Comparison of decarboxylation rates of acidic cannabinoids between secretory cavity contents and air-dried inflorescence extracts in Cannabis sativa cv. 'Cherry Wine'.

Studies with secretory cavity contents and air-dried inflorescence extracts of the CBD-rich hemp strain, Cannabis sativa cv. 'Cherry Wine', were conducted to compare the decarboxylation rates of acidic cannabinoids between two groups. The secretory cavity contents acquired from the capitate-stalked glandular trichomes by glass microcapillaries, and inflorescence samples air-dried for 15 days of storage in darkness at room temperature were analysed by high-pressure liquid chromatography. The ratio of acidic cannabinoids to the total cannabinoids was ranging from 0.5% to 2.4% lower in the air-dried inflorescence samples compared to the secretory cavity samples as follows. In the secretory cavity content, the percentage of acidic cannabinoids to the total cannabinoids was measured as 86.4% cannabidiolic acid (CBDA), 6.5% tetrahydrocannabinolic acid (THCA), 4.3% cannabichromenic acid (CBCA), 1.4% cannabigerolic acid (CBGA), and 0.6% cannabidivarinic acid (CBDVA), respectively. In the air-dried inflorescence, however, the acidic cannabinoids were detected with 84% CBDA, 4.8% THCA, 3.3% CBCA, 0.8% CBGA, and 0.3% Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), respectively. The ratio of cannabidiol (CBD) to cannabidiolic acid (CBDA) was close to 1:99 (w/w) in secretory cavity contents, however, it was roughly 1:20 (w/w) in the air-dried inflorescence. In addition, Δ9-tetrahydrocannabivarin (Δ9-THCV) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) were only detected in the air-dried inflorescence sample, and the ratio of Δ9-THCV to Δ9-THCVA was about 1:20 (w/w). Besides, cannabidivarinic acid (CBDVA) was only observed in the secretory cavity content.

Cannabidiol Alleviates Chronic Prostatitis and Chronic Pelvic Pain Syndrome via CB2 Receptor Activation and TRPV1 Desensitization.

PURPOSE: This study elucidates the mechanism of the physiological effect of cannabidiol (CBD) by assessing its impact on lipopolysaccharide (LPS)-induced inflammation in RWPE-1 cells and prostatitis-induced by 17ß-estradiol and dihydrotestosterone in a rat model, focusing on its therapeutic potential for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). MATERIALS AND METHODS: RWPE-1 cells were stratified in vitro into three groups: (1) controls, (2) cells with LPS-induced inflammation, and (3) cells with LPS-induced inflammation and treated with CBD. Enzyme-linked immunosorbent assays and western blots were performed on cellular components and supernatants after administration of CBD. Five groups of six Sprague-Dawley male rats were assigned: (1) control, (2) CP/CPPS, (3) CP/CPPS and treated with 50 mg/kg CBD, (4) CP/CPPS and treated with 100 mg/kg CBD, and (5) CP/CPPS and treated with 150 mg/kg CBD. Prostatitis was induced through administration of 17ß-estradiol and dihydrotestosterone. After four weeks of CBD treatment, a pain index was evaluated, and prostate tissue was collected for subsequent histologic examination and western blot analysis. RESULTS: CBD demonstrated efficacy in vivo for CP/CPPS and in vitro for inflammation. It inhibited the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by activating the CB2 receptor, reducing expression of interleukin-6, tumor necrosis factor-alpha, and cyclooxygenase-2 (COX2) (p<0.01). CBD exhibited analgesic effects by activating and desensitizing the TRPV1 receptor. CONCLUSIONS: CBD inhibits the TLR4/NF-κB pathway by activating the CB2 receptor, desensitizes the TRPV1 receptor, and decreases the release of COX2. This results in relief of inflammation and pain in patients with CP/CPPS, indicating CBD as a potential treatment for CP/CPPS.

