Immune and glial cell alterations in the rat brain after repeated exposure to the synthetic cannabinoid JWH-018.

The use of synthetic cannabinoid receptor agonists (SCRAs) poses major psychiatric risks. We previously showed that repeated exposure to the prototypical SCRA JWH-018 induces alterations in dopamine (DA) transmission, abnormalities in the emotional state, and glial cell activation in the mesocorticolimbic DA circuits of rats. Despite growing evidence suggesting the relationship between substance use disorders (SUD) and neuroinflammation, little is known about the impact of SCRAs on the neuroimmune system. Here, we investigated whether repeated JWH-018 exposure altered neuroimmune signaling, which could be linked with previously reported central effects. Adult male Sprague-Dawley (SD) rats were exposed to JWH-018 (0.25 mg/kg, i.p.) for fourteen consecutive days, and the expression of cytokines, chemokines, and growth factors was measured seven days after treatment discontinuation in the striatum, cortex, and hippocampus. Moreover, microglial (ionized calcium-binding adaptor molecule 1, IBA-1) and astrocyte (glial fibrillary acidic protein, GFAP) activation markers were evaluated in the caudate-putamen (CPu). Repeated JWH-018 exposure induces a perturbation of neuroimmune signaling specifically in the striatum, as shown by increased levels of cytokines [interleukins (IL) -2, -4, -12p70, -13, interferon (IFN) γ], chemokines [macrophage inflammatory protein (MIP) -1α, -3α], and growth factors [macrophage colony-stimulating factor (M-CSF), vascular endothelial growth factor (VEGF)], together with increased IBA-1 and GFAP expression in the CPu. JWH-018 exposure induces persistant brain region-specific immune alterations up to seven days after drug discontinuation, which may contribute to the behavioral and neurochemical dysregulations in striatal areas that play a role in the reward-related processes that are frequently impaired in SUD.

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design.

Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)-dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.

Antimicrobial properties and biotransforming ability of fungal endophytes from Ficus carica L. (Moraceae).

The endophytic fungal community associated with leaves of Ficus carica L. (Moraceae) from Argentina was investigated. Fifteen fungal isolates were isolated and identified by molecular methods into the genera Alternaria, Cladosporium, Curvularia, Diaporthe, Epicoccum, Myrothecium, Neofusicoccum, Nigrospora, Preussia and Ustilago. Cladosporium cladosporioides and Curvularia lunata were the most frequently isolated species. The fungal metabolic profiles were obtained by automated TLC and NMR and analysed by PC Analysis. Antifungal and antibacterial activity was assessed by bioautographic assays. In addition, the biotransforming ability of the fungal isolates was tested on F. carica extracts. Five isolates (33.3%) exhibited inhibitory activity against at least one of the microorganisms tested. Most of the fungal endophytes were able to metabolise the flavonoid rutin 1, and the coumarin psoralen 3 present in F. carica extracts. Further investigations of the psoralen biotransforming ability performed by the selected endophyte Alternaria alternata F8 showed the accumulation of the 6,7-furan-hydrocoumaric acid derivative 4 as the main biotransformation product. Our results corroborate that F. carica can live symbiotically with rich and diverse endophytic communities adding insights about their ecological interactions.

Sustainable Refining of Vegetable Oil Made Easy with a Designer Phospholipase C Enzyme.

The increasing demand pressures the vegetable oil industry to develop novel refining methods. Degumming with type C phospholipases (PLCs) is a green technology and provides extra oil. However, natural PLCs are not active under the harsh conditions used in oil refining plants, requiring additional unit operations. These upfront capital expenditures and the associated operational costs hinder the adoption of this method. Here, we present a process based on ChPLC, a synthetic PLC obtained by consensus sequence design, possessing superior thermal stability and catalytic properties. Using ChPLC, crude soybean oil degumming was completed at 80 °C in 30 min, the temperature and residence time imposed by the design of existing oil refining plants. Remarkably, an extra yield of oil of 2% was obtained using 60% of the dose recommended for PLCs marketed today, saving upfront investments and reducing the operational cost of degumming. A techno-economic analysis indicates that, for medium size plants, ChPLC reduces the overall cost of soybean oil enzymatic degumming by 58%. The process presented here facilitates the implementation of enzymatic technologies to oil producers, regardless of their processing capacity, bringing potential annual benefits in the billion-dollar range for the global economy.

