT clinic has provided medical attention to hundreds of patients often stigmatized by the health care system. This experience enhanced my understanding of humanistic medicine. It allowed me to address the needs of a population that is often overlooked. Spending time to understand primary health concerns will allow more focused medical attention and assist in breaking down the barriers to medical care in Tijuana, Mexico. We must not forget the power of empathy in all relationships: physician to patient, “consejera” to support group, and peerto-peer. Nor should we forget how knowledge can empower individuals to take ownership of their health. It is experiences like these that remind us why we chose the field of medicine. Pomegranate is an ancient fruit and a commodity favored by people worldwide for its unique shape and flavor. Pomegranate peel is a major by-product from the juicing process with rich health-promoting bio-active compounds but remains underutilized or disposed of. The main goal of this dissertation was to study the extraction of functional ingredients from pomegranate peel waste, evaluate their nutritional benefits, and develop value-added nutritional products. A novel green process for antioxidant extraction from wet pomegranate peel was developed. The effects of various extraction conditions on polyphenol yield and quality were investigated, including particle size, time, temperature, and solvent ratios. WPP extraction at 20°C for 6 min with a solvent ratio of 4:1 is recommended as an economic and sustainable process, resulting in 10.53% total phenolic yield with 6.35 g g-1 tannic acid equivalent and 88.93% punicalagin purity. Plant bio-active compounds have demonstrated promising effects in promoting health. Therefore, the hypolipidemic effects of pomegranate peel powder and pomegranate peel extract were researched via Lakeview Golden Syrian hamsters with a high-fat diet. PPP and PPE significantly improve obesity-related indices, including adjusting microbiota composition, increasing the microbiota diversity towards a leaner type, and down-regulating the expression of 2 genes to reduce cholesterol ingestion and LDL uptake. However, further research on potential toxicity was required,square plastic plant pot as adverse plasma LDL-elevating effects were observed at a higher dose of PPP and PPE intake. As functional food fortified with plant bio-active compounds became more popular, the potential of PPE as ingredients for fortified food supplementation was evaluated. A PPEfortified Greek Style yogurt was developed using a top-down formulation method based on USDA dietary guidelines. According to the results from response surface methodology, the optimum formula for a 130 g product consisted of 10.4 g of protein and 77 g of PPE, which could satisfy the polyphenol daily need while maintaining pleasant product properties. An organic acid-based ultrasound-assisted co-extraction process of polyphenol and pectin was investigated for thorough WPP utilization and broader industry applications.

Citric acid was applied for extraction since it’s a common GRAS acid in food applications. Process conditions, including pH, extraction temperature, extraction time, ultrasound intensity, and solvent ratio, were evaluated via Box-Behnken design to study the effects on extraction performance, including polyphenol/pectin yield, antioxidant activity, and pectin characteristics. High methylated pectin yield was as high as 13.99%, which demonstrated that WPP could be utilized as a reliable pectin source. The extraction kinetics were characterized for future industry application guidance. In conclusion, this dissertation demonstrated the potential of pomegranate peel to be utilized as a source of healthy and bio-active compounds. With the depletion of natural resources and growing populations, there is a great motivation for recovering valuable compounds from fruit and vegetable wastes and converting them into functional ingredients as a part of sustainable agricultural practices. Pomegranate is an important California commodity and pomegranate by-products are a large portion of the process. These by-products could be repurposed into valuable compounds, but they are not unutilized or underutilized, ending up in landfills or animal feeds. In this dissertation, physiochemical properties and extraction methods of bio-active compounds in pomegranate peel were comprehensively studied to determine the feasibility of converting these pomaces into healthful food ingredients. The research objectives were to identify the physiochemical characteristics of pomegranate peel and assess different size reduction methods to obtain suitable peel particles for effective extraction by using water as a solvent; evaluate the effects of extraction temperature, time, and water usage on the phenolic yield, phenolic composition, and antioxidant activity to optimize the extraction conditions; study the bio-active properties and nutritional benefits of pomegranate peel powder and extract through animal models; investigate the characteristics of food products developed with pomegranate peel powder and extract; and study the co-extraction mechanism of pectin and polyphenol to improve extract yield and phenolic stability. There are 6 chapters in this dissertation. Chapter 1 includes an introduction, research objectives, and literature review on the processing and waste utilization of pomegranate fruits, with a focus on the current waste management practices and effects of punicalagin, which is the most unique polyphenol in pomegranate. In Chapter 2, the physicochemical properties of pomegranate fruits were investigated to explore the potential of recovering bio-active food ingredients from the inedible fruit peel. Phenolic extraction parameters and effects of wet and dried pomegranate peel were studied, aiming to increase the extraction sustainability with reduced cost. The study in Chapter 3 investigated the potential of developing functional food fortified with a value-added ingredient from pomegranate peel extract, specifically Greek Style Yogurt. Products with different levels of protein content and phenolic content were formulated to study the chemical and sensory properties, which provided solid guidance for future product development. In the research of Chapter 4, the hypolipidemic properties of pomegranate peel and extract were investigated in vivo using hamster models. Currently, no literature is available on the functionality of complete pomegranate peel and extract. In this study, it was found that the pomegranate peel and extract were effective in lowering blood cholesterol in hamsters fed with high-fat diets. The study in Chapter 5 studied the co-extraction mechanism of pectin and polyphenol for improved extract yield and phenolic stability. Ultrasound extraction of pectin and polyphenol in citric acid from pomegranate peel was tested and the corresponding physicochemical properties were evaluated. Unlike most of the research using inorganic acid for extraction, this research utilized GRAS organic acid and provided functional ingredients from fruit by-products without safety concerns. Conclusions and future directions on reutilizing the pomegranate waste as food ingredients are detailed in Chapter 6. Pomegranate belongs to the family Punicaceae. It has been grown since ancient times for its delicious fruit and as an ornamental plant for its red, orange, or occasionally creamy yellow flowers. The estimated global cultivation area for pomegranate is about 300,000 ha, with fruit production of 3.0 million metric tons . Spanish missionaries brought pomegranate to the Americas in the 1500s . Wonderful, a primary cultivar in the U.S., was discovered in Florida and brought to California in 1896. Since then, pomegranate has been grown abundantly in California and Arizona, where mild winters enable the fruits to reach the quality necessary for successful commercial production.

Membranes that separate compartments are rendered as gray bars, with both sides labelled, and transporters are shown as breaks in the gray bar with pairs of brown ovals on either side to suggest a channel. This new feature makes intracellular transport within pathways clearer and easier to visualize. PMN 15 is an extensive and regularly-updated database of compounds, pathways, reactions, and enzymes for 126 plant and green algae species and subspecies as well as a pan-species reference database called PlantCyc. We examined the qualityof the data contained in the databases by assessing the accuracy of pathway prediction via manual validation of a randomly-selected subset of predicted pathways. Using two publicly available transcriptomics datasets, we demonstrated how PMN resources can be leveraged to characterize and gain insights from omics data. The present work demonstrates that the Plant Metabolic Network can be a useful tool for various analyses of plant metabolism across species. PMN 15 differs from other metabolic pathway databases in several ways: the quantity of curated and computational information,25 liter plant pot its comprehensive set of tools, and its specific focus on plants. Other, comparable databases include KEGG , Plant Reactome , and Wiki Pathways .

Like PMN, these databases contain metabolic pathways along with their associated reactions, compounds, and enzymes. KEGG pathways represent broad metabolic reactions shared among many organisms, and it is common to map genes or compounds to KEGG pathways alongside Gene Ontology annotations for enrichment analyses. However, because KEGG pathways represent a generalized set of reactions leading to many possible compound classes , it lacks the granularity to analyze metabolism on a species-specific level . For example, a recent study identified enriched KEGG pathways among genes belonging to gene families that were expanded in Senna tora compared with its relatives . Enrichment analysis of the same genes using PMN’s StoraCyc 1.0.0 identified individual phenylpropanoid biosynthetic pathways enriched among the gene set, such as coumarin biosynthesis. PMN and MetaCyc feature structured data that is both human- and machine-readable, making it possible for users to obtain pathway structure and other data for their own offline analysis and enabling features such as the Pathway Co-Expression Viewer to be easily incorporated. Wiki Pathways is another pathway centric database. Wiki Pathways is not plant-focused, and takes a crowd-sourced approach, in contrast with PMN’s focus on expert curation. Plant Reactome, another metabolism database, is specific to plants and green algae as PMN is. However, Plant Reactome uses Oryza sativa as a reference species to predict reactions and pathways to the 106 other species currently in the database and uses gene orthology to predict the presence of a pathway, where a pathway is predicted in a species if at least one rice ortholog for an enzyme in that pathway is present in that species . Pathway prediction in PMN, on the other hand, is more stringent via its implementation through the PathoLogic and SAVI pipelines.

The ability of PMN to enable research is dependent on the accuracy of its data. We therefore evaluated the performance of PMN’s metabolic reconstruction pipeline both in its entirety and using only computational prediction. The manual pathway validation revealed a number of pathways predicted to be present outside of their known taxonomic range, such as momilactone’s predicted presence across Poaceae despite being known to exist only in rice and a few other species, some outside of Poaceae . While some of these results may reflect compounds that are, in fact, more widely distributed than currently thought, many such cases likely result from inaccurate prediction of enzymatic function by E2P2. The performance of enzyme function prediction using a sequence similarity approach can suffer when dealing with highly similar enzymes of a shared family . In cases like momilactone, where the pipeline has predicted the pathway in species closely related to species known to possess it, it may be the case that the predicted species do have most of the enzymes necessary to catalyze the pathway, but that one or a few of the predicted enzymes actually have a different function in vivo. This may draw attention to cases where enzymes have gained new functions and allow for exploration of how enzymes evolve. Meanwhile, cases of universal plant pathways being predicted only in Brassicaceae may indicate the pitfalls of an overemphasis on Arabidopsis in curation and research, as key enzymes might be predicted less reliably outside of this clade. This might be the case if there are Brassicaceaespecific variations that may result in a failure to reliably predict orthologs. A focus on curating information from diverse species may improve the accuracy of the computational prediction, requiring less semi-automated curation to fix such errors. Additionally, incorporating evidence from recently published species-specific metabolomics reference datasets may help corroborate PMN’s prediction of metabolites, for which there is currently little experimental support . Pathway misannotation in the naïve prediction pipeline could also be the result of PathoLogic’s incorrect integration of enzyme annotation with reference reactions.