Extracorporeal Shockwave Therapy Alleviates Inflammatory Pain by Down-Regulating NLRP3 Inflammasome in Experimental Chronic Prostatitis and Chronic Pelvic Pain Syndrome.

PURPOSE: To evaluate the anti-inflammatory and antioxidative effects of extracorporeal shockwave therapy (ESWT) on prostatitis and explore the mechanism of alleviating pain. MATERIALS AND METHODS: For in vitro testing, RWPE-1 cells were randomly divided into 5 groups: (1) RWPE-1 group (normal control), (2) LPS group (lipopolysaccharide inducing inflammation), (3) 0.1ESWT group (treated by 0.1 mJ/mm² energy level), (4) 0.2ESWT group (treated by 0.2 mJ/mm² energy level), and (5) 0.3ESWT group (treated by 0.3 mJ/mm² energy level). After ESWT was administered, cells and supernatant were collected for ELISA and western blot. For in vivo testing, Sprague-Dawley male rats were randomly divided into 3 groups: (1) normal group, (2) prostatitis group, and (3) ESWT group (n=12 for each). Prostatitis was induced by 17 beta-estradiol and dihydrotestosterone (DHT) administration. Four weeks after ESWT, the pain index was assessed for all groups and prostate tissues were collected for immunohistochemistry, immunofluorescence, apoptosis analysis and, western blot. RESULTS: Our in vitro studies showed that the optimal energy flux density of ESWT was 0.2 mJ/mm². In vivo, ESWT ameliorated discomfort in rats with prostatitis and inflammation symptoms were improved. Compared to normal rats, overexpressed NLRP3 inflammasomes triggered apoptosis in rats with prostatitis and this was improved by ESWT. TLR4-NFκB pathway was overactive after experimental prostatitis, compared to normal and ESWT groups, and prostatitis induced alterations in BAX/BAK pathway were inhibited by ESWT. CONCLUSIONS: ESWT improved CP/CPPS by reducing NLRP3 inflammasome and ameliorated apoptosis via inhibiting BAX/BAK pathway in a rat model. TLR4 may play a key role in bonding NLRP3 inflammasome and BAX/BAK pathways. ESWT might be a promising approach for the treatment of CP/CPPS.

Degeneration of oil bodies by rough endoplasmic reticulum -associated protein during seed germination in Cannabis sativa.

Oil bodies serve as a vital energy source of embryos during germination and contribute to sustaining the initial growth of seedlings until photosynthesis initiation. Despite high stability in chemical properties, how oil bodies break down and go into the degradation process during germination is still unknown. This study provides a morphological understanding of the mobilization of stored compounds in the seed germination of Cannabis. The achenes of fibrous hemp cultivar (Cannabis sativa cv. 'Chungsam') were examined in this study using light microscopy, scanning electron microscopy and transmission electron microscopy. Oil bodies in Cannabis seeds appeared spherical and sporadically distributed in the cotyledonary cells. Protein bodies contained electron-dense globoid and heterogeneous protein matrices. During seed germination, rough endoplasmic reticulum (rER) and high electron-dense substances were present adjacent to the oil bodies. The border of the oil bodies became a dense cluster region and appeared as a sinuous outline. Later, irregular hyaline areas were distributed throughout oil bodies, showing the destabilized emulsification of oil bodies. Finally, the oil bodies lost their morphology and fused with each other. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area. Some storage proteins are considered emulsifying agents on the surface region of oil bodies, enabling them to remain stable and distinct within and outside cotyledon cells. At the early germination stage, rER appeared and dense substances aggregated adjacent to the oil bodies. Certain proteins were synthesized within the rER and then translocated into the oil bodies by crossing the half membrane of oil bodies. Our data suggest that rER-associated proteins function as enzymes to lyse the emulsifying proteins, thereby weakening the emulsifying agent on the surface of the oil bodies. This process plays a key role in the degeneration of oil bodies and induces coalescence during seed germination.