Maternal immune activation impairs endocannabinoid signaling in the mesolimbic system of adolescent male offspring.

Prenatal infections can increase the risk of developing psychiatric disorders such as schizophrenia in the offspring, especially when combined with other postnatal insults. Here, we tested, in a rat model of prenatal immune challenge by the viral mimic polyriboinosinic-polyribocytidilic acid, whether maternal immune activation (MIA) affects the endocannabinoid system and endocannabinoid-mediated modulation of dopamine functions. Experiments were performed during adolescence to assess i) the behavioral endophenotype (locomotor activity, plus maze, prepulse inhibition of startle reflex); ii) the locomotor activity in response to Δ9-Tetrahydrocannabinol (THC) and iii) the properties of ventral tegmental area (VTA) dopamine neurons in vivo and their response to THC; iv) endocannabinoid-mediated synaptic plasticity in VTA dopamine neurons; v) the expression of cannabinoid receptors and enzymes involved in endocannabinoid synthesis and catabolism in mesolimbic structures and vi) MIA-induced neuroinflammatory scenario evaluated by measurements of levels of cytokine and neuroinflammation markers. We revealed that MIA offspring displayed an altered locomotor activity in response to THC, a higher bursting activity of VTA dopamine neurons and a lack of response to cumulative doses of THC. Consistently, MIA adolescence offspring showed an enhanced 2-arachidonoylglycerol-mediated synaptic plasticity and decreased monoacylglycerol lipase activity in mesolimbic structures. Moreover, they displayed a higher expression of cyclooxygenase 2 (COX-2) and ionized calcium-binding adaptor molecule 1 (IBA-1), associated with latent inflammation and persistent microglia activity. In conclusion, we unveiled neurobiological mechanisms whereby inflammation caused by MIA influences the proper development of endocannabinoid signaling that negatively impacts the dopamine system, eventually leading to psychotic-like symptoms in adulthood.

Regulation of Tumor and Metastasis Initiation by Chemokine Receptors.

Tumor-initiating cells (TICs) are a rare sub-population of cells within the bulk of a tumor that are major contributors to tumor initiation, metastasis, and chemoresistance. TICs have a stem-cell-like phenotype that is dictated by the expression of master regulator transcription factors, including OCT4, NANOG, and SOX2. These transcription factors are expressed via activation of multiple signaling pathways that drive cancer initiation and progression. Importantly, these same signaling pathways can be activated by select chemokine receptors. Chemokine receptors are increasingly being revealed as major drivers of the TIC phenotype, as their signaling can lead to activation of stemness-controlling transcription factors. Additionally, the cell surface expression of chemokine receptors provides a unique therapeutic target to disrupt signaling pathways that control the expression of master regulator transcription factors and the TIC phenotype. This review summarizes the master regulator transcription factors known to dictate the TIC phenotype, along with the complex signaling pathways that can mediate their expression and the chemokine receptors that are most upstream of this phenotype.

Adolescent self-administration of the synthetic cannabinoid receptor agonist JWH-018 induces neurobiological and behavioral alterations in adult male mice.

