Rooted in therapeutics: comprehensive analyses of Cannabis sativa root extracts reveals potent antioxidant, anti-inflammatory, and bactericidal properties.

Following the legalization of recreational Cannabis in Canada in 2018, the associated waste, including Cannabis roots, has significantly increased. Cannabis roots, comprising 30%-50% of the total plant, are often discarded despite their historical use in Ayurvedic medicine for treating inflammatory and infectious disorders. This study evaluates the phytochemical and therapeutic properties of Cannabis root extracts from a high tetrahydrocannabinolic acid, low cannabidiolic acid cultivar (variety Alien Gorilla Glue). We performed ultra high-performance liquid chromatography coupled with mass spectrometry (UPLC-QTOF-MS) to identify the chemical components of the Cannabis roots. Extracts using water, ethanol and acid-base solvents were tested for antioxidant activity through free radical scavenging, metal chelation, and lipoperoxidation inhibition assays. Mitochondrial membrane protection was assessed using flow cytometry with the MitoPerOx probe in THP-1 monocytic leukemia cells. Anti-inflammatory potential was evaluated by measuring interleukin-6 levels in lipopolysaccharide-stimulated THP-1 cells. Bactericidal/fungicidal efficacy against Escherichia coli, Staphylococcus aureus, and Candida albicans was determined using the p-iodonitrophenyltetrazolium assay. Additionally, we investigated the anticholinesterase activity of Cannabis root extracts, given the potential role of plant alkaloids in inhibiting cholinesterase, an enzyme targeted in Alzheimer's disease treatments. UPLC-QTOF-MS analysis suggested the presence of several phenolic compounds, cannabinoids, terpenoids, amino acids, and nitrogen-containing compounds. Our results indicated significant antioxidant, bactericidal, and anticholinesterase properties of Cannabis root extracts from both soil and hydroponic cultivation. Extracts showed strong antioxidant activity across multiple assays, protected mitochondrial membrane in THP-1 cells, and exhibited anti-inflammatory and bactericidal/fungicidal efficacy. Notably, soil-cultivated roots displayed superior anti-inflammatory effects. These findings demonstrate the remarkable antioxidant, anti-inflammatory, and anti-microbial activities of Cannabis roots, supporting their traditional uses and challenging their perception as mere waste. This study highlights the therapeutic potential of Cannabis roots extracts and suggests avenues for further research and application.

Evaluation of the effects of blue-enriched white light on cognitive performance, arousal, and overall appreciation of lighting.

Introduction: Light's non-visual effects on the biological clock, cognitive performance, alertness, and mental health are getting more recognized. These are primarily driven by blue light, which triggers specific retinal cells containing melanopsin. Traditionally, research on light has relied on correlated color temperature (CCT) as a metric of its biological influence, given that bluer light corresponds to higher Kelvin values. However, CCT proves to be an inadequate proxy of light's biological effects. A more precise metric is melanopic Equivalent Daylight Illuminance (mel-EDI), which aligns with melanopsin spectrum. Studies have reported positive cognitive impacts of blue-enriched white light. It's unclear if the mixed results are due to different mel-EDI levels since this factor wasn't assessed. Method: Given recent recommendations from experts to aim for at least 250 mel-EDI exposure daily for cognitive benefits, our aim was to assess if a 50-minute exposure to LED light with 250 mel-EDI could enhance concentration and alertness, without affecting visual performance or comfort compared to conventional lighting producing around 150 mel-EDI. To ensure mel-EDI's impact, photopic lux levels were kept constant across conditions. Conditions were counterbalanced, parameters included subjective sleepiness (KSS; Karolinska Sleepiness Scale), concentration (d2-R test), visual performance (FrACT; Freiburg Visual Acuity and Contrast Test), general appreciation (VAS; Visual Analogous Scale), preferences and comfort (modified OLS; Office Lighting Survey). Results: The experimental light significantly reduced sleepiness (p = 0.03, Cohen's d = 0.42) and also decreased contrast sensitivity (p = 0.01, Cohen's d = 0.50). The conventional light was found to be more comfortable (p = 0.002, Cohen's d = 0.62), cheerful (p = 0.02, Cohen's d = 0.46) and pleasant (p = 0.005, Cohen's d = 0.55) while the experimental light was perceived as brighter (p = 0.004, Cohen's d = 0.58) and tended to be more stimulating (p = 0.10). Notably, there was a preference for conventional lighting (p = 0.004, Cohen's d=0.56) and concentration was equally improved in both conditions. Discussion: Despite the lack of further improvement in concentration from exposure to blue-enriched light, given the observed benefits in terms of vigilance, further research over an extended period would be justified. These findings could subsequently motivate cognitive optimization through lighting for workers that would benefit from artificial lighting such as in northern regions.