In Vitro and In Vivo Anti-Inflammatory Potential of Cannabichromene Isolated from Hemp.

Cannabichromene (CBC), a non-psychoactive cannabinoid found in Cannabis sativa, has recently been shown to possess several medicinal properties. However, how CBC produces anti-inflammatory effects and the mechanisms of this remain poorly studied. Therefore, we extracted and purified the CBC from the Cannabis sativa cv. pink pepper (hemp cultivar). The efficacy of CBC in reducing inflammation in RAW 264.7 macrophages and a λ-carrageenan-induced mouse model was then evaluated. CBC had no cytotoxicity up to a concentration of 20 µM and inhibited nitric oxide production by approximately 50% at a concentration of 20 µM. In addition, CBC treatment significantly inhibited causes of inflammation such as inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) at both the mRNA and protein levels. Moreover, CBC suppressed LPS-stimulated inflammation in RAW 264.7 cells by downregulating the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase pathways (MAPK). Furthermore, our in vivo experiments confirmed that the λ-carrageenan-induced increase in the levels of the cytokines iNOS, IL-1ß, and IL-6 was abrogated following treatment with CBC. Therefore, CBC has potential anti-inflammatory effects and may be useful for preventing or treating inflammation.

Cannabidiol, a Regulator of Intracellular Calcium and Calpain.

Cannabidiol (CBD) is one of the most abundant components of Cannabis and has long been used in Cannabis-based preparations. Recently, CBD has become a promising pharmacological agent because of its beneficial properties in the pathophysiology of several diseases. Although CBD is a kind of cannabinoid and acts on cannabinoid receptors (CB1 and CB2), molecular targets involved in diverse therapeutic properties of CBD have not been identified because CBD also interacts with other molecular targets. Considering that CBD alters the intracellular calcium level by which calpain activity is controlled, and both CBD and calpain are associated with various diseases related to calcium signaling, including neurological disorders, this review provides an overview of calpain and calcium signaling as possible molecular targets of CBD. As calpain is known to play an important role in the pathophysiology of neurological disease, a deeper understanding of its relationship with CBD will be meaningful. To understand the role of CBD as a calpain regulator, in silico structural analysis on the binding mode of CBD with calpain was performed.

Cervi Parvum Cornu complex for men with lower urinary tract symptoms: a multicenter, randomized, double-blind, placebo-controlled trial.

Background: To evaluate the efficacy and safety of Cervi Parvum Cornu, Angelicae Gigantis Radix and Glycyrrhizae Radix complex (CAG) in men with moderate lower urinary tract symptoms (LUTS). Materials and methods: From November 2020 to January 2022, participants with International Prostate Symptom Score (IPSS) of 12-19 in two centers were recruited and randomize into three groups: a CAG 500 mg/day group (CAG 500), a CAG 1000 mg/day group (CAG 1000), and a placebo group (PG). They were treated for 12 weeks. The primary endpoint was change of IPSS at the end of study from baseline. Secondary end points included change of prostate specific antigen (PSA), testosterone, dihydrotestosterone (DHT), maximum urinary flow rate (Q max), post-void residual volume (PVR), International Index of Erectile Function (IIEF), and drug safety. Results: A total of 103 patients were able to finish the study according to the study protocol. Total IPSS and sub-scores (residual urine sensation, frequency, weak stream, hesistancy, nocturia, and quality of life) in CAG 500 and CAG 1000 were significantly improved at the 12th week compared to those of the PG. Changes of serum PSA, DHT, and testosterone levels at the 12th week from baseline did not show significant differences among the three groups. Q max and PVR changes did not show significant differences among the three groups either. Total IIEF and sub-scores (erectile function, orgasmic function, sexual desire, intercourse satisfaction) in CAG 1000 were significantly improved at 12th week compared to those in PG. No significant adverse events were found. Conclusions: CAG is well tolerated in patients with moderate LUTS. Treatment with CAG for 12 weeks has a therapeutic effect on moderate LUTS.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Comparison of Auxenochlorella protothecoides and Chlorella spp. Chloroplast Genomes: Evidence for Endosymbiosis and Horizontal Virus-like Gene Transfer.

