Identification of minimum essential therapeutic mixtures from cannabis plant extracts by screening in cell and animal models of Parkinson's disease.

Medicinal cannabis has shown promise for the symptomatic treatment of Parkinson's disease (PD), but patient exposure to whole plant mixtures may be undesirable due to concerns around safety, consistency, regulatory issues, and psychoactivity. Identification of a subset of components responsible for the potential therapeutic effects within cannabis represents a direct path forward for the generation of anti-PD drugs. Using an in silico database, literature reviews, and cell based assays, GB Sciences previously identified and patented a subset of five cannabinoids and five terpenes that could potentially recapitulate the anti-PD attributes of cannabis. While this work represents a critical step towards harnessing the anti-PD capabilities of cannabis, polypharmaceutical drugs of this complexity may not be feasible as therapeutics. In this paper, we utilize a reductionist approach to identify minimal essential mixtures (MEMs) of these components that are amenable to pharmacological formulation. In the first phase, cell-based models revealed that the cannabinoids had the most significant positive effects on neuroprotection and dopamine secretion. We then evaluated the ability of combinations of these cannabinoids to ameliorate a 6-hydroxydopmamine (OHDA)-induced change in locomotion in larval zebrafish, which has become a well-established PD disease model. Equimolar mixtures that each contained three cannabinoids were able to significantly reverse the OHDA mediated changes in locomotion and other advanced metrics of behavior. Additional screening of sixty-three variations of the original cannabinoid mixtures identified five highly efficacious mixtures that outperformed the original equimolar cannabinoid MEMs and represent the most attractive candidates for therapeutic development. This work highlights the strength of the reductionist approach for the development of ratio-controlled, cannabis mixture-based therapeutics for the treatment of Parkinson's disease.

Biomimetic hydrogels designed for cartilage tissue engineering.

Cartilage regeneration and repair remain a clinical challenge due to the limited capability of cartilage to self-regenerate. Worldwide, the costs associated with cartilage regeneration per patient are estimated on average £30 000 for producing and supplying cells. Regenerative approaches may include the use of cell therapies and tissue engineering by combining relevant cells, scaffolds and instructive biomolecules to stimulate or modulate cartilage repair. Hydrogels have been of great interest within these fields to be used as 3D substrates to cultivate and grow cartilage cells. Currently, biomimetic hydrogels with adequate biological and physicochemical properties, such as mechanical properties, capable of supporting load-bearing capability, are yet to succeed. In this review, biomaterials' advantages and disadvantages for the manufacturing of biomimetic hydrogels for cartilage regeneration are presented. Different studies on the formulation of cartilage-like hydrogels based on materials such as gelatin, chondroitin sulfate, hyaluronic acid and polyethylene glycol are summarised and contrasted in terms of their mechanical properties (e.g. elastic modulus) and ability to enhance cell function such as cell viability and GAG content. Current limitations and challenges of biomimetic hydrogels for cartilage regeneration are also presented.

Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples.

Introduction: Medical cannabis patients receive clinical benefits from the secondary metabolites of the plant, which contain a variety of cannabinoids and terpenoids in combinations that can be used to classify the chemovars. State-regulated medical cannabis programs rely on breeder-reported "strain" names both within diversion control systems and to describe the medical cannabis products that are sold to patients in medical cannabis dispensaries. In state-regulated medical cannabis programs, there is no conventional nomenclature system that correlates the breeder-reported names with their profiles of active ingredients, and these "strain" names are invalid as they refer to chemical differences properly referred to as to chemovars. Materials and Methods: To determine the actual levels of chemical diversity represented in 2662 samples of Cannabis flower collected between January 2016 and June of 2017 in Nevada, chemical profile data were measured from these samples by a state-qualified third-party testing laboratory. Principal component analysis (PCA) was used to define clusters in data sets representing both cannabinoids and terpenoids, cannabinoids only, or terpenoids only. Results: The PCA of the terpenoid only data set revealed three well-defined clusters. All three terpenoids only data clusters had high tetrahydrocannabinolic acid synthase, but the terpene profiles listed in reverse-order of abundance best defined these chemovars. The three chemovars in Nevada were labeled with 396 breeder-reported sample names, which overestimate the diversity and do not inform patients regarding chemical properties. Representative DNA samples were taken from each chemovar to determine whether the genetic diversity was greater than the chemical diversity. The limited genotyping experiment was based on DNA sequence polymorphisms. The genetic analysis revealed twelve distinct genetic clades, which still does not account for the entirety of the 396 reported sample names. The finite genotypes did not correlate with the chemotypes determined for the samples. This suggests that either the DNA-markers used were too narrowly restricted for factual separation or that environmental factors contributed more significantly to the chemical profiles of cannabis than genetics. Conclusion: The three chemovars and twelve genotypes reflect low medical diversity on the market in Nevada during its "medical use only" phase. Furthermore, the 396 breeder-reported sample names within this set imply a false sense of diversity of products in Nevada dispensaries.

O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel Responses.