In addition to incorporating enzyme predictions, PathoLogic can infer pathways for a given species based on a number of additional considerations. For example, if a species contains an enzyme which catalyzes a reaction unique only to one pathway in the PGDB, the pathway is likely to be predicted to be present. Additionally, if all reactions of a pathway are predicted to be present, the pathway is likely to be predicted as. Using PathoLogic without taxonomic pruning thus provides increased prediction sensitivity while also increasing false positives . By design, SAVI removes false-positive and adds false-negative pathways predicted by PathoLogic. Our analyses indicate that the predominant function of SAVI and PathoLogic’s taxonomic pruning currently is to remove false-positives and consequently restrict the taxonomic range of predicted pathways, consistent with previous analyses of SAVI’s performance . Interestingly, our manual pathway assessment revealed that, in certain cases, SAVI should have increased the range of a predicted pathway and added it to more species than it was predicted for by PathoLogic. For example, the phytol salvage pathway is predicted to be present in all photosynthetic organisms . While PathoLogic incorrectly restricted the predicted range of this pathway to include only angiosperms even without taxonomic pruning, SAVI did not correct this incorrect taxonomic restriction, nor did it assign the pathway to the few angiosperm species not predicted by PathoLogic to contain the pathway. Examples like this may represent errors in the manual curation decisions used by SAVI to make its correction, or it may reflect new information added to the literature after those curation decisions were made. Both possibilities represent important information in accurately representing metabolism across species and highlight the need to regularly update the curation rules upon which SAVI operates. We therefore reclassified the final pathway assignments in PMN 15 for each pathway whose classification after SAVI implementation was determined to be anything other than “Expected”. Through the continual process of introducing new species — and thus new pathways — into PMN, along with regular curation of those new pathway predictions, SAVI’s correction performance,black plastic plant pots and thus the overall value of data in PMN, should continue to improve over time. The results of the manual pathway validation suggest that additional systematic manual checks and validation may be productive. The manual validation reported here focused on the phylogenetic distribution of pathways, but this is only one aspect of the data found in PMN. Future reviews will focus on reviewing the previously-curated data in plant-specific pathways, both to review for accuracy and to check for research published after the pathway was last updated that may have been missed by curators when it was published. Semi-automated curation could also play a role; nearly half of PMN compounds, for example, do not have ChEBI links, and scripts could be written to identify ChEBI and other external links like this that should be added, to be vetted by curators before inclusion in PMN. PMN is organized primarily by species, and a significant component of the expansion over its history has been in the form of adding new species and subspecies to it. In order for this to be a worthwhile endeavor and useful to the plant biology research community, the species databases need to be meaningfully differentiated from one another in ways that accurately reflect their metabolic differences. Multiple correspondence analysis was therefore performed to determine whether related species would cluster together, an indication that underlying biology is driving the differences in their database contents.

The analysis revealed that some plant groups such as Brassicaceae, Poaceae, the green algae, and non-flowering plants each clustered together, showing that these major groups of plants can be readily differentiated based on their metabolic complements. Within the eudicots, however, there was little separation apart from the grouping of Brassicaceae. Other groups such as Rosaceae and Solanaceae did not separate from the other eudicots, even though both groups are known to have unique metabolism, suggesting that more research and curation on members of these groups is needed. This analysis also indicated that despite being represented by a number of PMN species, the unique metabolisms of these groups remain understudied. The separation of Brassicaceae from the other groups may reflect a more comprehensive body of knowledge about the metabolism of Arabidopsis due to its status as a model plant and, as a result, a larger number of Brassicaceae specific pathways being known than for compounds specific to other clades. This is reflected in the large percentage of pathways and enzymes in PMN that are curated to the species. The same might be true of the grasses, a clade that contains economically important crops such as maize, rice, wheat, and switch grass. These results suggest that study of representative members of a group could help differentiate the group as a whole and suggest that much of current knowledge is limited to common pathways. The focus on Arabidopsis in the database also carries a risk of biasing studies that utilize the PlantCyc database as a source, though as this reflects a similar bias in plant research in general this issue may not be limited to PlantCyc and PMN. More detailed studies of the metabolism of other groups are needed to understand what makes them unique. PMN has been used in a variety of ways by the plant research community. One common use is to find metabolic information about a specific area of metabolism, such as finding sets of biosynthesis genes for a particular compound or sets of compounds under study, or finding pathways associated with a set of genes highlighted by an experiment. Clark and Verwoerd used AraCyc to determine different biosynthetic routes for anthocyanin pigments and to predict minimal sets of genes which could be mutated to eliminate pigment production. Pant et al. performed metabolite profiling on phosphorus-deprived Arabidopsis wild type plants and phosphorus-signaling mutants. PMN was used to find genes in the biosynthetic pathways of metabolites which showed altered concentration in the mutants and P-deprived plants. Saptari and Susila examined the expression of hormone biosynthesis genes during somatic embryogenesis in Arabidopsis and rice. The authors used PMN to identify hormone biosynthetic genes and performed expression analysis on the identified gene set. Kooke et al. used AraCyc to identify genes involved in glucosinolate and flavonoid metabolism, and then examined the relationship between methylation of these genes and metabolic trait values. Uhrig et al. examined diurnal changes in protein phosphorylation and acetylation, and used PMN’s pathway enrichment feature to identify AraCyc pathways enriched for proteins associated with these protein modification events. A second common use of PMN is to study broader patterns in plant metabolism. Hanada et al. explored two rival hypotheses which attempt to explain the large number of Arabidopsis metabolic genes for which single mutantsshow weak or no phenotypes, and used data from PMN to determine the connectivity of different metabolites in the network. Chae et al. compared primary and specialized metabolism in plants and green algae and found that specialized metabolism genes have different evolutionary patterns from primary metabolism genes. Moore et al. used AraCyc in assembling lists of enzymecoding genes involved in primary and specialized metabolism, and then explored associations between various qualities and metrics of the genes and their involvement in primary or specialized metabolism. The PlantClusterFinder software was also used in that analysis. Song et al. set out to test the hypothesis that stoichiometric balance imposes selection on gene copy number. AraCyc pathways were used as a source of functionally-related gene groups to test for reciprocal retention.

We set up the analysis in Section 2 and provide a brief background on the data sets in Section 3. In Section 4.1, we compare results from the sub-canopy fires and in Section 4.2, we highlight the attributes that distinguish the grassland fire from the sub-canopy surface fires, thereby synthesizing the key insights from each of the three scenarios considered here. As with most data collected from prescribed burns, the current data were also occasionally influenced by wind variability. We take the opportunity to comment on the effect such variability may have on the fire while simultaneously attempting to summarize the general features that broadly characterize each scenario.We take this opportunity to comment on the choice of 1 hour moving averages in the analysis of the sub-canopy surface fires. Moving averages are computed in this work, instead of block averages, in order to preserve the resolution of the data and the smoothness of the signal. The selection of a 1 hour time period for computing the moving averages can be explained as follows. It has been documented in the literature that relatively long time periods are reasonable in computing eddy fluxes. To quote from Chapter 2 of the Handbook of Micrometeorology : ‘‘Eddy fluxes need to be formed over a sufficiently long time that any motions that contribute to the transport can be sampled adequately.

In practice,drainage collection pot this has meant that eddy fluxes have been calculated over time periods up to an hour in duration, sufficient for several of the largest planetary–boundary–layer scale to be sampled by the measuring system.’’ While this argument was made in the context of turbulence in the absence of a fire, it provides a reasonable estimate for the choice of the time period of averaging in the present study. Furthermore, for the backing fires of 2011 and 2012 discussed in this study, it was found that while the time required for the fire lines to pass the tower locations was of the order of minutes, the influence of the turbulence induced by the fire lines at the towers lasted for approximately an hour . One-hour moving averages generate a sufficiently smooth slowly-varying component for the velocity and temperature signals, which can be treated as the larger-scale atmospheric flow upon which the fire-induced fluctuations ‘‘ride’’. However, it is later seen from the MKE budget terms that the fire noticeably affects the mean flow even if we use a time window as conservative as 1 hour to separate the effects of the fire from the ambient flow. This, incidentally, gives us the advantage of being able to track the ‘‘new’’ mean state during FFP. Note that, in contrast to the present study, previous studies have computed block averages on the scale of a minute. Furthermore, they computed perturbations during the FFP period based on pre-FFP block means. That approach, in effect, treated all the coherent high- and low frequency velocity and temperature variations during the FFP period as fire-induced turbulence, thereby presuming no change in the ambient atmospheric conditions during the FFP period. Moreover, the over bar terms can be very sensitive to the averaging scheme and time window.

We have, therefore, included some information on the possible differences arising from using different averaging time windows in the form of a sensitivity analysis in the attached Supplementary Information. Only 1 hour of experimental data are available for the grassland fire. This makes it difficult to compute the slowly-varying parts of the velocity components and temperature as done for the sub-canopy surface fires, which is in turn used to compute the turbulent fluctuations. Another approach to compute the fluctuations would be to remove the 1 hour block mean obtained from the entire time series. However, as seen in Table 1, the spread-rate and intensity of the grassland fire is one to two orders of magnitude higher than the spread-rates and intensities of the sub-canopy surface fires. Moreover, the fire-induced turbulence intensity was found to be four to five times greater than the ambient atmospheric turbulence . Removing the 1 hour block mean to obtain the fire-induced turbulent fluctuations would have resulted in their underestimation during FFP. The relatively high intensity and spread-rate of the grassland fire suggests that the time scale of the influence of the fire at the measuring tower is comparatively much shorter , so that fire-induced changes can be considered entirely as turbulence without attributing any change to the mean state of the atmosphere. As mentioned above, tower-based data from four experimental fires are used in this study. We refer to the 2011 and 2012 backing surface fires beneath the canopy in the New Jersey Pinelands National Reserve as NJ2011 and NJ2012, the 2019 heading surface fire beneath the canopy in the NJPNR as NJ2019, and the heading surface fire from the FireFlux experiment in the grasslands of Texas as TX2006.