Resequencing of the chloroplast genome (cpDNA) of Auxenochlorella protothecoides UTEX 25 was completed (GenBank Accession no. KC631634.1), revealing a genome size of 84,576 base pairs and 30.8% GC content, consistent with features reported for the previously sequenced A. protothecoides 0710, (GenBank Accession no. KC843975). The A. protothecoides UTEX 25 cpDNA encoded 78 predicted open reading frames, 32 tRNAs, and 4 rRNAs, making it smaller and more compact than the cpDNA genome of C. variabilis (124,579 bp) and C. vulgaris (150,613 bp). By comparison, the compact genome size of A. protothecoides was attributable primarily to a lower intergenic sequence content. The cpDNA coding regions of all known Chlorella species were found to be organized in conserved colinear blocks, with some rearrangements. The Auxenochlorella and Chlorella species genome structure and composition were similar, and of particular interest were genes influencing photosynthetic efficiency, i.e., chlorophyll synthesis and photosystem subunit I and II genes, consistent with other biofuel species of interest. Phylogenetic analysis revealed that Prototheca cutis is the closest known A. protothecoides relative, followed by members of the genus Chlorella. The cpDNA of A. protothecoides encodes 37 genes that are highly homologous to representative cyanobacteria species, including rrn16, rrn23, and psbA, corroborating a well-recognized symbiosis. Several putative coding regions were identified that shared high nucleotide sequence identity with virus-like sequences, suggestive of horizontal gene transfer. Despite these predictions, no corresponding transcripts were obtained by RT-PCR amplification, indicating they are unlikely to be expressed in the extant lineage.

Industry-Based Misconceptions Regarding Cross-Pollination of Cannabis spp.

Cross-pollination of commercial crops has been an ongoing issue in many species. Cannabis spp. encompasses the classifications of marijuana [high in Δ9-tetrahydrocannabinol (THC)] and hemp (below 0.3% THC). As such, cannabis is the most recent crop facing the dilemma of cross-pollination and is leading to litigation. These litigations are driven by the large misunderstanding of the impacts of cross-pollination within the cannabis industry. The misconception is that if hemp is cross-pollinated by high THC cannabis, the hemp will become "hot" (high in THC) thereby rendering the crop illegal under the 2018 Farm Bill. However, there are many factors that contribute to the amount of THC a plant may produce. This article examines and refutes the misconception of cross-pollination increasing THC levels by highlighting several methods of how THC may become high in a given hemp crop.

Effects of short-term environmental stresses on the onset of cannabinoid production in young immature flowers of industrial hemp (Cannabis sativa L.).

