UNASSIGNED: Cannabidiol (CBD), as an important therapeutic property of the cannabis plants, is mainly produced in the flower organs. Auxin response factors (ARFs) are play a crucial role in flower development and secondary metabolite production. However, the specific roles of ARF gene family in cannabis remain unknown.
UNASSIGNED: In this study, various bioinformatics analysis of CsARF genes were conducted using online website and bioinformatics, quantitative real time PCR technology was used to investigate the expression patterns of the CsARF gene family in different tissues of different cannabis varieties, and subcellular localization analysis was performed in tobacco leaf.
UNASSIGNED: In this study, 22 CsARF genes were identified and found to be unevenly distributed across 9 chromosomes of the cannabis genome. Phylogenetic analysis revealed that the ARF proteins were divided into 4 subgroups. Duplication analysis identified one pair of segmental/whole-genome duplicated CsARF, and three pairs of tandemly duplicated CsARF. Collinearity analysis revealed that two CsARF genes, CsARF4 and CsARF19, were orthologous in both rice and soybean. Furthermore, subcellular localization analysis showed that CsARF2 was localized in the nucleus. Tissue-specific expression analysis revealed that six genes were highly expressed in cannabis male flowers, and among these genes, 3 genes were further found to be highly expressed at different developmental stages of male flowers. Meanwhile, correlation analysis between the expression level of CsARF genes and CBD content in two cultivars \'H8\' and \'Y7\' showed that the expression level of CsARF13 was negatively correlated with CBD content, while the expression levels of six genes were positively correlated with CBD content. In addition, most of CsARF genes were responsive to IAA treatment.
UNASSIGNED: Our study laid a foundation for the further studies of CsARFs function in cannabis, and provides candidate genes for breeding varieties with high CBD yield in cannabis production.

The diversity of cannabinoid isomers and complexity of Cannabis products pose significant challenges for analytical methodologies. In this study, we developed a method to analyze 14 different cannabinoid isomers in diverse samples within milliseconds by leveraging the unique adduct-forming behavior of silver ions in advanced cyclic ion mobility spectrometry-mass spectrometry. The developed method achieved the separation of isomers from four groups of cannabinoids: Δ3-tetrahydrocannabinol (THC) (1), Δ8-THC (2), Δ9-THC (3), cannabidiol (CBD) (4), Δ8-iso-THC (5), and Δ(4)8-iso-THC (6) (all MW = 314); 9α-hydroxyhexahydrocannabinol (7), 9β-hydroxyhexahydrocannabinol (8), and 8-hydroxy-iso-THC (9) (all MW = 332); tetrahydrocannabinolic acid (THCA) (10) and cannabidiolic acid (CBDA) (11) (both MW = 358); Δ8-tetrahydrocannabivarin (THCV) (12), Δ8-iso-THCV (13), and Δ9-THCV (14) (all MW = 286). Moreover, experimental and theoretical traveling wave collision cross section values in nitrogen (TWCCSN2) of cannabinoid-Ag(I) species were obtained for the first time with an average error between experimental and theoretical values of 2.6%. Furthermore, a workflow for the identification of cannabinoid isomers in Cannabis and Cannabis-derived samples was established based on three identification steps (m/z and isotope pattern of Ag(I) adducts, TWCCSN2, and MS/MS fragments). Afterward, calibration curves of three major cannabinoids were established with a linear range of 1-250 ng·ml-1 for Δ8-THC (2) (R2 = 0.9999), 0.1-25 ng·ml-1 for Δ9-THC (3) (R2 = 0.9987), and 0.04-10 ng·ml-1 for CBD (4) (R2 = 0.9986) as well as very low limits of detection (0.008-0.2 ng·ml-1). Finally, relative quantification of Δ8-THC (2), Δ9-THC (3), and CBD (4) in eight complex acid-treated CBD mixtures was achieved without chromatographic separation. The results showed good correspondence (R2 = 0.999) with those obtained by gas chromatography-flame ionization detection/mass spectrometry.