The 2019 burn unit is located at the Silas Little Experimental Forest within NJPNR, New Lisbon, New Jersey. For the sub-canopy burns, averaged half-hourly ambient wind velocity data in the stream wise and cross-stream directions are taken from nearby AmeriFlux towers: the Cedar Bridge Tower for NJ2011 and the Silas Little Experimental Forest Tower for NJ2012 and NJ2019. These data are provided by Clark and Heilman et al. ; they are shown in Fig. 1 for reference and comparison with the 1 hour moving means of the measured horizontal velocity components. For NJ2011, NJ2012, and TX2006, experimental burn data obtained from a single meteorological tower are studied in each case. For NJ2019, data obtained from two meteorological towers, i.e. the West Tower and a Control Tower, are studied for the entirety of this paper. The Control Tower is located outside the burn unit, 185 m away from its northern edge . This can also be seen in Fig. 1 of ref. Heilman et al. , which describes the burn unit and the location of the measurement towers. Measurement heights for each burn are shown in Table 1. It must be noted that the fuel consumption during the forest burns differed from each other. While NJ2011 occurred in a pine-dominated region, NJ2012 and NJ2019 occurred in an oak-dominated region. Although fuel loading of the forest floor was similar in both cases, consumption was relatively low in the oak-dominated burns. This is typical of hardwood-dominated forests: fuel consumption is usually less than that in forests with more pine trees and saplings. For a complete description of the burn experiments, including but not limited to detailed illustrations of the burn plots, their ignition lines, and the respective directions of fire line propagation relative to the ambient winds,10 liter pot we refer the reader to the works of Clements et al. , and Heilman et al. . Most of the important features of these four sets of data are summarized in Table 1. Note that although the pre-, during, and post-FFP periods indicated in Table 1 bear some similarity to the corresponding periods defined in the works mentioned above, they are not exactly the same. While the 1 hour moving means explored in the previous section are useful in studying the evolution of the slowly-varying mean, a first-order statistical analysis of the horizontal velocity components is required for insights into the frequency and direction of the strongest turbulent fluctuations a few minutes before, during, and after FFP. This is achieved with the help of a 2D histogram plotted on a wind compass, plotted using WindRose in MATLAB . This function groups velocity data from a time series into classes based on their magnitude, while preserving their direction relative to the positive stream wise direction. Note that zero degrees represents the positive stream wise direction on the wind rose in each scenario. Interesting observations can be made from the wind-rose statistics for the backing surface fires. As seen from Figs. 2–, a shift is seen at ℎ = 20 m in 2011 from high stream wise variability pre-FFP to high cross-stream variability during FFP and post FFP . This corroborates well with the increase in the magnitude of ��20 as seen in Fig. 1. In fact, the cross-stream winds show considerable variability in the range of 6 to 12 m/s during FFP as opposed to post-FFP suggesting that this effect is due to the presence of the flame. Comparison with the ambient cross-stream wind velocity, which shows a decreasing trend during and after FFP at the measurement tower, also emphasizes that the increased cross-stream variability is attributed to fire-induced entertainment.

We can also attribute this to the diversion of momentum into the cross-stream direction as the stream wise wind competes with the air entrained by the fire from the burnt region. Additionally, crown torching in the stand, especially within 15–20 m of the measurement tower, would have also induced strong horizontal velocity gradients near the canopytop, leading to fire-induced entertainment in the cross-stream direction near ℎ = 20 m. Note that the shift in the cross-stream direction happens late at ℎ = 3 m since it takes a while for the cross-stream wind near the canopy top to force the wind near the ground surface. At both ℎ = 3 m and 20 m in 2012, we see a noticeable shift from high variability in the cross-stream direction pre-FFP to high variability mostly in the stream wise direction during FFP and post FFP . This is attributed to the high ambient mean stream wise wind velocity as observed in Section 4.1.1. Furthermore, the relatively lower fire intensity in 2012 does not cause noticeable entertainment in the cross-stream direction during FFP. Contrast this with the FFP period in 2011 and 2 during which the cross-stream winds attain speeds in the range of 6 to 12 m/s. We now focus on the wind-rose statistics for the heading fire, i.e. NJ2019. At the West Tower, the pre-FFP, FFP, and post-FFP times are 1450 to 1520 LT, 1520 to 1550 LT, and 1550 to 1620 LT, respectively . An increase in stream wise variability is seen at the expense of cross-stream variability at ℎ = 20 m from pre-FFP to FFP times possibly because of entertainment of air from the upwind side of the fire at the West Tower. The considerable variability in the cross-stream direction post-FFP is, again, attributed to the ambient wind, which undergoes a southerly shift from the FFP time to post FFP time and has a strong cross-stream component. However, we expect the strong ambient cross-steam wind to also divert some of the stream wise eddies and momentum into the cross-stream direction. This would mean that while the cross-stream gust adds its own momentum to the canopy from aloft, it also potentially diverts some of the fire induced turbulence near the canopy top into the cross-stream direction. Moreover, the wind statistics near the surface seems to mimic that of the higher height suggesting a strong forcing by the ambient post-FFP cross-stream wind.Studies on turbulence during management-scale experimental burns conducted in differing conditions of wind and vegetation are relatively isolated. The integrated contextual framework provided in this work represents a major stride towards the synthesis of and comparison among such studies. Through the comparison, we have reinvestigated fire-induced turbulence dynamics with the backdrop of canopy turbulence or ABL turbulence from a fundamental standpoint. The effects of changes in ambient wind conditions on the measured data have been taken into account because of their linkage to fire behavior. While the inferences drawn here have also been informed by local meteorological conditions at the time of the burn experiments, we have successfully encapsulated the coherent patterns that broadly characterize fire-induced turbulent flow in the two environments primarily by examining turbulent fluxes and the TKE budget terms. We have also examined the terms of the MKE budget equation for all of the sub-canopy surface fire experiments. The sinking of air under gravitational acceleration or the rising of air due to buoyant updrafts accounts for most of the variability in the MKE tendency during and after fire-front passage. The ‘‘slowly-varying’’ mean flow responds noticeably to the presence of the fire and cannot be equated to the pre-FFP mean flow. In this study, major differences among three scenarios of surface fires have been highlighted: sub-canopy heading fires, sub-canopy backing fires, and heading grassland fires.

The explanation that I find most likely is a reduction in water intake. Weight loss can indicate a reduction in fat mass or lean mass, but it can also indicate a reduction in water mass, or dehydration. The food in the experimental condition was purposely designed to provide sufficient calories while being lower in mass and volume than everyday food. One of the best ways to create energy-dense food is to reduce water content . Consequently, the food in the experimental condition had lower water content than the food in the control condition. It is therefore likely that participants consumed less water from food during the experimental condition, and that they did not sufficiently make up for this difference by drinking more fluids. This hypothesis is supported by the results of the post hoc analyses, described below. In the post hoc analyses, it was found that participants in the experimental condition reported consuming 2,800.82 total grams of food + fluid each day, whereas participants in the control condition reported consuming 3,180.42 total grams of food + fluid each day. Therefore, participants in the experimental condition reported consuming an average of 379.6 grams less over the course of a day, even though they did not consume fewer macro-nutrients .

Aside from macro-nutrients,container vertical farming the weight of food usually results from a substantial amount of water and a small amount of fiber and alcohol, along with a relatively small amount of ash . Consequently, it appears likely that participants in the experimental condition consumed less water. This would not be surprising, given that the experimental food was designed to be energy dense. In support of this theory, participants in the experimental condition lost an average of 2.157 pounds, or 1.25% of their body weight, which can easily occur from dehydration. Some researchers have defined mild dehydration as a 1-2% decrease in body weight, moderate dehydration as a 2-5% decrease in body weight, and severe dehydration as a 5% or greater decrease in body weight . It is possible that during the experimental condition, participants consumed less water from food and fluids, resulting in mild dehydration that contributed a 1.25% decrease in body weight. For comparison, when participants were in the control condition they lost 0.04% of their body weight. If participants were, indeed, mildly dehydrated during the experimental condition, is this a cause for concern? During the experimental condition, participants consumed approximately 2,410.82 grams from non-energy-yielding sources . Even if the ash and fiber comprised 15% of this mass, participants would have consumed over 2,000 grams of water – the minimum intake required by NASA, based on participants’ average caloric intake in this study .

Additionally, decrements in cognitive or physical performance usually do not occur until individuals have lost 2% or more of their body weight . Indeed, during the experimental condition, participants showed no significant changes in psychological or physical outcomes other than decreased weight. These factors would indicate that any mild dehydration experienced by participants is little cause for concern. However, although NASA’s ISS requirements stipulate astronauts should receive at least 2 liters of water a day, the Institute of Medicine recommends more than that . Furthermore, numerous factors make it impossible to determine whether sufficient water was consumed in the experimental condition: participants’ fluid reports were questionable, participants were allowed to consume caffeine, and participants did not record their exercise. Nevertheless, at present, the data do not indicate participants were worrisomely dehydrated while eating the experimental diet. In future studies of commercial space food, incorporating measures that can differentiate between fat mass and lean mass and measures that can assess changes in dehydration would help to determine the cause of weight loss.NASA’s ISS requirement is that astronauts be provided with 12-15% of their calories from protein, 30-35% of their calories from fat, and 50-55% of their calories from carbohydrates . The Institute of Medicine sets forth adequate macro-nutrient proportions of 10-35% of calories from protein, 25-35% of calories from fat, and 45-65% of calories from carbohydrate. During the experimental condition, participants met both NASA and the Institute of Medicine’s recommended macro-nutrient proportions.

During the control condition, participants exceeded NASA’s protein and fat requirements, and failed to meet the lower limit of NASA’s carbohydrate requirement. During the control condition, participants also consumed fat in excess of the Institute of Medicine’s recommendations. In summary, during the experimental condition, participants consumed macro-nutrients in the recommended proportions, whereas during the control condition they did not.Participants expressed a desire for more animal protein, indicating that satisfaction with the diet may increase if more animal protein were added. Increasing the amount of animal protein in the diet could also assist with assembling a low fiber diet, which I aimed to achieve but which was difficult to attain, as many “healthy” prepackaged foods are high in fiber. Increasing the amount of protein in the experimental diet would not necessarily be unhealthy, as 14.5% of calories in the diet came from protein, and the Institute of Medicine’s upper limit for protein is 35% . However, NASA has an upper limit of 15% because a high protein diet, especially one from animal sources, could lead to hypercalcemia and increased risk of developing renal stones . Renal stones are especially a concern for astronauts as physiological changes during spaceflight may already lead to increased risk of renal stone formation . Consequently, there is rationale for keeping protein intake relatively low. As discussed in Table 1, given that many of the foods in the experimental diet contained a mix of animal and vegetable proteins, and that I did not have access to a calorimeter, it was not possible for me to calculate the experimental diet’s percentage of protein from animal vs. vegetable sources. However, based on a visual analysis of the types of food in the experimental diet, it is possible that diet did not meet NASA’s recommendation to have 2/3 of protein come from animal sources . It therefore seems advisable to at least shift away from plant-based protein bars and toward more substantial animal sources, such as peppered beef jerky. Various brands and varieties should be investigated to identify a meat source that is tender, salty, spicy, and substantial enough to provide a “meal-like” sensation,hydroponic vertical garden without exceeding the sodium limit and without having small pieces that could flake off and float away in a microgravity environment.NASA only includes items that score, on average, 6.0 or higher on the 9-point Hedonic Scale. In this study, participants rated 50% of the experimental diet items as 6.0 or higher. Because these items were so well liked, I recommend keeping these items in the diet. See all green-colored items in Table 10 for a list of these foods. Nearly one-third of the items in the experimental diet were rated between 5.0 and 5.9. These foods were rated below NASA’s acceptable score of 6.0 and their elimination should therefore be considered. However, it’s not clear that participants’ ratings in this study were equivalent to NASA’s food ratings because in this study, participants ate and rated the food in a naturalistic setting, rather than in a laboratory. Research has shown that the environment can greatly impact food ratings; individuals rate food differently depending upon whether they are in a laboratory, a restaurant, or a cafeteria . In this study, participants may have eaten in the cafeteria with colleagues, alone at their desk, or at home.