BACKGROUNDS: Cannabis sativa L. produces at least 120 cannabinoids. Although genetic variation is the main factor in cannabinoid production, the effects of short-term environmental stresses in the early flowering stage remains largely unknown. METHODS: To investigate the effects of short-term environmental stresses on the onset of cannabinoid production in young immature flowers, a hemp variety, Green-Thunder (5-8% CBD/mg of dry weight), was treated with mechanical damage, insect herbivory, extreme heat, or drought stress for 5-7 days during the first 2 weeks of flowering. Three hemp tissues, including flowers, leaves, and stems, were collected from hemp grown under these stress conditions at multiple time points during the first 2 weeks after transition to the short photoperiod and analyzed using high pressure liquid chromatography to quantify phytocannabinoids including cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), Δ-tetrahydrocannabinolic acid (THCA), Δ-tetrahydrocannabinol (THC), and cannabinol (CBN). RESULTS: The 5 days of mechanical wounding did not affect the production of any of the cannabinoids during the initial stage of flowering. However, after 5 days of herbivore treatment, there was a significant difference in concentration between day 1 and day 6 of CBGA (control: 308 µg/g; treatment - 24 µg/g), CBG (control: 69 µg/g; treatment: 52 µg/g), and CBD (control: 755 µg/g; treatment: 194 µg/g) between the control and treatment plants. The 7 days of heat treatment at 45-50 oC significantly reduced the production of CBGA during this observed window (control: 206 µg/g; treatment: 182 µg/g) and CBG (control: 21 µg/g; treatment: - 112 µg/g). Notably, the largest change was observed after 7 days of drought stress, when plants showed a 40% greater accumulation of CBG (control: 336 µg/g; treatment: 622 µg/g), and a significant decrease (70-80%) in CBD (control: 1182 µg/g; treatment: 297 µg/g) and THC amounts (control: 3927 µg/g; treatment: 580 µg/g). CONCLUSIONS: Although this observation is limited in the early flowering stage, the common field stresses are adequate to induce changes in the cannabinoid profiles, particularly drought stress being the most impactful stress for hemp flower initiation with the altering the cannabinoid production by decreasing CBD and THC accumulation while increasing CBG by 40%.

Employing Aeroponic Systems for the Clonal Propagation of Cannabis.

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings were excised from two hemp varieties, named "Cherry Wine" and "Red Robin" (17-20% w/w CBD), that served as 'mother plant'. An auxin precursor (indole-3-butyric acid) was applied to stimulate root development in the basal portion of the excised cuttings prior to placement in the system. Cuttings were lightly misted with the nutrient mist solution every three days to provide nutritional support as the solution contains the essential macronutrients, including nitrogen, phosphorus, and potassium. The aeroponic system water reservoir maintained a pH range between 5.0-6.0 and a water temperature between 20-22 °C. A submersible water pump was used to deliver water to the cuttings. The shoot tip cuttings were provided with 24 h of light per day for 10 days until root development occurred, upon which the rooted cuttings were transplanted for research purposes. These aeroponic systems have proven to generate desirable results for Cannabis propagation. The method described here alleviates potential time constraints that arise from traditional methods to allow for a more efficient means for the asexual propagation of Cannabis.

The thickening and modification of the galactan-enriched layer during primary phloem fibre development in Cannabis sativa.

Primary phloem fibres (PPFs) have higher fibre quality and are economically more important for the textile sector than secondary phloem fibres. Both the chemical composition and mechanical structure of the secondary cell wall mainly influence the quality of bast fibres. We investigated the thickening of the galactan-enriched (Gn) layer and its modification process into a gelatinous (G)-layer, which is the largest portion of the secondary cell wall, during the development of the PPF in Cannabis sativa. Stem segments of hemp collected at 17, 29, 52 and 62 days after sowing were comparatively examined using light microscopy, scanning electron microscopy and transmission electron microscopy. The initial cells of PPF started the proliferation and differentiation at 17 days, but the secondary cell wall thickening had already commenced before the 29 days. Both the G- and Gn-layer were rapidly added onto the S-layer of PPFs; thus, the secondary cell wall thickness increased approximately 2-fold at 52 days (from the 29-day mark), and 8-fold at 62 days. The cortical microtubule arrays appeared adjacent to the plasma membrane of PPF cells related to the cellulose synthesis. Additionally, cross-sectioned microfibrils were observed on Gn-layer as the cluster of tiny spots. At 62 days, the specific stratification structure consisting of several lamellae occurred on the G-layer of the secondary cell wall. The secondary cell wall thickened remarkably at 52 days through 62 days so that the mature secondary cell wall consisted of three distinctive layers, the S-, G- and Gn-layer. Cortical microtubule arrays frequently appeared adjacent to the plasma membrane together with cellulose microfibrils on secondary cell wall. The G-layer of PPF at 62 days exhibited the characteristic stratification structure, which demonstrates the modification of the Gn-layer into the G-layer.