RATIONALE: The use of synthetic cannabinoid receptor agonists (SCRAs) is growing among adolescents, posing major medical and psychiatric risks. JWH-018 represents the reference compound of SCRA-containing products. OBJECTIVES: This study was performed to evaluate the enduring consequences of adolescent voluntary consumption of JWH-018. METHODS: The reinforcing properties of JWH-018 were characterized in male CD1 adolescent mice by intravenous self-administration (IVSA). Afterwards, behavioral, neurochemical, and molecular evaluations were performed at adulthood. RESULTS: Adolescent mice acquired operant behavior (lever pressing, Fixed Ratio 1-3; 7.5 µg/kg/inf); this behavior was specifically directed at obtaining JWH-018 since it increased under Progressive Ratio schedule of reinforcement, and was absent in vehicle mice. JWH-018 IVSA was reduced by pretreatment of the CB1-antagonist/inverse agonist AM251. Adolescent exposure to JWH-018 by IVSA increased, at adulthood, both nestlet shredding and marble burying phenotypes, suggesting long-lasting repetitive/compulsive-like behavioral effects. JWH-018 did not affect risk proclivity in the wire-beam bridge task. In adult brains, there was an increase of ionized calcium binding adaptor molecule 1 (IBA-1) positive cells in the caudate-putamen (CPu) and nucleus accumbens (NAc), along with a decrease of glial fibrillary acidic protein (GFAP) immunoreactivity in the CPu. These glial alterations in adult brains were coupled with an increase of the chemokine RANTES and a decrease of the cytokines IL2 and IL13 in the cortex, and an increase of the chemokine MPC1 in the striatum. CONCLUSIONS: This study suggests for the first time that male mice self-administer the prototypical SCRA JWH-018 during adolescence. The adolescent voluntary consumption of JWH-018 leads to long-lasting behavioral and neurochemical aberrations along with glia-mediated inflammatory responses in adult brains.

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming.

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.

Considerations for addressing anti-vaccination campaigns: How did we get here and what can we do about it?

A course on vaccine development asked students to write a blog addressing general anti-vaccination strategies and their significance today, in the context of the resistance seen against novel SARS-CoV-2 mRNA vaccines. This perspective explores how and why these efforts are successful at reducing vaccine uptake and why, for the most part, efforts to combat the movement have been unsuccessful. This summary of the collective view of the class provides recommendations for combatting current and future campaigns of misinformation. It is hoped that this perspective will serve as a call to action for clinical pharmacologists and translational scientists to do their part to educate the lay community and promote the science in an open and transparent manner to ensure that current and future vaccines fulfill their potential.

Subsets of cancer cells expressing CX3CR1 are endowed with metastasis-initiating properties and resistance to chemotherapy.

Metastasis-initiating cells (MICs) display stem cell-like features, cause metastatic recurrences and defy chemotherapy, which leads to patients' demise. Here we show that prostate and breast cancer patients harbor contingents of tumor cells with high expression of CX3CR1, OCT4a (POU5F1), and NANOG. Impairing CX3CR1 expression or signaling hampered the formation of tumor spheroids by cell lines from which we isolated small subsets co-expressing CX3CR1 and stemness-related markers, similarly to patients' tumors. These rare CX3CR1High cells show transcriptomic profiles enriched in pathways that regulate pluripotency and endowed with metastasis-initiating behavior in murine models. Cancer cells lacking these features (CX3CR1Low) were capable of re-acquiring CX3CR1-associated features over time, implying that MICs can continuously emerge from non-stem cancer cells. CX3CR1 expression also conferred resistance to docetaxel, and prolonged treatment with docetaxel selected CX3CR1High phenotypes with de-enriched transcriptomic profiles for apoptotic pathways. These findings nominate CX3CR1 as a novel marker of stem-like tumor cells and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation.

Does the Montreal Cognitive Assessment (MoCA) identify cognitive impairment profiles in Parkinson's disease? An exploratory study.