Sometimes, when they ate, they saw just their own food, and atother times they saw a plethora of freshly prepared food that their colleagues were eating, yet which they could not eat. Consequently, it is not clear how the multiple environments in this study should be categorized and how participants’ ratings would have been different, had they been doing a taste test in a laboratory. Furthermore, some items that received a mean rating between 5.0 and 5.9 were controversial, meaning that some participants loved them and some hated them. Consequently, when designing future versions of the experimental diet, I recommend that these 5.0- to 5.9- scored items be put back into the pool of options for consideration. Instead of eliminating all these items, perhaps reducing their frequency would be wiser. For instance, all three noodle/potato entrées received mean ratings between 5.0 and 5.9. Yet one participant specifically requested more of these items, indicating a strong preference for them. Keeping just one noodle entrée in the menu may be a better choice than eliminating them completely. However, eliminating all 10 items that were rated lower than 5.0 is advised. Because participants were provided with over 3,000 calories a day, these 10 items could be completely removed from the diet while still providing over 2,400 calories per day – well over the 1,810.71 calories that participants, on average, consumed each day of the experimental condition. Removing these 10 items from the diet would still provide crew with 38 different food items for their 4-day trip and would, if the ratings for the remaining food items did not change, raise the mean food rating of the diet to 6.18 on the Hedonic Scale. All experimental food had to be eaten at room temperature. Room temperature food is less odiferous than heated food, and I had therefore anticipated the smell of food would not be a strong factor in food choice. Consequently, participants were not asked to rate the smell of foods. However, the strong odor of certain foods became apparent to me while evaluating leftovers. This experience highlighted a major difference between the study environment and the Crew Transportation Vehicle: in the study, the open-air setting likely muted the smell of foods, whereas in the enclosed Crew Transportation Vehicle, the smell of foods would be easily noticeable. This is a concern because smells could exacerbate the symptoms of space motion sickness, which is commonly experienced by astronauts during early flight. Former astronauts have recalled that, “Even if the sick people wisely stuck to broth and crackers, odors from the meals being enjoyed by those fortunate enough to feel better could be overwhelming. Some commanders made the law of ‘no stinky food on day 1 or 2.’ That precluded such odiferous items as tuna, salmon, or beef with barbeque sauce” . It would therefore be wise to remove strongly odiferous foods from the diet. The odor of foods could be evaluated in the same way that materials used in spaceflight are evaluated, by having panelists rate the smell from 0 = “undetectable” to 4 = “revolting” . Foods that are rated 2.5 or higher could be eliminated from the diet . Another factor that became quickly apparent was the large variation in participants’ food preferences. Some participants craved the noodle entrées whereas others said the noodles were inedible. Food choice is impacted by not only the qualities of the food itself, but also demographic , sociocultural, and economic variables . When NASA initially designed the Space Shuttle menu, a standard menu was developed for all missions . After four missions, the menu was changed to be crew-selected, so that crew could provide input but all crew members were ultimately provided with the same food. However, crew members expressed a desire for even more personalized menus, and consequently starting with the seventh Space Shuttle mission , individualized menus were provided for each crew member. Crew members were allowed to select the food they wanted from a menu of over 350 food items. Crew members’ choices were then analyzed by a dietitian, who would recommend substitutions to obtain a balanced diet. Based on the large variation in food preferences observed in this study, it seems the individualized menu would be better for achieving high food satisfaction than the standard menu. However, most commercial aerospace companies hope to fly not just eight astronauts a year, but hundreds. Personalizing the diet for each astronaut could become onerous. Instead, I recommend letting crew members pick the food they want from a collection of 100+ food items. Each crew member could be provided with a container and be allowed to select whatever food they wanted as long as it fit in that container and met certain mass restrictions.

The sclerotia of Polyporus umbellatus Fries is widely used to promote urination and prevent dampness.Our previous studies systematically demonstrated that ergosta‐4,6,8,22‐tetraen‐3‐one isolated from Polyporus umbellatus Fries showed significant antitumor, diuretic, and renoprotective effects.Our recent study demonstrated that ergone inhibited NF‐κB signaling and α‐SMA expression in 5/6 nephrectomised and unilateral ureteral obstruction rats.The Wnt signaling pathway is an evolutionarily conserved developmental signaling cascade that plays a critical role in regulating organ development and tissue homeostasis. Leucine‐rich repeat‐containing G protein‐coupled receptor 5 is a novel functional marker in glioma stem cells that promotes EMT by activating the Wnt/β‐catenin signaling pathway.Sry‐like high‐mobility group box 8 regulated cancer stem‐like properties and cisplatin‐induced EMT by the Wnt/β‐catenin signaling pathway in tongue squamous cell carcinoma.In addition,stackable planters bone marrow mesenchymal stromal cells suppressed EMT by inhibiting the Wnt/β‐catenin signaling pathway in silica‐induced pulmonary fibrosis.

Our previous study showed the activation of the canonical Wnt/β‐catenin signaling pathway accompanied by the upregulation of proinflammatory and pro‐oxidative protein expression in the NF‐κB signaling pathway and downregulation of the anti‐inflammatory Nrf2 signaling pathway in patients with chronic kidney disease compared with the Nrf2 signaling pathway in healthy controls.Many compounds inhibit the Wnt signaling pathway, and these compounds may suppress EMT in cancer and fibrosis .FH535, a β‐catenin/Tcf inhibitor, not only increased radio‐sensitivity but also suppressed EMT in the radio resistant KYSE‐150R esophageal cancer cell line, which indicated that inhibitors of the Wnt signaling pathway might be effective anticancer agents with the potential to be anticancer drugs.In addition, it was reported that FH535 alleviated multiple types of cancer, including colorectal cancer, gastric cancer, and hepatocellular carcinoma.Isoquercitrin, a bio-active flavonoid from Bidens bipinnata L., inhibited the Wnt/β‐catenin signaling pathway and hepatocyte growth factor/scatter factor‐induced EMT in NBT‐II cells.In addition, salinomycin was previously used as an antibiotic and also showed significant anticancer activity by suppressing EMT.Salinomycin‐inhibited EMT by suppressing the Wnt/β‐catenin signaling pathway in epithelial ovarian cancer cells, indicating that small molecules targeting the Wnt/β‐catenin signaling pathway might have anticancer properties by reversing EMT.Poria cocos is a well‐known fungus that exhibits an effective therapeutic effect to improve kidney function in clinic.Our previous studies have confirmed that extracts of the surface layer of Poria cocos show remarkable antihyperlipidemic, diuretic, and renoprotective effects.

Recently, our group isolated more than 90 triterpenoid compounds from the surface layer of Poria cocos, some of which exhibited significant anti-fibrotic properties.New triterpenoids, including poricoic acid ZC, poricoic acid ZD, and poricoic acid ZE, significantly down regulated the expression of Wnt1, active β‐catenin, Snail, Twist, MMP‐7, PAI‐1, and FSP1 in HK‐2 cells induced by TGF‐β1 and angiotensin II and mice with unilateral ureteral occlusion. Moreover, new triterpenoids, including poricoic acid ZG and poricoic acid ZH, improved renal fibrosis by targeting the phosphorylation of Smad3 signaling and the Wnt/β‐catenin signaling pathwa . Alismatis rhizome , the dried rhizome of Alisma orientale Juzep, exhibited diuretic, antihyperlipidemic, and renoprotective effects that were also confirmed by our previous studies.Our recent study demonstrated that triterpenoid was the main component of AR,and further study showed that the novel tetracyclic triterpenoid 25‐O‐methylalisol F inhibited EMT by suppressing the Wnt/β‐catenin signaling pathway as well as the phosphorylation of Smad3 signaling in both NRK‐52E and NRK‐49F cells . Additionally, it was also observed that ergone inhibited extracellular matrix accumulation in HK‐2 cells and attenuated podocyte injury through inhibiting the activation of the Wnt/β‐catenin signaling pathway induced byangiotensin II.Taken together, these data indicate that tetracyclic triterpenoid and steroid compounds show significant anti-fibrotic properties in renal fibrosis.Akt is a serine/threonine‐specific protein kinase that plays a key role in various cellular activities. Many cellular signals are transduced through the Akt signaling pathway, and Akt is involved in EMT in cooperation with other proteins. For example, tripartite motif‐containing 14 is an oncogene that regulated EMT and the metastasis of human gastric cancer by activating Akt signaling.M3 muscarinic acetylcholine receptors regulated EMT, perineural invasion, and metastasis in cholangiocarcinoma via the Akt pathway.