CONCLUSIONS: We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics studies of both hemp-type and drug-type cannabis. Cannabis (Cannabis sativa L.) holds great promise in the medical and food industries due to its diverse chemical composition, including specialized cannabinoids. However, the study of key genes involved in various biological processes, including secondary metabolite biosynthesis, has been hampered by the lack of efficient in vivo functional analysis methods. Here, we present a novel, short-cycle, high-efficiency transformation method for cannabis seedlings using Agrobacterium tumefaciens. We used the RUBY reporter system to monitor transformation results without the need for chemical treatments or specialized equipment. Four strains of A. tumefaciens (GV3101, EHA105, LBA4404, and AGL1) were evaluated for transformation efficiency, with LBA4404 and AGL1 showing superior performance. The versatility of the system was further demonstrated by successful transformation with GFP and GUS reporter genes. In addition, syringe infiltration was explored as an alternative to vacuum infiltration, offering simplicity and efficiency for high-throughput applications. Our method allows rapid and efficient in vivo transformation of cannabis seedlings, facilitating large-scale protein expression and high-throughput characterization studies.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and β-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.

Cannabis sativa has long been used for neurological and psychological healing. Recently, cannabidiol (CBD) extracted from cannabis sativa has gained prominence in the medical field due to its non-psychotropic therapeutic effects on the central and peripheral nervous systems. CBD, also acting as a potent antioxidant, displays diverse clinical properties such as anticancer, antiinflammatory, antidepressant, antioxidant, antiemetic, anxiolytic, antiepileptic, and antipsychotic effects. In this review, we summarized the structural activity relationship of CBD with different receptors by both experimental and computational techniques and investigated the mechanism of interaction between related receptors and CBD. The discovery of structural activity relationship between CBD and target receptors would provide a direction to optimize the scaffold of CBD and its derivatives, which would give potential medical applications on CBD-based therapies in various illnesses.

UNASSIGNED: Cannabis use may be increasing as countries legalize it and it becomes socially acceptable. A history of cannabis use may increase risk of complications after various kinds of surgery and compromise functional recovery. Here we systematically reviewed and meta-analyzed available evidence on how history of cannabis use affects recovery after hip or knee arthroplasty (THA/TKA).
UNASSIGNED: The PubMed, EMBASE, and Web of Science databases were comprehensively searched and studies were selected and analyzed in accordance with the PRISMA guidelines. The methodological quality of included studies was assessed based on the Newcastle-Ottawa Scale, while quality of evidence was evaluated according to the \"Grading of recommendations assessment, development, and evaluation\" system. Data on various outcomes were pooled when appropriate and meta-analyzed.
UNASSIGNED: The systematic review included 16 cohort studies involving 5.91 million patients. Meta-analysis linked history of cannabis use to higher risk of the following outcomes: revision (RR 1.68, 95% CI 1.31-2.16), mechanical loosening (RR 1.77, 95% CI 1.52-2.07), periprosthetic fracture (RR 1.85, 95% CI 1.38-2.48), dislocation (RR 2.10, 95% CI 1.18-3.73), cardiovascular events (RR 2.49, 95% CI 1.22-5.08), cerebrovascular events (RR 3.15, 95% CI 2.54-3.91), pneumonia (RR 3.97, 95% CI 3.49-4.51), respiratory failure (RR 4.10, 95% CI 3.38-4.97), urinary tract infection (RR 2.46, 95% CI 1.84-3.28), acute kidney injury (RR 3.25, 95% CI 2.94-3.60), venous thromboembolism (RR 1.48, 95% CI 1.34-1.63), and deep vein thrombosis (RR 1.42, 95% CI 1.19-1.70). In addition, cannabis use was associated with significantly greater risk of postoperative transfusion (RR 2.23, 95% CI 1.83-2.71) as well as higher hospitalization costs.
UNASSIGNED: History of cannabis use significantly increases the risk of numerous complications and transfusion after THA or TKA, leading to greater healthcare costs. Clinicians should consider these factors when treating cannabis users, and pre-surgical protocols should give special consideration to patients with history of cannbis use.