An important proportion of patients with Parkinson's Disease (PD) present signs of cognitive impairment, although this is heterogeneous. In an attempt to classify this, the dual syndrome hypothesis distinguishes between two profiles: one defined by attentional and executive problems with damage in anterior cerebral regions, and another with mnesic and visuospatial alterations, with damage in posterior cerebral regions. The Montreal Cognitive Assessment (MoCA) is one of the recommended screening tools, and one of the most used, to assess cognitive impairment in PD. However, its ability to specifically identify these two profiles of cognitive impairment has not been studied. The aim of this study was, therefore, to analyze the capacity of the MoCA to detect cognitive impairment, and also to identify anterior and posterior profiles defined by the dual syndrome hypothesis. For this purpose, 59 patients with idiopathic PD were studied with the MoCA and a neuropsychological battery of tests covering all cognitive domains. Results of logistic regression analysis with ROC (Receiver Operating Characteristic) curves showed that MoCA detected cognitive impairment and identified patients with a profile of anterior/attentional and executive deficit, with acceptable sensibility and specificity. However, it did not identify patients with a posterior/mnesic-visuospatial impairment. We discuss the reasons for the lack of sensitivity of MoCA in this profile, and other possible implications of these results with regards the usefulness of this tool to assess cognitive impairment in PD.

Cloning and Production of Thermostable Enzymes for the Hydrolysis of Steryl Glucosides in Biodiesel.

Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.

Repeated exposure to JWH-018 induces adaptive changes in the mesolimbic and mesocortical dopaminergic pathways, glial cells alterations, and behavioural correlates.

BACKGROUND AND PURPOSE: Spice/K2 herbal mixtures, containing synthetic cannabinoids such as JWH-018, have been marketed as marijuana surrogates since 2004. JWH-018 has cannabinoid CB1 receptor-dependent reinforcing properties and acutely increases dopaminergic transmission selectively in the NAc shell. Here, we tested the hypothesis that repeated administration of JWH-018 (i) modulates behaviour, (ii) affects dopaminergic transmission and its responsiveness to motivational stimuli, and (iii) is associated with a neuroinflammatory phenotype. EXPERIMENTAL APPROACH: Rats were administered with JWH-018 once a day for 14 consecutive days. We then performed behavioural, electrophysiological, and neurochemical evaluation at multiple time points after drug discontinuation. KEY RESULTS: Repeated JWH-018 exposure (i) induced anxious and aversive behaviours, transitory attentional deficits, and withdrawal signs; (ii) decreased spontaneous activity and number of dopamine neurons in the VTA; and (iii) reduced stimulation of dopaminergic transmission in the NAc shell while potentiating that in the NAc core, in response to acute JWH-018 challenge. Moreover, (iv) we observed a decreased dopamine sensitivity in the NAc shell and core, but not in the mPFC, to a first chocolate exposure; conversely, after a second exposure, dialysate dopamine fully increased in the NAc shell and core but not in the mPFC. Finally, selected dopamine brain areas showed (v) astrogliosis (mPFC, NAc shell and core, VTA), microgliosis (NAc shell and core), and downregulation of CB1 receptors (mPFC, NAc shell and core). CONCLUSION AND IMPLICATIONS: Repeated exposure to JWH-018 may provide a useful model to clarify the detrimental effects of recurring use of Spice/K2 drugs.

Fungal endophytes in Peperomia obtusifolia and their potential as inhibitors of chickpea fungal pathogens.

Chickpea (Cicer arietinum L., Fabaceae) is the second most important legume after common bean (Phaseolus vulgaris L., Fabaceae) and third in production among the legumes grains worldwide. Ascochyta blight and Fusarium wilt are among the main fungal infections which cause the major losses of chickpea crop. In this work we report the phyto-pathogen controlling properties of 24 endophyte Phomopsis/Diaporthe isolates on the chickpea fungal pathogens Ascochyta rabiei, Fusarium oxysporum and Fusarium solani. The Phomopsis/Diaporthe strains were isolated amongst a total of 62 endophytic fungi from the aerial parts of the herbaceous perennial American plant Peperomia obtusifolia (Piperaceae) along with Fusarium, Septoria, Colletotrichum, Alternaria and Roussoella genera among others. Phomopsis/Diaporthe isolates were identified as Diaporthe infecunda (12 isolates), Diaporthe sackstoni (1 isolate), Diaporthe cf. brasiliensis (4 isolates) and Phomopsis cf. tuberivora (7 isolates). All the Phomopsis/Diaporthe strains antagonized A. rabiei strain AR2 with a mean of inhibition (% I) of 86.59 ± 1.49% in dual cultures. The metabolic characterization of the Phomopsis/Diaporthe strains showed groups in three clusters which were in agreement with the taxonomic identification. Bioautographic evaluation of organic extracts showed that those of D. cf. brasiliensis and D. infecunda were better as inhibitors. Strain Po 45 was one of the most active (cluster 1, 96.87% I), and its ethyl acetate extract inhibited A. rabiei growth in a bioautographic assay until at least 10 µg/mm applied showing a specific chromatographic band as the responsible of the A. rabiei inhibition.