Canopy homologue 2 promoted EMT via activating the Akt/GSK3 pathway in non–small‐cell lung cancer.In addition, Akt also promoted EMT cooperation with miRNAs. MiR‐944 inhibited EMT and the metastasis of gastric cancer by the metastasis‐associated in colon cancer 1 /Met/Akt signaling pathway.MiR‐1296 inhibited EMT and the metastasis of hepatocellular carcinoma via the serine/threonine‐protein kinase 1/PI3K/Akt signaling pathway.Taken together, these data suggest that Akt plays a key role in EMT cellular signal transduction and that targeting Akt might be an effective approach to treat fibrosis and tumors . Simvastatin is a 3‐hydroxy‐3‐methylglutaryl coenzyme A inhibitor that was originally used to treat cardiovascular diseases. Recently, it was reported that simvastatin had a therapeutic effect in several cancers. The administration of simvastatin suppressed EMT through the phosphatase and tensin homologue /PI3K/Akt pathway in EC9706‐R cells, suggesting a new therapeutic function for simvastatin in cancers.Trichostatin A, a histone deacetylase inhibitor, is an antifungal antibiotic. In addition to its antibiotic properties, trichostatin A alleviated EMT in bleomycin‐ induced lung injury in mice via inhibiting the Akt signaling pathway.Ubenimex inhibits multiple proteases, including arginyl aminopeptidase, leukotriene A4 hydrolase, alanyl aminopeptidase, and leucyl/cysteinyl aminopeptidase, a membrane dipeptidase used to treat acute myelocytic leukemia and lymphedema. Ubenimex alleviated acquired sorafenib resistance in renal cell carcinoma via suppressing the Akt pathway.Luteolin is widely distributed in plants and has shown anti inflammatory, antioxidant, antimicrobial, and antitumor properties. Luteolin attenuated TGF‐β1‐induced EMT by mediating the PI3K/Akt/NF‐κB‐Snail pathway in lung cancer cells. In addition, luteolin attenuated the progression of gastric cancer by reversing EMT via the Notch signaling pathway.Moreover, luteolin also suppressed the metastasis of triple‐negative breast cancer via blocking EMT by the down regulation of β‐catenin.Furthermore, luteolin suppressed EMT by down regulating the expression of cyclic AMP‐responsive element binding protein 1175 in colorectal cancer cells.Collectively, luteolin showed therapeutic effects in both tissue fibrosis and tumors through various signaling pathways and might be a promising candidate to treat fibrosis and tumors. α‐Mangostin derived from the pericarp of the mangosteen fruit has been shown to have various cellular functions, such as arresting the cell cycle, inhibiting cell viability, inducing apoptosis, and differentiation, reducing inflammation and decreasing adhesion.

α‐Mangostin suppressed viability and EMT by downregulating the PI3K/Akt pathway in pancreatic cancer.Icaritin, a hydrolytic product of icariin that is isolated from members of the Epimedium genus, induced the trans‐differentiation of embryonic stem cells into cardiomyocytes, prevented steroid‐ associated osteonecrosis and stimulated neuronal differentiation.Icaritin inhibited invasion and EMT via targeting the PTEN/Akt/HIF‐1α signaling pathway.As a highly conserved cell signaling system, the Notch signaling pathway is widely involved in cell proliferation and differentiation during embryonic and adult development. Extensive studies have demonstrated that the Notch signaling pathway is also critical for EMT in tumorigenesis and fibrogenesis. The over expression of Notch1‐induced EMT in PC‐3 cells, and claudin‐1 contributed to EMT by the Notch signaling pathway in human bronchial epithelial cells.In addition, Notch and TGF‐β1 generated a reciprocal positive regulatory loop and cooperatively regulated EMT in epithelial ovarian cancer cells,stackable flower pots which provided new insight into the mechanism of EMT.Moreover, miR‐34a downregulation induced by hypoxia enhanced EMT via the Notch signaling pathway in tubular epithelial cells, which indicated that the Notch signaling pathway is critical for EMT during fibrosis.These cases suggest that a targeted intervention in the Notch signaling pathway might be an effective strategy to treat cancer and fibrosis. DAPT, a γ‐secretase inhibitor, decreased the expression of Snail and vimentin and increased E‐cadherin expression in two oral squamous cell carcinoma cell lines, Tca8113 and CAL27, indicating that the targeted inhibition of the Notch signaling pathway might be a new therapeutic strategy to treat cancer. Another γ‐secretase inhibitor, RO4929097, not only inhibited EMT, invasion, and metastasis in cervical cancer HeLa and CaSki cells but also exerted significant therapeutic effects in patients with recurrent malignant glioma, cervical and colon cancer and advanced solid tumors in clinical trials.3,6‐Dihydroxyflavone is ubiquitous in vegetables and fruits and blocks EMT in breast cancer cells through suppressing the Notch signaling pathway.Moreover, luteolin inhibited EMT in gastric cancer by the Notch signaling pathway.In addition, emodin is a main bioactive component of Polygonum cuspidatum that suppressed EMT in alveolar epithelial cells via the Notch signaling pathway. Therefore, it is a promising prospect in treating pulmonary fibrosis.Furthermore, berberine was isolated from Berberis vulgaris and reversed EMT by blocking the Notch/Snail signaling pathway in mice with diabetic nephropathy.The RAS signaling pathway not only regulates blood pressure and fluid balance but also is involved in many kinds of diseases, including cancer and fibrosis. Recently, emerging evidence has suggested that the RAS signaling pathway plays a key role in EMT during fibrosis and tumorigenesis. Angiotensin II promoted EMT by the interaction between hematopoietic stem cells and the stromal cell‐derived factor‐1/CXR4 axis in intrahepatic cholangiocarcinoma.In addition, the overexpression of angiotensin II type 1 receptor induced EMT and promoted tumorigenesis in human breast cancer cells, and the silencing of angiotensin II type 1 receptor suppressed EMT that was induced by high glucose through inactivating the mTOR/p70s6k signaling pathway in the human proximal tubular epithelial HK‐2cell line.

Based on these results, it was suggested that suppressing RAS might be an effective therapy to treat cancer and fibrosis. Losartan is an AT1R antagonist that improved renal fibrosis by suppressing EMT in rats with hyperglycemia.Although many RAS inhibitors showed beneficial effects in tumors and fibrosis, few were reported to inhibit EMT in the treatment of cancer and fibrosis.MiRNAs are endogenous small noncoding RNAs that bind the 3′‐untranslated region of messenger RNAs to regulate gene expression. Recently, many miRNAs have been found to promote or suppress EMT in fibrosis and tumors. For example, miRNA‐497/Wnt3a/c‐Jun regulated growth and EMT, and miR‐497 served as a tumor suppressor in glioma cells.Moreover, miR‐205 inhibited tumor growth, invasion, and EMT via targeting semaphorin 4C in hepatocellular carcinoma.Furthermore, the upregulation of miR‐183‐5p induced apoptosis and inhibited EMT, proliferation, invasion, and migration by the downregulation of ezrin in human endometrial cancer cells.Collectively, EMT is inhibited by many miRNAs, including miR‐145, miR‐497, miR‐145‐5p, miR‐138, miR‐200a, miR‐200b, miR‐655, miR‐30‐5p, and miR‐32.In addition, EMT is also promoted by many miRNAs, including miR‐221, miR‐222, miR‐214‐3p, and miR‐181a.The flavonoids rhamnetin from cloves, berries and cirsiliol from Cirsium lineare Sch.‐Bip. showed anti inflammatory and antitumor properties. Both rhamnetin and cirsiliol induced radio‐senitization and inhibited EMT by miR‐34a/Notch1 signaling in non–small‐cell lung cancer cells.Quercetin alleviated TGF‐β1‐induced fibrosis in HK‐2 cells via downregulating miR‐21 expression and upregulating PTEN and TIMP metallopeptidase inhibitor 3 expression.Sophocarpine from Sophora alopecuroides L. inhibited tumor progression and reversed EMT by targeting miR‐21 in head and neck cancer.In addition, sophocarpine exerted a profound antitumor effect through inhibiting EMT induced by TGF‐β. Zerumbone from Zingiber zerumbet Smith exhibited anti‐inflammatory and anticancer properties. Recently, it was reported that zerumbone inhibited the β‐catenin pathway via miR‐200c to block EMT and cancer stem cells.In addition, nicotine upregulated FGFR3 and RB1 and promoted EMT by the downregulation of miR‐99b and miR‐192 in non–small‐cell lung cancer cells.50 Moreover, osthole alleviated EMT‐mediated metastasis by inhibiting miR‐23a‐3p.Furthermore, resveratrol inhibited proliferation, invasion, and EMT via the upregulation of miR‐200c in HCT‐116 colorectal cancer cells.RNA‐binding proteins such as RNA‐binding Fox protein 2 , epithelial splicing regulatory protein 1 , and ESRP2 control the splicing of many gene transcripts and splice nascent RNAs to functionally and structurally different miRNAs to regulate the process of EMT. For example, bleomycin inhibited ESRP1 expression, leading to the increased alternative splicing of FGFR2 to its mesenchymal isoform IIIc, which induced EMT in lung fibrosis.In addition, over expressed ESRP1 contributed to EMT in ovarian cancer, inducing a cell‐specific variant of CD44 and a protein‐enabled homologue.Moreover, Rbfox2 was upregulated during the EMT, and the depletion of Rbfox2 suppressed the expression of mesenchymal marker genes.Several studies have revealed that small molecules regulate the expression of RNA‐binding proteins to mediate EMT. For example, caffeine, an alkaloid in tea and coffee, reduced p53α expression and upregulated p53β expression through altering the expression of serine/arginine‐rich splicing factor 3 to regulate EMT.Both tissue fibrosis and tumors lead to high morbidity and mortality worldwide; thus, effective therapeutic strategies are urgently needed. Mounting studies have demonstrated that EMT plays a critical role in fibrosis and tumors, suggesting that drugs targeting EMT may be an effective therapy against fibrosis and tumors. Although TGF‐β1 is a potent inducer of EMT, new targets are needed due to the controversial role of TGF‐β1, which has been shown to have multiple beneficial roles in various bio-activities. As summarized above, myriad mediators, including many transcription factors , signaling pathways , RNA‐binding proteins and miRNAs, regulate EMT.

To determine acute mortality via oral exposure, A. pisum were treated by being individually dipped in a particular pesticide solution. They were removed from the solution with a fine paint brush, and placed on a paper towel in a fume hood until excess pesticide had dripped off and the aphids had dried sufficiently and were able to crawl. 10 topically-treated adult A. pisum were placed in a test arena, and a single untreated H. convergenswas added and allowed to feed for a period of 48 h. To test the effects of residual exposure, individual arenas were treated by adding 4 ml of a given pesticide solution and turning 360 degrees in order to coat the entire inside surface before the solution was poured out. Vials were inverted, suspended on a rack, and allowed to dry in a fume hood for a period of 24 h. Approximately 10 untreated adult A. pisum were then added as a food source to the treated arenas. A single untreated H. convergenswas added per arena and allowed to crawl on the treated surface for a period of 48 h. For topical exposure,hydroponic fodder system individual H. convergens were placed in an 8.5 cm petri dish with filter paper lining and treated in a Potter spray tower set to 68.9 kPa. For each pesticide solution, the spray volume per application was 1.4 ml, resulting in a spray deposit of 2.50 mg cm-2 .