Hypoxia sensors are essential for regulating local oxygen (O2) homeostasis within the body. This is especially pertinent within the CNS, which is particularly vulnerable to O2 deprivation due to high energetic demand. Here, we reveal hypoxia-monitoring function exerted by astrocytes through an O2-regulated protein trafficking mechanism within the CNS. Strikingly, cultured mouse astrocytes isolated from the parafacial respiratory group (pFRG) and retrotrapezoid nucleus (RTN) region are capable of rapidly responding to moderate hypoxia via the sensor cation channel transient receptor potential (TRP) A1 but, unlike multimodal sensory neurons, are inert to hyperoxia and other TRPA1 activators (carbon dioxide, electrophiles, and oxidants) in normoxia. Mechanistically, O2 suppresses TRPA1 channel activity by protein internalization via O2-dependent proline hydroxylation and subsequent ubiquitination by an E3 ubiquitin ligase, NEDD4-1 (neural precursor cell-expressed developmentally down-regulated protein 4). Hypoxia inhibits this process and instantly accumulates TRPA1 proteins at the plasma membrane, inducing TRPA1-mediated Ca2+ influx that triggers ATP release from pFRG/RTN astrocytes, potentiating respiratory center activity. Furthermore, astrocyte-specific Trpa1 disruption in a mouse brainstem-spinal cord preparation impedes the amplitude augmentation of the central autonomic respiratory output during hypoxia. Thus, reversible coupling of the TRPA1 channels with O2-dependent protein translocation allows astrocytes to act as acute hypoxia sensors in the medullary respiratory center.

Insulin-induced lipid body accumulation is accompanied by lipid remodelling in model mast cells.

Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory responses in tissue. Mast cell lines, primary bone marrow-derived mast cells and peripheral blood basophils present a 'steatotic' phenotype in response to chronic insulin exposure, where cells become loaded with lipid bodies. Here we show this state is associated with reduced histamine release, but increased capacity to release bioactive lipids. We describe the overall lipid phenotype of mast cells in this insulin-induced steatotic state and the consequences for critical cellular lipid classes involved in stages of inflammation. We show significant insulin-induced shifts in specific lipid classes, especially arachidonic acid derivatives, MUFA and PUFA, the EPA/DHA ratio, and in cardiolipins, especially those conjugated to certain DHA and EPAs. Functionally, insulin exposure markedly alters the FcεRI-induced release of Series 4 leukotriene LTC4, Series 2 prostaglandin PGD2, Resolvin-D1, Resolvin-D2 and Resolvin-1, reflecting the expanded precursor pools and impact on both the pro-inflammation and pro-resolution bioactive lipids that are released during mast cell activation. Chronic hyperinsulinemia is a feature of obesity and progression to Type 2 Diabetes, these data suggest that mast cell release of key lipid mediators is altered in patients with metabolic syndrome.

"TRPV1 is a component of the atrial natriuretic signaling complex, and using orally delivered antagonists, presents a valid therapeutic target in the longitudinal reversal and treatment of cardiac hypertrophy and heart failure".

Activation of the atrial natriuretic signaling pathway is intrinsic to the pathological responses associated with a range of cardiovascular diseases that stress the heart, especially those involved in sustained cardiac pressure overload which induces hypertrophy and the pathological remodeling that frequently leads to heart failure. We identify transient receptor potential cation channel, subfamily V, member 1, as a regulated molecular component, and therapeutic target of this signaling system. Data show that TRPV1 is a physical component of the natriuretic peptide A, cGMP, PKG signaling complex, interacting with the Natriuretic Peptide Receptor 1 (NPR1), and upon binding its ligand, Natriuretic Peptide A (NPPA, ANP) TRPV1 activation is subsequently suppressed through production of cGMP and PKG mediated phosphorylation of the TRPV1 channel. Further, inhibition of TRPV1, with orally delivered drugs, suppresses chamber and myocyte hypertrophy, and can longitudinally improve in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction, reversing pre-established hypertrophy induced by pressure load while restoring chamber function. TRPV1 is a physical and regulated component of the natriuretic peptide signaling system, and TRPV1 inhibition may provide a new treatment strategy for treating, and reversing the loss of function associated with cardiac hypertrophy and heart failure.

Transcriptional and Functional Plasticity Induced by Chronic Insulin Exposure in a Mast Cell-Like Basophilic Leukemia Cell Model.