Cannabis Roots: Therapeutic, Biotechnological and Environmental Aspects.

Since the legalization of recreational cannabis in Canada in 2018, the number of licenses for this crop has increased significantly, resulting in an increase in waste generated. Nevertheless, cannabis roots were once used for their therapeutic properties, indicating that they could be valued today rather than dismissed. This review will focus on both traditional therapeutic aspects and potential use of roots in modern medicine while detailing the main studies on active phytomolecules found in cannabis roots. The environmental impact of cannabis cultivation and current knowledge of the root-associated microbiome are also presented as well as their potential applications in biotechnology and phytoremediation. Thus, several high added-value applications of cannabis roots resulting from scientific advances in recent years can be considered to remove them from discarded residues.

Afadin couples RAS GTPases to the polarity rheostat Scribble.

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS complex with AFDN are unknown. We used proximity-based proteomics to generate an interaction map for two isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with AFDN and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in epithelial cells disrupts MAPK and PI3K activation kinetics and inhibits motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects and implicate AFDN as a genuine RAS effector.

Enhancing repair of full-thickness excisional wounds in a murine model: Impact of tissue-engineered biological dressings featuring human differentiated adipocytes.

Promotion of skin repair for acute or chronic wounds through the use of tissue-engineered products is an active field of research. This study evaluates the effects mediated by tissue-engineered biological dressings containing human in vitro-differentiated adipocytes and adipose-derived stromal cells (ASCs). Re-epithelialization, granulation tissue formation and neovascularization of full-thickness cutaneous wounds were specifically assessed using a murine model featuring a fluorescent epidermis. In comparison with wounds that did not receive an adipocyte-containing biological dressing, treated wounds displayed a slight but significantly faster wound closure based on macroscopic observations over 18 days. Non-invasive imaging of GFP-expressing keratinocytes determined that the kinetics of re-epithelialization were similar for both groups. Treated wounds featured thicker granulation tissues (1.7-fold, P < 0.0001) enriched in collagens (1.3-fold, P < 0.0104). In addition, wound cryosections labeled for detection of CD31-expressing cells indicated a 2.2-fold (P < 0.0002) increased neovascularization for the treated wounds at the time of terminal biopsy. This is in accordance with the secretion of pro-angiogenic factors detected in media conditioned by the dressings. Taken together, these results establish that a new type of engineered substitutes featuring a mixture of adipocytes and ASCs can promote cutaneous healing when applied as temporary dressings, suggesting their potential relevance for chronic wound management studies.

Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications.

INTRODUCTION: Monosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation. METHODS: The expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line. RESULTS: MSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression. CONCLUSIONS: Our findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs.

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3 µM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5 µM, ≥2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).

CIN85 modulates the down-regulation of Fc gammaRIIa expression and function by c-Cbl in a PKC-dependent manner in human neutrophils.

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-ß-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.

Human angiomotin-like 1 associates with an angiomotin protein complex through its coiled-coil domain and induces the remodeling of the actin cytoskeleton.

Angiostatin is a potent inhibitor of angiogenesis. One mechanism through which angiostatin inhibits angiogenesis is by binding to the cell surface protein p80-angiomotin. The p80-angiomotin protein promotes angiogenesis, in part, by conferring a hypermigratory phenotype to endothelial cells. Although p80-angiomotin is extensively characterized, less is known about the related protein angiomotin-like 1. We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly suggest that angiomotin-like 1 is involved in actin-cytoskeleton-based processes, in part, via its interaction with a p80-angiomotin-containing complex and the actin cytoskeleton. These findings have important implications for angiogenesis-driven disease since angiomotin and angiomotin-like 1 are both expressed in capillaries.

Signal peptide peptidase promotes the formation of hepatitis C virus non-enveloped particles and is captured on the viral membrane during assembly.

The maturation of the core protein (C) of Hepatitis C virus (HCV) is controlled by the signal peptidase (sp) and signal peptide peptidase (spp) of the host. To date, it remains unknown whether spp cleavage influences viral infectivity and/or the assembly process. Here, evidence is provided that cleavage by spp is not required for assembly of nucleocapsid-like particles (NLPs) in yeast (Pichia pastoris). The immature NLPs (not processed by spp) show a density of 1.11 g ml(-1) on sucrose gradients and a diameter of 50 nm. Co-expression of human spp (hspp) with C generates the 21 kDa mature form of the protein and promotes the accumulation of non-enveloped particles. The amount of non-enveloped particles accumulating in the cell was correlated directly with the expression level of hspp. Furthermore, immunocapture studies showed that hspp was embedded in the membranes of enveloped particles. These results suggest that maturation of the C protein can occur after formation of the enveloped particles and that the abundance of hspp influences the types of particle accumulating in the cells.

The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation.

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.