This deposit is similar to that used in other studies on non- target insects as well as by the IOBC Working Group “Pesticides and Beneficial Organisms” . The treated insects were allowed to dry, and were placed individually into an untreated arena with 10 untreated A. pisum adults as a food source for a period of 48 h. The statistical program R was used for all analyses. Generalized linear models with binomial errors, or quasibinomial errors in cases where there was over dispersion, were used to analyze the effects of pesticide, pesticide concentration , life stage , and exposure route on the acute mortality of H. convergens. For all GLMs, model reduction and log likelihood ratio tests were used to assess the statistical significance of each factor and their interactions on acute mortality at α = 0.05. In a first analysis, the uncorrected acute mortality data were analyzed. Starting with a full model that included all four factors and interactions, the data set was finally subdivided into eight separate models with no significant interaction between factors to compare the relative toxicity of the different pesticides tested. From these models, the pesticides and pesticide concentrations with similar effects on acute mortality were grouped , and their toxicity was characterized according to the criteria developed by the International Organisation for Biological Control for classifying pesticide selectivity on beneficial insects in a laboratory bio-assay: <30% mortality is harmless, 30-79% mortality is slightly harmful, 80-99% mortality is moderately harmful, and mortality > 99% is harmful . Although uncorrected mortality was used in our analysis, the IOBC classification remains applicable due to the fact that control mortalities were low. This classification scheme has been widely adopted for the comparison of differential toxicities of pesticides to natural enemies in a number of laboratory studies .

In a second analysis, all acute mortality data were first corrected to account for variability among control groups , and then used to examine comparative effects of life stage and route of exposure. An initial full model included all four factors, pesticide, pesticide concentration, life stage and exposure route, and their interactions as categorical variables. Due to significant interactions, the corrected data set was then further separated by pesticide and pesticide concentration to examine the effects of life stage and exposure route on acute mortality of H. convergens. Additionally, the interaction for effects between pesticide and gender were tested for adults. Of the seven pesticides tested, only lambda-cyhalothrin and cyantraniliprole were either moderately harmful or harmful, causing greater than 80% mortality. Lambda cyhalothrin was either moderately harmful or harmful to both life stages of H. convergens when topically exposed at the 100% concentration or when residually exposed at either concentration. However, for oral exposure at both concentrations, it was moderately harmful or harmful to larvae, but not to adults. Cyantraniliprole was moderately harmful to larvae, but not to adults at the 100% concentration when orally exposed, but it was harmless or slightly harmful at the 10% concentration and for other exposure routes. Chlorantraniliprole, copper+mancozeb, novaluron, spinetoram, and sulfur were either harmless or slightly harmful to both life stages at both concentrations, regardless of exposure route. The acute toxicities of the seven pesticides to H. convergens in this study were classified according to guidelines initially established by the IOBC for laboratory bio-assays due to the widespread use of this classification.

However, a more recent revision of these guidelines for classifying pesticide selectivity may more accurately describe their potential toxicity to natural enemies in the field . The revision suggests reducing the number of categories from four to three; the harmless category remaining unchanged at less than 30% acute mortality, pesticides responsible for 30-79% acute mortality being changed from slightly to moderately harmful, and the top two categories of 80-99% and > 99% acute mortality being combined into a single harmful category. Although these revised categories have yet to be widely adopted, they offer a valuable improvement over the original classification as LC50 values would no longer be considered only slightly harmful and the artificial separation of > 99% mortality as a distinct category would be dropped. Under these revised guidelines, lambda-cyhalothrin would be considered harmful rather than moderately harmful to larvae for both concentrations and all three routes of exposure, and to adults at both concentrations for residual exposure and at the 100% concentration for topical exposure. Additionally, lambda-cyhalothrin would change from slightly harmful to moderately harmful for adults orally exposed to the 100% concentration and for adults topically exposed to the 10% concentration. Cyantraniliprole would also be reclassified from slightly harmful to moderately harmful at the 100% concentration for adults orally exposed and for larvae topically exposed, and from moderately harmful to harmful for larvae orally exposed to the 100% concentration. Spinetoram and novaluron would also change from slightly harmful to moderately harmful for topical exposure of larvae at the 100% concentration. Copper+mancozeb and sulfur would remain in the harmless category for all life stages, concentrations, and exposure routes. All further discussion of toxicity will be used in reference to the revised classification system. For corrected acute mortality, life stage had no significant effect for chlorantraniliprole, cyantraniliprole, spinetoram and sulfur at the 100% concentration or for copper+mancozeb at both concentrations. The most pronounced life stage effects were for cyantraniliprole, lambdacyhalothrin, novaluron, and spinetoram at the 100% concentration, and with larvae being more susceptible than adults in all cases. Similarly, novaluron has been shown to have a significant acute effect on larvae of C. carnea, but not on adults . In contrast, while sulfur had limited effects on both adults and larvae of H. convergens in this study,hydroponic nft gully for G. occidentalis, it had significant effects on larvae while being harmless to adults . However, immature life stages are not always more susceptible than adults to pesticide exposure. For instance, phosmet and dimethoate were harmless to eggs and pupae of C. carnea, but harmful to adults .

For instance, spinosad was harmless to both adults and nymphs of the predatory mirid bugs Macrolophus pygmaeusand Nesidiocoris tenuis, while indoxacarb was harmless to M. pygmaeus nymphs, but moderately harmful to adults . Though testing a single life stage can be a good starting point for assessing pesticide toxicity, it is important to identify the most susceptible life stage. For H. convergens, larvae were more susceptible to pesticide exposure than adults at the 100% concentrations, but not necessarily for the more dilute 10% concentrations. A similar pattern was found for exposure route; effects were more frequent at the 100% concentration, for larvae than for adults, and for cyantraniliprole, novaluron, lambdacyhalothrin and spinetoram rather than the other pesticides tested. For cyantraniliprole at the 100% concentration, oral exposure caused the greatest mortality for both larvae and adults, whereas for the other pesticides, when there were significant effects of exposure route, topical exposure caused the greatest mortality. Lambda-cyhalothrin proved to be an exception to this pattern with residual exposure having the greatest effect on adults at the 10% concentration, residual and topical exposure having a similar effect for adults at the 100% concentration, and all exposure routes having a similar effect for larvae at both concentrations. Other studies have also found that exposure route can affect mortality rates of natural enemies. For instance, the effects of spinetoram on adult females of the parasitoid Neochrysocharis formosawere classified differently for all exposure routes: harmful via oral exposure, harmless via topical exposure, and moderately harmful via residual exposure . Additionally, lambda-cyhalothrin was harmless to N. formosa females via oral exposure, but moderately harmful via topical or residual exposure. For spirotetramat, topical exposure was moderately harmful to adults of the parasitoid Tamarixia radiata, while residual exposure was harmless . At the maximum label rate, novaluron was harmful to nymphs of Podisus maculiventris Say via residual or oral exposure . In contrast, in our study, novaluron was moderately harmful to H. convergens larvae via topical exposure, but was harmless via residual or oral exposure. Given the variable effects of exposure route on mortality, bio-assays for the effects of reduced risk pesticides on natural enemies should include multiple routes of exposure rather than being restricted to residual exposure alone, the approach that is recommended in the first tier of the IOBC testing guidelines . As testing for all individual exposure routes can be considerably more time consuming, a more practical approach would be to use a combined exposure scenario, where insects are exposed via multiple routes simultaneously rather than each route individually. Another potential benefit to testing multiple exposure routes simultaneously rather than individually is that it would allow for any additive or synergistic effects. assessed the toxicity of reduced-risk insecticides to adults and larvae of two lacewing species, C. carnea and C. johnsoni, by simultaneously treating insects topically, residually, and orally. From measures of survivor ship 10 days after exposure, chlorantraniliprole and cyantraniliprole proved to be harmless to larvae, but harmful to adults. In addition, from the same study, spinetoram and lambda-cyhalothrin were moderately harmful to larvae and harmful to adults. In a similar study of the survivor ship of the predatory mite G. occidentalis from simultaneous routes of pesticide exposure, showed chlorantraniliprole and cyantraniliprole to be harmless and spinetoram and lambda-cyhalothrin to be harmful to both larvae and adults. While our study with H. convergens differed in being focused on acute mortality from individual exposure routes, it does help to explain the variation in toxicity among natural enemy species and life stages observed from these earlier studies on mortality effects from simultaneous routes of exposure. Chlorantraniliprole and cyantraniliprole were harmless to lacewing larvae and to larval and adult stages of G. occidentalis, but were harmful to lacewing adults and moderately harmful to harmful for larvae and adults of H. convergens. From our study, we found that for H. convergens the most important exposure route for these two insecticides was via oral ingestion, suggesting that natural enemy species or life stages with sucking mouth parts may escape direct oral exposure, whereas those with chewing mouth parts may experience more toxic effects. In contrast, for spinetoram and lambda-cyhalothrin the most important exposure routes for H. convergens were either topical or residual, which helps to explain the more consistent harmful effect of these two insecticides to a broader range of natural enemy species and active life stages. From our study with H. convergens, we have found that even reduced-risk insecticides can, in some cases, cause substantial acute mortality effects in laboratory bio-assays. We have also been able to demonstrate that pesticide, pesticide concentration, predator life stage and exposure route can all influence the acute mortality response of H. convergens in laboratory bio-assays. Of the seven pesticides tested, lambda-cyhalothrin caused the greatest acute mortality for H. convergens, and was classified as either moderately harmful or harmful at the full field rate concentration. Both cyantraniliprole and spinetoram were also either moderately harmful or harmful to H. convergens larvae at the full field rate concentration.

Accurate and time- or cost-efficient methods of diagnosing infected plants are important elements of a disease management program, both with respect to roguing to reduce pathogen spread , and the efficacy of pruning to clear plants of infection . Accurate diagnosis of PD in grapevines is complicated by quantitative and qualitative differences in symptoms among cultivars and other aspects of plant condition . Our results suggest that a well-trained observer can accurately diagnose PD based on visual symptoms, particularly for advanced cases of the disease. The small number of false positives in disease category 1 and 2 vines may have been due to misdiagnosis of other biotic or abiotic factors . Alternatively, false positives might indicate bacterial populations that are near the detection limit; conventional PCR has at least as low a detection threshold as other methods that rely on the presence of live bacterial cells . Regardless, although scouting based on visual symptoms clearly captured most cases of PD in the current study,hydroponic dutch buckets some caution should be used when trying to diagnose early disease stages to ensure that vines are not needlessly removed. There is no cure for grapevines once infected with X. fastidiosa, except for recovery that can occur in some overwintering vines .