OBJECTIVE: Secretory granules (SG) and lipid bodies (LB) are the primary organelles that mediate functional responses in mast cells. SG contains histamine and matrix-active proteases, while LB are reservoirs of arachidonic acid and its metabolites, precursors for rapid synthesis of eicosanoids such as LTC4. Both of these compartments can be dynamically or ontologically regulated, with metabolic and immunological stimuli altering lipid body content and granule numbers responding to contextual signals from tissue. We previously described that chronic in vitro or in vivo hyperinsulinemia expands the LB compartment with a concomitant loss of SG capacity, suggesting that this ratio is dynamically regulated. The objective of the current study is to determine if chronic insulin exposure initiates a transcriptional program that biases model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. METHODS: We used a basophilic leukemic cell line with mucosal mast cell-like features as a model system. We tested the hypothesis that chronic insulin exposure initiates a transcriptional program that biases these model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. Transcriptional arrays were used to map gene expression patterns. Biochemical, immunocytochemical and mediator release assays were used to evaluate organelle numbers and functional responses. RESULTS: In a mucosal mast cell model, the rat basophilic leukemia line RBL2H3, mast cell granularity and SG numbers are inversely correlated with LB numbers. Chronic insulin exposure appears to modulate gene networks involved in both lipid body biogenesis and secretory granule formation. Western blot analysis confirms upregulation of protein levels for LB proteins, and decreases in proteins that are markers for SG cargo. CONCLUSIONS: The levels of insulin in the extracellular milieu may modify the phenotype of mast cell-like cells in vitro.

Prevalence of Antibodies to Zika Virus in Mothers from Hawaii Who Delivered Babies with and without Microcephaly between 2009-2012.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen. ZIKV infection is linked to the development of severe fetal abnormalities that include spontaneous abortion, stillbirth, hydranencephaly, and microcephaly. ZIKV outbreaks have been recorded in the United States. We recently demonstrated the first congenital ZIKV infection in the United States. In this study, we investigated archived blood samples from six mothers who gave birth to babies with microcephaly and 12 mothers who gave birth to healthy babies in Hawaii between 2009 and 2012. We tested maternal blood for the presence of ZIKV IgM and IgG antibodies using commercially available human ZIKV IgM and IgG ELISA kits. Blood from one mother who delivered babies with microcephaly tested positive for ZIKV IgM antibody (16.6%) and blood from three mothers tested positive for ZIKV IgG antibody (50%). ZIKV showed a trend toward significance with microcephaly. ZIKV IgG antibody positive mothers were more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgG antibodies (Odds ratio [OR] = 11.0, 95% confidence interval [CI] = 0.8-147.9, p = 0.083). Similarly, ZIKV IgM antibody positive mothers were also more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgM antibody (OR = 6.8, 95% CI = 0.2-195.1). These data provide further evidence of a link between ZIKV infection and microcephaly and suggests presence of ZIKV positive cases and associated microcephaly in the United States as early as 2009.

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae).

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High-content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.

Comparative genomics explains the evolutionary success of reef-forming corals.

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years.

Two-pore channel 1 interacts with citron kinase, regulating completion of cytokinesis.

Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.

Lipid body accumulation alters calcium signaling dynamics in immune cells.

There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcɛRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signaling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcɛRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signaling pathway and its downstream targets.

Single-walled carbon nanotube exposure induces membrane rearrangement and suppression of receptor-mediated signalling pathways in model mast cells.

Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and pro-inflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified 'interactome' isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together, these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism.

The transmembrane channel-like protein family and human papillomaviruses: Insights into epidermodysplasia verruciformis and progression to squamous cell carcinoma.

Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by increased sensitivity to infection by the ß-subtype of human papillomaviruses (ß-HPVs), causing persistent, tinea versicolor-like dermal lesions. In a majority of affected individuals, these macular lesions progress to invasive cutaneous squamous cell carcinoma (CSCC) in sun-exposed areas. While mutations in transmembrane channel-like 6 (TMC6 / EVER1) and 8 (TMC8 / EVER2) have been causally linked to EV, their molecular functions are unclear. It is likely that their protective effects involve regulation of the ß-HPV life cycle, host keratinocyte apoptosis vs. survival balance and/or T-cell interaction with infected host cells.

The calcium release-activated calcium channel Orai1 represents a crucial component in hypertrophic compensation and the development of dilated cardiomyopathy.

As exceptionally calcium selective store-operated channels, Orai channels play a prominent role in cellular calcium signaling. While most studied in the immune system, we are beginning to recognize that Orai1 provides unique calcium signaling pathways in numerous tissue contexts. To assess the involvement of Orai1 in cardiac hypertrophy we used transverse aortic constriction to model pressure overload cardiac hypertrophy and heart failure in Orai1 deficient mice. We demonstrate that Orai1 deficient mice have significantly decreased survival in this pressure overload model. Transthoracic echocardiography reveals that Orai1 deficient mice develop rapid dilated cardiomyopathy, with greater loss of function, and histological and molecular data indicate that this pathology is associated with significant apoptosis, but not major differences in cellular hypertrophy, fibrosis, and some major hypertrophic makers. Orai1 represents a crucial calcium entry mechanism in the compensation of the heart to pressure overload over-load, and the development of dilated cardiomyopathy.

Beyond apoptosis: the mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells.

Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation.

Successful TRPV1 antagonist treatment for cardiac hypertrophy and heart failure in mice.

Heart failure is becoming a global epidemic. It exerts a staggering toll on quality of life, and substantial medical and economic impact. In a pre-clinical model of cardiac hypertrophy and heart failure, we were able to overcome loss of heart function by administering the TRPV1 antagonist BCTC (4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide). The results presented here identify TRPV1 antagonists as new treatment options for cardiac hypertrophy and heart failure.