CONCLUSIONS: Integrated omics analyses outline the cellular and metabolic events of hemp plants in response to salt stress and highlight several photosynthesis and energy metabolism related pathways as key regulatory points. Soil salinity affects many physiological processes of plants and leads to crop yield losses worldwide. For hemp, a crop that is valued for multiple aspects, such as its medical compounds, fibre, and seed, a comprehensive understanding of its salt stress responses is a prerequisite for resistance breeding and tailoring its agronomic performance to suit certain industrial applications. Here, we first observed the phenotype of salt-stressed hemp plants and found that under NaCl treatment, hemp plants displayed pronounced growth defects, as indicated by the significantly reduced average height, number of leaves, and chlorophyll content. Next, we conducted comparative proteomics and metabolomics to dissect the complex salt-stress response mechanisms. A total of 314 proteins and 649 metabolites were identified to be differentially behaving upon NaCl treatment. Functional classification and enrichment analysis unravelled that many differential proteins were proteases associated with photosynthesis. Through metabolic pathway enrichment, several energy-related pathways were found to be altered, such as the biosynthesis and degradation of branched-chain amino acids, and our network analysis showed that many ribosomal proteins were involved in these metabolic adaptations. Taken together, for hemp plants, influences on chloroplast function probably represent a major toxic effect of salinity, and modulating several energy-producing pathways possibly through translational regulation is presumably a key protective mechanism against the negative impacts. Our data and analyses provide insights into our understanding of hemp\'s stress biology and may lay a foundation for future functional genomics studies.

Cannabis grains are frequently reported from archaeological sites in Asia, and hypothesized centers of origins are China and Central Asia. Chinese early cannabis remains are often interpreted as evidence of hemp fabric production, in line with early textual evidence describing ritualistic hemp cloth use and hemp cultivation as a grain crop. Modern measurements on cannabis varieties show distinct sizes between fibre or oil/fibre and psychoactive varieties, the former having larger seeds on average than the latter. This paper reviews the current macro-botanical evidence for cannabis across East, Central and South Asia and builds a comparative framework based on modern cannabis seed measurements to help identify cannabis use in the past, through the metric analysis of archaeologically preserved seeds. Over 800 grains of cannabis were retrieved from the 2008 excavation of Haimenkou, Yunnan, Southwest China, dating to between 1650 and 400 bc. These are compared with other known archaeological cannabis and interpreted through the metric framework. This offers a basis for exploration of the seed morphometrics potential to infer cannabis cultivation and diversification in uses. At Haimenkou, cannabis seeds size mostly plot in the range of overlapping psychoactive/fibre types; we therefore suggest that the cannabis assemblage from Haimenkou is indicative of a crop beginning to undergo evolution from its early domesticated form towards a diversified crop specialized for alternative uses, including larger oilseed/fibre adapted varieties.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s00334-023-00966-6.

This work aimed to enhance hemp seed oil encapsulation within a hemp seed protein-alginate complex by optimizing parameters in the solution-enhanced dispersion process, employing supercritical carbon dioxide (SEDS) without reliance on organic solvents or elevated temperatures. By response surface methodology (RSM), the microencapsulation efficacy (MEE), particle size (PS) and peroxide value (PV) was determined with respect to three parameters; temperature (°C), pressure (bar) and feed flow rate (mL/min). The optimum conditions were predicted at temperature (40 °C), pressure (150 bar) and feed flow rate (2 mL/min) to offer an MEE of 89.47%, PS of 7.81 μm and PV of 2.91 (meq/kg oil). In addition, the SEDS method was compared with spray- and freeze-drying for encapsulating hemp seed oil. The findings demonstrated SEDS\' superiority, exhibiting exceptional attributes such as the highest MEE, smallest PS and the production of spherical, smooth microcapsules. This highlights its effectiveness in comparison to spray- and freeze-drying methods.

This study aimed to investigate the chemical components and potential health benefits of the fruits of Cannabis sativa L. Fourteen new phenylpropanamides designated as cannabisin I-XIV (1-14) and 40 known analogs were isolated and characterized via nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism. In vitro bioassay using H2O2-induced PC12 cell damage models demonstrated that hempseeds extract and compounds 1, 3, 15, 26, 30, 36, 41, and 48 exhibited neuroprotective properties. 3,3\'-Demethylgrossamide (30) displayed encouraging protection activity, which was further investigated to relieve the oxidative stress and apoptosis of PC12 cells treated with H2O2. The isolation and characterization of these neuroprotective phenylpropanamides from the fruits of C. sativa provide insights into its health-promoting properties as a healthy food and herbal medicine for preventing and treating neurodegenerative diseases, especially Alzheimer\'s disease.