Wild resources are gathered for ritual purposes or to provide nutrients inadequately supplied by cultigens; other categories of use, such as medicinal plants, are of constant but low-intensity demand. In India today, parts of the acacia species Acacia nilotica are used as medicine , as are the leaves and juice of chenopodium . Other medicinal plants include Acacia catechu, used as an astringent, Acacia leucophloea, which provides a medicinally-useful gum, and Achyranthes aspera . Nineteenth-century documents from the Deccan region show that forest resources and other non-cultigens were also used as fodder, fuel, resins, dyes, and tannins, sources of lac and wax, and timber for crafts and structures . These flora would have been available within a 20-kilometer radius of Kaundinyapura in Early Historic times, when forest resources were more abundant than at present . The presence of Acacia arabica and Acacia cf. nilotica in the archaeobotanical record of Early Historic India confirms that the plant was known, available, and used. Acacia nilotica, also known as “Indian gum arabic,” is a multipurpose source of fuel as well as being suitable for load-bearing components like handles and cart-axles. Ethnographic observations show that the pods are eaten by cattle, goats, and sheep; the gum is used in printing and dyeing of cotton and silk; the bark can be treated to render a substitute for soap; and unripe pods are used for ink . Applying these examples as a model for the premodern era at Kaundinyapura, tannin extracted from Acacia leucophlaea, Acacia nilotica, and Anogeissus latifolia could have been used to convert surplus domestic animals into hides suitable for exchange,blueberry grow pot while cloth from locally-produced cotton could have been enhanced by dyes from wild plants such as Carthamus tinctoris as well as several Acacia species.

Agricultural workers are at increased risk for developing various respiratory diseases including chronic bronchitis, asthma, and COPD, due in part to exposure to respirable organic dusts associated with these environments . Individuals that work in concentrated animal feeding operations, such as those housing swine, have appreciably increased risk for negative lung health outcomes . Therapeutic options for affected individuals are limited, with no current treatments to reverse lung function decline associated with these ailments . Thus, novel treatment strategies that harness and/or promote reparative processes in the lung are necessary. It is increasingly appreciated that inflammation resolution is an active process and regulated by a variety of pathways and mediators, some of which involve omega-3 and omega-6 polyunsaturated fatty acids . As ω-3 PUFA are essential fatty acids that cannot be synthesized de novo by humans, dietary consumption of ω-3 PUFA dictates the tissue availability for these fatty acids and mediators derived from them. In a typical Western diet, ω-3 PUFA intakes are below recommended guidelines, while ω-6 PUFA intakes are high . Conversely to ω-3 PUFA, ω-6 PUFA are metabolized into lipid mediators that are largely involved in the induction of inflammatory processes . Thus, individuals consuming a diet with a high ω-6: ω-3 PUFA ratio may be at increased risk for inadequate control of inflammatory processes, with increased substrate to produce pro-inflammatory lipid mediators and a dearth of substrate for the production of specialized pro-resolving mediators . We have recently assessed the efficacy of dietary supplementation with the ω-3 PUFA docosahexaenoic acid on altering the lung inflammatory response and recovery following acute and repetitive organic dust exposure . Mice were fed a mouse chow supplemented with DHA for four consecutive weeks prior to challenge with a single DE exposure or DE challenge over 3 weeks . In these investigations, we identified impacts of a high DHA diet on lung inflammation, including alterations in macrophage activation, that were overall protective against the deleterious impacts of DE exposure. However, these studies were limited in that they only assessed the impacts of one ω-3 PUFA, DHA, on male sex and on a limited dietary regimen of 4–7 weeks .

Sex-specific differences in respiratory symptoms are observed among the asthmatic individuals and agricultural workers with asthma being more common in women than men and respiratory symptoms being more prevalent in men than women among the farmers . To better assess the impacts of a high ω-3 PUFA diet on the lung inflammatory response to DE and achieve a total tissue ω-6: ω-3 PUFA ratio of ∼1:1 that is considered ideal, we have now utilized the Fat-1 mouse transgenic model to better assess the sex-specific impacts of ω-3 PUFA on DEinduced inflammation. These mice express the Caenorhabditis elegans fatty acid desaturase gene that converts ω-6 PUFA to ω-3 PUFA, thus yielding an overall tissue ratio of ∼1:1. We hypothesized that use of this model would enhance the protective effects identified in initial studies utilizing only DHA supplementation, while also overcoming study limitations that plague fatty acid supplementation investigations, including ambiguous outcomes due to different fatty acid sources, purity, doses, and duration of supplementation . In addition, we have also tested a strategy to further enhance the efficacy of ω-3 PUFAs through the use of a therapeutic inhibitor of soluble epoxide hydrolase an enzyme that metabolizes lipid mediators such as SPM into inactive or less active forms . Through these investigations, we have clarified a role for ω-3 PUFA in regulating the initiation of lung inflammation following DE inhalation and identified differentially regulated genes in repair and recovery following these exposures. These studies warrant consideration of ω-3 PUFA supplementation as a complementary therapeutic strategy for protecting against the deleterious lung diseases associated with environmental dust exposures, such as those experienced by agriculture workers.Settled dusts in closed swine confinement facilities were collected one foot above the ground and kept at −20°C. Dust extracts were prepared as previously described . Briefly, 5 g dust was mixed with 50 ml Hank’s Balanced Salt Solution at room temperature for 1 h. The mixture was then centrifuged at 2,500 rpm for 20 min at 4° C, supernatant was centrifuged one more time and resultant supernatant was sterile filtered using a 0.22 μm filter. Extracts were aliquoted, labeled as 100% dust extract and kept frozen at −20° C. A 12.5% DE solution was prepared for use in mouse intranasal instillations by diluting the 100% extract with sterile saline. Detailed analyses of the DE have been performed previously .