The virulent nature of X. fastidiosa in grapevines, and the corresponding high mortality rate for early season infections, increases the potential value of any cultural practices that can cure vines of infection. Moreover, new vines replanted into established vineyards generally take longer to develop compared to vines planted in newly developed vineyards, potentially due to vine-to-vine competition for resources that limits growth of replacement vines. As a result, vines replanted in mature vineyards may never reach full productivity . Thus, management practices that speed the regeneration of healthy, fully developed, and productive vines may reduce the economic loss caused by PD . A multinomial logistic regression showed significant differences in the relative frequency of different grapevine growth outcomes between the two restoration methods . Chip-budded vines showed significantly lower frequency of strong growth and significantly higher frequencies of vines with developing growth and, especially, of no growth . Nearly 30% of chip-budded vines showed no growth in the following season, compared to 0% of vines on which established shoots were trained. These results indicate that training newly produced shoots from the remaining section of the scion was more likely to result in positive regrowth outcomes. As a result, of the two methods we evaluated, training of shoots that emerge from the scion of a severely pruned trunk is recommended for restoring growth.

However, it is important to note that the current study did not estimate the amount of time required for severely pruned vines to return to full productivity. Moreover, the study did not include mature vines, in which growth responses may differ from young vines. Additional studies may be needed to quantify vine yield, and perhaps fruit quality, in severely pruned vines over multiple seasons.The usefulness of pruning for disease management depends on its ability to clear plants of pathogen infection . A comparison of symptom prevalence among severely pruned and control vines from different disease severity categories showed significant effects of the number of years after pruning , pruning treatment , and initial disease symptom category . The analysis also showed significant interactions between year and treatment and between treatment and symptom category , a non-significant interaction between year and symptom category , and a marginally significant three-way interaction . Overall, more vines had symptoms in the second year compared to the first , and there was a higher prevalence of returning symptom in vines from higher initial disease categories . Severe pruning showed an apparent benefit to reducing symptoms of PD after the first year, but this effect weakened substantially by the second year, with no differences for category 1 or 3 vines, and a slightly lower disease prevalence for severely pruned category 2 vines .

A survival analysis of severely pruned category 3 vines showed a significant difference in the rate of symptom return among plots . All vines in plots 1 to 3 had symptoms by autumn 2000, two years after pruning . In plots 4 and 5, more than 80% of vines showed symptoms after three years. Only plot 6 showed markedly lower disease prevalence; in plot 6, ~70% and 50% of severely pruned category 3 vines showed no symptoms after two and four years, respectively, versus ~36% of control vines overall, after two years. It is important to note that at the time of this study, disease pressure may not fully explain the return of symptoms in severely pruned vines. Surveys conducted during the first two years of the study throughout the entirety of the six research blocks showed that the prevalence of PD in control vines actually declined slightly from the first to the second year , but not due to an increase in replanting efforts or vine death , Rather, this decline in prevalence likely reflects overwinter recovery of mild cases of the disease . Thus, the observed return of symptoms in most severely pruned vines does not appear to be explained by reinfection with X. fastidiosa after clearing of infection during the severe-pruning process. Our results indicate that the apparent effectiveness of severe pruning depended on the initial disease severity, and the effectiveness weakened over time. This suggests at least two constraints exist regarding the general utility of pruning as a PD management tool. First, severe pruning does not appear to be useful for mild cases of PD, as many of those same vines would recover from the infection over the winter . Second, there appears to be little value in pruning severely diseased vines; the high frequency of symptom return within a few years indicates that even severe pruning does not clear most vines of X. fastidiosa infection. That leaves a statistically significant window with respect to intermediate severity cases, which may benefit from severe pruning. The apparent benefit for this category of diseased vines would stem from infections that are not so localized that they are highly susceptible to natural recovery over the winter, but also not fully systemic such that the infection has developed below the pruning point . Reliable identification of this narrow class of diseased vines may require substantial experience with PD scouting, detailed record keeping, and an appreciation for variability in symptoms or infection dynamics based on grapevine cultivar and environmental conditions . Research in other bacterial plant pathosystems has evaluated the potential benefit of pruning and whether pruning extent is related to its effectiveness at clearing hosts of infection . A study of the citrus disease huanglongbing, associated with infection by Candidatus Liberibacter spp.,bato bucket evaluated two levels of pruning severity, neither of which showed promise as a disease management tool . In this pathosystem, it is plausible that a very protracted incubation period may undermine the effectiveness of pruning, because by the time the first symptoms are visible, the infection may have already moved throughout much of the tree. Collectively, our results are more similar to a study of citrus variegated chlorosis . In this study, the presence of X. fastidiosa in plant tissues at different distances from symptomatic leaves was determined for varying levels of disease severity. X. fastidiosa was more widely distributed in trees with severe disease symptoms compared to those with early stage foliar symptoms. Although ColettaFilho et al. did not test whether pruning at various distances proximal to symptomatic leaves would eliminate X. fastidiosa infections, the current recommendation is to prune citrus material if early symptoms are present, and to not prune plants with severe disease symptoms . Citrus plant age is also an important consideration; Coletta-Filho and de Souza recommend that symptomatic citrus trees up to three-years-old be removed rather than pruned, whereas trees four-years-old or older should be pruned.

We did not examine vine age as a factor in this study, but the biology of citrus and grape differ in terms of the overwinter recovery that can occur in grape and the apparently slower movement of X. fastidiosa in citrus compared to grape. Anecdotally, the two most mature plots in our study showed the most rapid return of disease, and the youngest plot showed the slowest return. More studies of the effect of vine age are needed before concluding that interactive effects of plant age and pruning differ between the PD and citrus variegated chlorosis pathosystems.The plant microbiota, defined here as the community of bacteria, fungi, archaea, viruses, and other microscopic organisms that live on or in plant tissues , confer many services as well as disservices to their hosts, including disease development and defense , protection against herbivory , tolerance of abiotic stress , and aid in nutrient uptake . These microbial communities associate with all plant tissues , including seeds . Seeds play a major role in plant communities as agents of dispersal, genetic diversity, and regeneration , and they have significant economic and social value through agriculture . Seeds also are a major bottleneck in natural plant populations, as they face heightened mortality from abiotic stressors, pests, pathogens, and predators . As the initial source of inoculum in a plant’s life cycle, seed microbes are can be transmitted across plant generations and have lifelong impacts . Consequently, understanding how seeds acquire and interact with their microbiota, for example, via priority effects or according to the Primary Symbiont Hypothesis , has implications for improving seed health, seedling establishment, and plant community structure. Previous work on seed microbiota has primarily taken a pattern-based approach to studying assembly processes . Such an approach uses culturing and/or next-generation sequencing to compare, contrast, and correlate patterns in microbial community composition, diversity, and species co-occurrences. Typically, however, these community data provide limited insights into processes such as dispersal, microbe-plant interactions, and microbemicrobe interactions.

Given that seed microbial communities are highly variable across individual plants, plant species, and locations , such pattern-based data cannot always be used to predict assembly outcomes. Moreover, such studies often consider how these assembly processes occur at a single spatial scale . We hypothesize that a mechanistic, multi-scale approach would provide a more complete understanding of how microbial communities assemble in seeds, with the field of meta community ecology providing a theoretical framework for such an approach. Meta community theory accounts for the interaction between ecological processes and habitat heterogeneity across spatiotemporal scales to impact community patterns . This emphasis on multiple scales and heterogeneity can help explain the main drivers of community assembly and patterns of biodiversity and co-occurrence . Plant-associated microbial communities vary widely across environmental gradients and host genetics from the levels of tissues to populations . As such, treating individual plants as heterogeneous habitats for microorganisms that are embedded in a larger, heterogeneous landscape of multiple plants representing different species provides a new approach to observing, testing, and modeling drivers of microbial community variation . However, the study of microbiota through a meta community lens is still relatively new, both for animals and plants , and the plant seed represents a relatively understudied microbiome in this context. In this review, we address how mechanisms of seed microbial community assembly have been studied at different spatial micro-, meso-, and macro-scales , and advocate for a meta community-based approach to seed microbiology in future work. For this review, we use the definition of community assembly from Fukami : “the construction and maintenance of local communities through sequential, repeated immigration of species from the regional species pool.” Additionally, most studies that we cover in our review will be focused on fungi and bacteria . We acknowledge that archaea, viruses, and protists are frequent members of plant-associated microbial communities , many plant viruses are seed transmitted , and viruses can play a major role in the diversity and function of soil microbial communities . However, the ecological roles of these microbes in plant microbial communities, including those of seeds, are still largely unknown. As such, we cannot speak on their contributions to seed microbiota assembly here and recommend new research on these microbes in seeds. We will first summarize the modes of microbial acquisition into seeds, and how meta community ecology frames this assembly process. We then discuss studies of seed microbiome assembly which examine the processes of filtering, species interactions, dispersal, and ecological drift.

These data are consistent with the hypothesis that the adaptation of invertebrate-infecting nsRNA ancestor viruses to plants happened several times through independent events during the evolutionary history of nsRNA viruses infecting plants. It is worthy of note that rubodviruses, laulaviruses, and coguviruses, although phylogenetically related, have divergent genome structures and gene expression strategies. In fact, the members of the first two genera have a genome composed of three monocistronic nsRNAs encoding different proteins;RdRp, NP, and putative MP in the case of rubodviruses, and RdRp, NP and a protein of unknown function in the case of laulaviruses. Instead, coguviruses have a bipartite genome consisting of one nsRNA encoding the RdRp , and one ambisense RNA , in which the ORFs encoding the NP and the MP are separated by a long intergenic region . It has been shown that such an IR is AU-rich and self-complementary, thus assuming in both polarity strands a compact conformation containing a long hairpin predicted to serve as a transcription termination signal during the expression of viral genes.