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 °C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

Cannabis Contaminants Limit Pharmacological Use of Cannabidiol.

For nearly a century, Cannabis has been stigmatized and criminalized across the globe, but in recent years, there has been a growing interest in Cannabis due to the therapeutic potential of phytocannabinoids. With this emerging interest in Cannabis, concerns have arisen about the possible contaminations of hemp with pesticides, heavy metals, microbial pathogens, and carcinogenic compounds during the cultivation, manufacturing, and packaging processes. This is of particular concern for those turning to Cannabis for medicinal purposes, especially those with compromised immune systems. This review aims to provide types of contaminants and examples of Cannabis contamination using case studies that elucidate the medical consequences consumers risk when using adulterated Cannabis products. Thus, it is imperative to develop universal standards for cultivation and testing of products to protect those who consume Cannabis.

Cannabidiol Drugs Clinical Trial Outcomes and Adverse Effects.

This review aims to present completed clinical trial data surrounding the medicinal benefits and potential side effects of the increasingly popular cannabidiol (CBD)-based drug products, specifically Epidiolex. The article is divided into two sections based on if the ailment being treated by this cannabinoid is classified as either physiological or neurological conditions. In addition to describing the current status, we also examined the different primary and secondary outcomes recorded for each study, which varies greatly depending on the funding source of the clinical trial. With the recent FDA-approval of Epidiolex, this review mainly focused on trials involving this specific formulation since it is the only CBD-based drug currently available to clinicians, although all other clinically trialed CBD(A) drugs were also examined. We hope this review will help guide future research and clinical trials by providing the various outcomes measured in a single review.

Contrasting Roles of Cannabidiol as an Insecticide and Rescuing Agent for Ethanol-induced Death in the Tobacco Hornworm Manduca sexta.

Cannabis sativa, also known as marijuana or hemp, produces a non-psychoactive compound cannabidiol (CBD). To investigate the defensive role of CBD, a feeding preference assay was performed with tobacco hornworm Manduca sexta. The larvae clearly show feeding preference towards the Cannabis tissue containing low CBD over high CBD. While the larva avoided the high CBD diet, we investigated detrimental effects of CBD in the insects' diet. Contrasted to the performance on low CBD-infused artificial diet (AD), larvae reared on the high CBD diet suffer significantly reduced growth and increased mortality. Through testing different carriers, we found that the increase of EtOH in the diet is negatively correlated with insect development and behaviors. Notably, CBD treatment significantly improved ethanol-intoxicated larval survival rate by 40% and also improved diet searching activity, resulting in increased diet consumption. Electrophysiology results revealed that the CBD-treated ganglia had delayed but much larger response with electric stimuli in comparison to the larvae reared on AD only and EtOH-added diet. Our results show CBDs' defensive role against pest insects, which suggests its possible use as an insecticide. We also provide evidence that CBD alleviates alcohol-induced stress; consequently, improving the performance and viability of M. sexta larvae.

Chemical defense responses of upland cotton, Gossypium hirsutum L. to physical wounding.