A previous study compared immune response to the agricultural dust administered via intranasal instillation and 100 µg LPS challenge in mice , which has been estimated to be approximately 250× more than the LPS in 12.5% DE. In this same study, the mean endotoxin levels have been reported to be 0.384 μg/ml. Given this finding, when we administer 50 µL DE via intranasal route, this would correspond to approximately 20 ng LPS. In addition, other studies report respirable LPS levels to be between 14–129 EU/mL .At the end of the three-week period, mice were euthanized, and trachea were cannulated to obtain bronchoalveolar lavage fluid from each mouse. Collection of BALF included three times washing with 1 ml PBS each time. All washes were centrifuged at 1,200 rpm for 5 min. While the first wash was kept separate, the second and third washes were combined before centrifugation. The supernatant from the first wash was aliquoted and stored in −80° C for cytokine profiling. The pelleted cells obtained from all the washes were combined and counted. Cytospin slides were prepared using 100,000 cells, stained with Diff-Quik kit and differential cell counts were obtained as described before .For histopathological assessments,hydroponic bucket lungs were inflated with 10% buffered formalin at 15 cm pressure. The same mouse lungs that were lavaged with PBS to obtain BALF was used for histology. Fixed lungs were transferred into 70% ethanol and then shipped to UC Irvine Pathology Research Services Core for paraffin embedding, sectioning, and Hematoxylin and Eosin staining. The observer was blinded to the identity of each slide. A lymphoid aggregate was defined as close aggregation of ≥20 lymphocytes. Alveolar cellularity was evaluated by the number of cells in the alveolar spaces in the lung parenchyma in a total of five images obtained throughout the whole lung using 40× objective with 150% optical zoom. The resulting five values were averaged per tissue section. Each histopathological evaluation was represented as percentiles and a score between 0-to-4 was assigned for each percentile.A mouse NanoString Immunology panel was purchased for direct counting of 561 RNA transcripts using a nCounter Sprint Profiler. Each mouse lung was immediately put into 1 ml of RNA Later, kept at 4°C overnight, then stored in RNA Later at −80° C until RNA extraction. A total of three male mouse lungs per group obtained from three independent studies were thawed for RNA extraction. After lung samples were rinsed in sterile PBS, they were homogenized in 1 ml of Trizol using a 7 cm polypropylene pellet pestle in a microtube, then the extraction was performed as per manufacturer’s instructions using a PureLink RNA mini kit . RNA integrity number was obtained for each sample at the UC Riverside Institute for Integrative Genome Biology Core Facility using an Agilent Bioanalyzer 2,100 . Samples were prepared by a 16-h hybridization step of 50 ng RNA with the codeset probe provided in the Immunology panel. At the end of the hybridization, samples were diluted with nuclease-free water to 35 μL, and 32 µL of each sample was loaded onto a nCounter Sprint Cartridge. Given each cartridge can hold up to 12 samples, a total of 24 samples were run on two cartridges. All samples passed the QC test without any QC flags. The data resulting from each run were combined and analyzed together using nSolver 4.0 and NanoString Advanced analysis. On the nSolver software, gene expression data were normalized using ten housekeeping genes that showed strong correlation with each other, these included Rpl19, Alas1, Ppia, Oaz1, Sdha, Eef1g, Gusb, Gapdh, Hprt and Tbp.

For the advanced analysis, at least three housekeeping genes whose expression correlated well with each other , and thus ideal for normalization of the data were used to normalize the raw data , and a 77 transcript counts were taken as “count threshold”, which is two times the highest background to-noise ratio . Advanced analysis produced differential expression analysis, gene set analysis, and pathway scores. Differential expression outcomes identified the top 20 most upregulated genes among all the treatments, while gene set analysis displayed which pathways those most upregulated genes are related to. To further explore the protein-protein interactions among differentially regulated proteins, we used the STRING database of genes that were statistically significant based on the unadjusted p-values . Genes with low counts were not included in any of the analyses. The lungs are continually exposed to harmful stimuli found in the air, including environmental dusts, diesel exhaust particles, and smoke exposures. The ability of the airways to respond to these stimuli and repair damage caused by the exposures is vital to respiratory health, because unrepaired damage can lead to debilitating airway diseases . Long-term particulate matter exposures have been consistently linked to negative cardiovascular and lung health outcomes and increased mortality . Disease susceptibility caused by chronic inhalation of particulates is clearly evidenced by occupational exposures such as those seen in agriculture workers; exposures to livestock farming operations are consistently linked to increased respiratory symptoms and inflammatory lung disease in not only workers, but in individuals living in the surrounding communities, including children and adults . Approximately two-thirds of agriculture workers report respiratory disease; 50% of agriculture industry workers experience asthma-like symptoms ,25–35% of individuals working in concentrated animal feeding operations experience chronic bronchitis , and the prevalence of chronic obstructive pulmonary disease among agriculture workers is doubled compared to non-farming working control subjects . Curative options are not available for these workers, with current therapeutic options aimed primarily at symptom management and the prevention of lung disease. To improve treatment options for this population, studies investigating therapeutic mechanisms to stimulate endogenous lung tissue repair mechanisms are warranted. To this end, we have assessed the impacts of a low ω-6: ω-3 PUFA total body tissue ratio on lung inflammation following repetitive exposure to inhaled environmental dusts, using a well described mouse model of DE inhalation. In addition, we have explored the therapeutic utility of an sEH inhibitor, TPPU, in enhancing the impacts of high ω-3 PUFA tissue levels, including exploring its effects in regulating SPM levels during inflammation resolution.