Taking this into consideration, the question arises of how viruses with such different genomic organizations may have evolved from the same ancestor virus. In this respect,planting gutter it can be speculated that a recombination event between the viral and the vc strand of two genomic RNAs with long, AU rich, and almost identical 50 UTRs could generate ambisense RNAs containing an IR similar to those of coguviruses. Since nsRNA viruses with genomic RNAs showing structural features compatible with this possibility were not known previously, such a possibility appeared unlikely. However, the very long, AU-rich, and highly conserved 50 UTRs reported here for RNA 2 and RNA 3 of GMRV and GGDV, and also observed in the corresponding RNAs of ARWV1 and ARWV2, are the first clear evidence that nsRNA viruses with the structural features compatible with this evolutionary scenario may exist. Based on these considerations, the possibility that the bipartite genomes of coguviruses originated from a tripartite ancestor with genomic RNAs containing 50 UTRs similar to those observed in the rubodviruses appears feasible. No glycoprotein is encoded by rubodviruses , a feature previously reported also for coguviruses that, according to electron microscopy observations, are flexuous, non-enveloped viruses.

In contrast, glycoproteins are expressed by most nsRNA plant viruses transmitted by arthropods . The lack of glycoprotein in the genome of rubodviruses and coguviruses opens the question on the existence of vectors, if any, involved in their transmission. In this respect, it is worthy of note that ophioviruses and varicosaviruses, which are plant-restricted or transmitted by fungi, also do not code for any glycoprotein. Vegetative propagation has been proposed as the prevalent transmission mechanism for ARWV1 and ARWV2. Whether, this is also the case for GMRV and GGDV needs further investigation. Most plant viruses code for viral suppressor proteins counteracting the plant antiviral defense mechanisms based on RNA silencing. Further studies are needed to ascertain whether one or more of the three proteins encoded by GGDV and GMRV and other rubodviruses may interfere with RNA silencing, thus showing multifunctional role, as already reported for VSRs of other viruses. GMRV and GGDV were identified in two different grapevines cultivars, Muscat rose and Garan dmak, that were tested by HTS; moreover, both viruses were found in association with other viruses and viroids. Interestingly, no obvious symptoms were observed in the two grapevines. Although infectivity of both viruses was ascertained by graft-transmission, only a Cabernet franc grapevine infected by GGDV developed symptoms post-grafting; further HTS analysis on this indicator plant reveals the presence of GRSPaV, GYSVd-1, and HSVd. Therefore, it was not possible to ascertain whether GGDV is associated with symptoms and additional studies on its pathogenicity are needed, likewise GMRV. An initial survey using several accessions of grapevine located in three different collections in California resulted in the identification of two and one plants infected by GGDV and GMRV, respectively; like the original sources of the viruses , these grapevines were symptomless.

A more extensive survey, including other grapevine-growing regions in California and the USA, is necessary to determine the real distribution of these novel viruses. In that sense, the detection method developed in this study could be useful for virus testing and certification programs. Finally, during the review process of this manuscript, two novel mycoviruses related to coguviruses and rubodviruses were reported, which extends the host range of phenui-like viruses.Fetal tumors are rare. Autopsy evaluation of stillborn fetuses has revealed a 0.5% rate of fetal tumors. Of liveborn children, prenatally diagnosed masses are most commonly lymphatic malformations and sacrococcygeal teratomas. Cervical teratomas, although accounting for only 3% of teratomas diagnosed in the neonatal period, can cause airway compromise and hydrops, and may be managed with ex-utero intrapartum treatment . Fetal renal tumors are less common, occurring in 7 of 100,000 live births, and there have been reports of fetal neuroblastoma as well. Fetal tumors with associated hydrops portend a particularly poor prognosis, with survival rates below 20%. Early prenatal diagnosis is essential for identifying patients who may benefit from fetal, rather than postnatal intervention, as is the case for certain teratomas. However, in cases where fetal intervention is unlikely to improve survival, knowledge of the wide differential diagnosis of a fetal tumor can significantly improve education and counseling for families.We report a case of a fetus diagnosed with a large renal mass with diffuse metastatic lesions and hydrops. A 34-year-old G5P2002 woman who presented to labor and delivery at 30 weeks and 4 days gestation with several days of abdominal pain and tightness. She had been referred for an ultrasound by her midwife after measuring larger than dates. The ultrasound was concerning for a fetal mass and polyhydramnios, and she was referred to Labor & Delivery for evaluation. Repeat ultrasound at our institution showed fetal hydrops with scalp edema, pericardial and pleural effusions, and a solid 10.4 × 8.2 cm mass with vascular flow extending from the right cheek into the right chest . There was evidence of polyhydramnios with an amniotic fluid index of 27.4 cm. The fetal stomach and airway were not visualized, raising concern for obstruction by the mass. Fetal magnetic resonance imaging confirmed a large 9.8× 8.9 × 8.4 cm soft tissue mass involving the right lateral neck, right axilla, right upper extremity, and right chest wall , with notable compression of the airway. Multiple additional small subcutaneous nodules were identified throughout the fetal torso and extremities, including a 1.8 × 2.8 cm lesion on the left thigh, a 2.7 × 2.5 cm lesion on the right cheek, and a 2.9 × 2.1 cm lesion on the right buttock. Hydrops was confirmed. A fetal echocardiogram was done which showed normal cardiac anatomy, normal biventricular systolic function, diffuse skin edema, bilateral pleural effusions, and a trivial pericardial effusion. The combined cardiac index was normal at 430 ml/min/kg. The differential diagnosis for this large soft tissue tumor presenting in a fetus was broad . Initially, there was concern primarily for a cervical teratoma or lymphatic malformation causing airway impingement and hydrops, and consideration was given to in utero resection to alleviate the hydrops. Ex-utero intrapartum treatment was also a potential management option. The findings of widely disseminated lesions on MRI, however, indicated that the fetus likely did not have a cervical teratoma or lymphangioma amenable to surgical resection. The multiplicity of the lesions raised concern for widely metastatic neoplasm or multiple vascular malformations. Although the fetus was not in imminent distress, the findings of fetal hydrops portended an extremely poor prognosis.

In addition, the rapidly accumulating polyhydramnios was physically disabling for the mother. Given the severity of these findings,gutter berries extensive multidisciplinary discussions were held between the patient, maternal fetal medicine, and the fetal surgery teams. There was no identifiable fetal or postnatal intervention for the fetus, and given the high likelihood of morbidity and fetal or neonatal death, it was decided to proceed with elective termination via intra-fetal cardiac potassium chloride injection followed by induction of labor to avoid the maternal morbidity of a Cesarean section. On hospital day 3, the fetus was delivered vaginally. Immediately notable were innumerable subcutaneous masses . On autopsy, the primary tumor was found to arise from the right kidney with extensive subcutaneous and internal metastatic disease. The largest metastatic focus was the originally identified tumor on the right chest, extending to the right back, measuring 10.5 cm in largest dimension. Additional metastatic subcutaneous disease was identified on the face, extremities, and abdominal wall . Microscopic examination revealed sheets of tumor cells invading the soft tissue, muscle, lymph nodes, right kidney, adrenal glands, thymus, heart, and liver. Tumor cells demonstrated epithelioid to spindle cell morphology in a loose fibrous stroma arranged in sheets and scattered nests. There were variable amounts of eosinophilic cytoplasm, round to oval nuclei, and prominent nucleoli. Additionally, rhabdoid cells were present with abundant eosinophilic cytoplasm, eccentric nuclei, and scattered intracytoplasmic inclusions. Intravascular and perineural tumor was noted in multiple sections. Immunohistochemistry revealed near diffuse positivity for cytokeratin , diffuse positivity for vimentin, focal CD34 staining, retained SMARCA4 and SMARCB1 . However, within the tumor cells there was focal loss of SMARCB1/ INI-1. Of note, WT1 had patchy positivity in the cytoplasm but was not found in the nucleus. Staining for synaptophysin, CD99, HMB45, myogenin, MYOD1, SMA, desmin, SALL4, Glypican-3, and PHOX2B were negative. The results of immunohistochemical analysis were consistent with the diagnosis of malignant primitive epithelioid sarcoma with features of malignant rhabdoid tumor. This is the first report of a fetal diagnosis of malignant primitive epithelioid sarcoma with features of malignant rhabdoid tumor in the literature. Additionally, retention of SMARCB1/ INI-1 expression is exceedingly rare in epithelioid sarcoma. Congenital malignancies are extremely uncommon, affecting fewer than 13 per 100,000 live births, and the most prevalent etiologies include teratomas, neuroblastoma, soft tissue tumors, or leukemia. Epithelioid sarcoma is a rare malignant neoplasm of mesenchymal origin most commonly seen in young adults and occasionally young children. It is most often slow-growing, painless, and difficult to diagnose given its ambiguous nature. Histologically, it demonstrates epithelioid morphology with deletion of SMARCB1/INI-1. SMARCB1/INI-1 is a tumor suppressor gene. Its native function is poorly understood, although it is known to be a member of a family of genes encoding chromatin-remodeling complex. Loss of SMARCB1/INI-1 expression is seen in the majority of epithelioid sarcomas and the vast majority of rhabdoid tumors. In one series of rhabdoid tumors, only 16% had retained expression of SMARCB1/INI-1. Another series of two siblings with classic rhabdoid tumor also retained expression of SMARCB1/INI-1, suggesting that rhabdoid tumor may arise by a separate mutational locus. One additional case of epithelioid sarcoma with retained SMARCB1/INI-1 expression has been reported in a 15-month old infant presenting with an isolated heel mass. Malignant rhabdoid tumor is a highly aggressive subtype of sarcoma. It is most commonly seen in young children and originates in the kidneys, central nervous system , or soft tissue, but has been previously reported in fetal and neonatal patients. When diagnosed in utero, the mean gestational age is around 33 weeks. Rhabdoid tumor is identified histologically by the presence of rhabdoid tumor cells with round vesicular nuclei, prominent nucleoli, and eosinophilic inclusions within the cytoplasm, and usually with deletion of the SMARCB1/INI-1 gene on chromosome 22q11. They exhibit positivity for cytokeratins AE1/AE3, vimentin, and epithelial membrane antigen , although there is some variability. In a 40-year review of the literature, 12 fetal cases and 60 neonatal cases were identifie. Most were of renal origin without CNS involvement, one-third were extrarenal tumors not involving the CNS, and 16.7% were of CNS origin. Overall survival was 9.7%. More than half had distant metastasis at diagnosis, and all patients with metastatic disease at diagnosis died. Multiple subcutaneous nodules resembling the “blueberry muffin” presentation of neuroblastoma were present in this case as well as several others. Placental metastases may also be seen, but all reports describe placental metastases of the tumor without evidence of maternal disease. This tumor is aggressive and rapidly fatal with survival ranging from a few minutes to 3 months in one case series of 8 fetuses and neonates with malignant rhabdoid tumor. Treatment options are extremely limited.