Upland cotton (Gossypium hirsutum L.) produces terpenoid aldehydes (TAs) that protect the plant from microbial and insect infestations. Foliar TAs include plus (+)- and minus (-)-gossypol, hemigossypolone, and heliocides. To examine foliar TAs' response to physical wounding, the four TA derivatives of a fully glanded G. hirsutum variety JACO GL were quantified by ultra-high performance liquid chromatography. The results show that foliar heliocides increased by 1.7-fold in younger leaves after wounding. While the hemigossypolone level was not affected by the physical wounding, the level of heliocides was significantly increased up to 1.8-fold in the younger leaves. Upland cotton accumulates concentrated carbohydrates, amino acids, and fatty acids in foliar extrafloral nectar (EFN) to serve as a nutrient resource, which attracts both beneficial insects and damaging pests. To better understand the nectar physiology, particularly to determine the temporal dynamics of EFN metabolites in response to the wounding, a gas chromatograph-mass spectrometer (GC-MS) was used to perform metabolic profiling analyses of a G. hirsutum variety Deltapine 383 that has fully developed extrafloral nectaries. A total of 301 compounds were monitored, specifically 75 primary metabolites, two secondary metabolites and 224 unidentified compounds. The physical wounding treatment changed the EFN composition and lowered overall production. The accumulation of 30 metabolites was altered in response to the wounding treatment and threonic acid levels increased consistently. GC-MS combined with Kovat's analysis enabled identification of EFN secondary metabolites including furfuryl alcohol and 5-hyrdomethoxyfurfural, which both have antioxidant and antimicrobial properties that may protect the nectar against microbial pathogens. This study provides new insights into the wounding response of cotton plants in terms of cotton metabolites found in leaf glands and extrafloral nectar as well as highlighting some protective functions of secondary metabolites produced in foliar glands and extrafloral nectaries.

Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

Despite the capacity to accumulate ~70% w/w of lipids, commercially produced unicellular green alga A. protothecoides may become compromised due to the high cost of phosphate fertilizers. To address this limitation A. protothecoides was selected for adaptation to conditions of 100× and 5× lower phosphate and peptone, respectively, compared to 'wild-type media'. The A. protothecoides showed initial signs of adaptation by 45-50 days, and steady state growth at ~100 days. The low phosphate (P)-adapted strain produced up to ~30% greater biomass, while total lipids (~10% w/w) remained about the same, compared to the wild-type strain. Metabolomic analyses indicated that the low P-adapted produced 3.3-fold more saturated palmitic acid (16:0) and 2.2-fold less linolenic acid (18:3), compared to the wild-type strain, resulting in an ~11% increase in caloric value, from 19.5kJ/g for the wild-type strain to 21.6kJ/g for the low P-adapted strain, due to the amounts and composition of certain saturated fatty acids, compared to the wild type strain. Biochemical changes in A. protothecoides adapted to lower phosphate conditions were assessed by comparative RNA-Seq analysis, which yielded 27,279 transcripts. Among them, 2,667 and 15 genes were significantly down- and up-regulated, at >999-fold and >3-fold (adjusted p-value <0.1), respectively. The expression of genes encoding proteins involved in cellular processes such as division, growth, and membrane biosynthesis, showed a trend toward down-regulation. At the genomic level, synonymous SNPs and Indels were observed primarily in coding regions, with the 40S ribosomal subunit gene harboring substantial SNPs. Overall, the adapted strain out-performed the wild-type strain by prioritizing the use of its limited phosphate supply for essential biological processes. The low P-adapted A. protothecoides is expected to be more economical to grow over the wild-type strain, based on overall greater productivity and caloric content, while importantly, also requiring 100-fold less phosphate.

Effects of magnetic field therapy after taping application on pain and function of patients with knee osteoarthritis.

[Purpose] The purpose of study was to investigate the effect of taping applied before magnetic field therapy. [Subjects and Methods] Thirty patients who diagnosed with knee arthritis by a specialist in the rehabilitation department were divided into three groups using the random allocation method. The control group received 15 min general physiotherapy. For the experimental group 1, non-elastic taping was applied to the medial side of the knee after general physiotherapy, followed by a 15 min magnetic field therapy. For the experimental group 2, elastic taping was applied to the medial side of the knee after general physiotherapy, followed by a 15 min magnetic field therapy. This study used visual analog scale to evaluate pain. This study used Western Ontario and McMaster Universities to evaluate the knee function. [Results] Experimental group 1 and experimental group 2 showed a significant difference visual analog scale and Western Ontario and McMaster Universities from the control group. However, no significant difference was observed between experimental group 1 and experimental group 2. [Conclusion] Choosing the applicable taping method according to the condition of the patients and applying it to therapy is necessary to reduce pain and improve the function of the patients with knee osteoarthritis.