Antibacterial activity of prenylated benzopyrans from Peperomia obtusifolia (Piperaceae).

Peperomia obtusifolia is a herbaceous perennial plant native to the Americas reported as a traditional medicine to treat snake bites and as a skin cleanser. The bioassay-guided fractionation of crude extracts from aerial parts of P. obtusifolia against a panel of clinically important fungi and bacteria, showed that hexane and dichloromethane extracts demonstrated selective bacterial inhibition, allowing the isolation of the known compounds peperobtusin A (1), and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3"-methyl-2"-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2H-1-benzopyran-6-carboxylic acid (2) from dichloromethane extract. Compound 2 was active against Gram-positive bacteria including community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates and an Enterococcus faecalis vancomycin-resistant strain, with minimal inhibitory concentration (MIC) values of 4 µg/mL (10.8 µM) and 8 µg/mL (21.6 µM) respectively. The interaction of compound 2 with the bacterial membrane was demonstrated by means of Zeta potential experiments on S. aureus, then confirming the membrane damage by fluorescent microscopy experiments.

Psychological factors of sibling caregivers of patients with severe mental disorders: an observational study.

The goal of the present study is to explore the perception of the relationship with parental figures, traumatic experiences, personality traits and psychosocial characteristics of the participant sibling caregivers. The sample was composed of 30 sibling caregivers recruited at psychiatric facilities in Italy, and of 30 control siblings. The battery of instruments administered included Parental Bonding Instrument (PBI), Minnesota Multiphasic Personality Inventory 2 (MMPI-2), and The Inventory of Traumatic Experiences (TEC). This research found that sibling caregivers of patients with severe psychiatric pathologies are distinctly different from the siblings of the control group with respect to the perception of their relationship with parental figures more frequently regarded as dysfunctional, and were also characterized by a higher presence of traumatic experiences. The problematic relationship with parental figures, some traumatic experiences, and the burden of taking care of a sibling with psychiatric disorders are probably important variables with regards to the individual's overall psychological condition.

Sex and Gender Differences in the Effects of Novel Psychoactive Substances.

Sex and gender deeply affect the subjective effects and pharmaco-toxicological responses to drugs. Men are more likely than women to use almost all types of illicit drugs and to present to emergency departments for serious or fatal intoxications. However, women are just as likely as men to develop substance use disorders, and may be more susceptible to craving and relapse. Clinical and preclinical studies have shown important differences between males and females after administration of "classic" drugs of abuse (e.g., Δ9-tetrahydrocannabinol (THC), morphine, cocaine). This scenario has become enormously complicated in the last decade with the overbearing appearance of the new psychoactive substances (NPS) that have emerged as alternatives to regulated drugs. To date, more than 900 NPS have been identified, and can be catalogued in different pharmacological categories including synthetic cannabinoids, synthetic stimulants (cathinones and amphetamine-like), hallucinogenic phenethylamines, synthetic opioids (fentanyls and non-fentanyls), new benzodiazepines and dissociative anesthetics (i.e., methoxetamine and phencyclidine-derivatives). This work collects the little knowledge reached so far on the effects of NPS in male and female animal and human subjects, highlighting how much sex and gender differences in the effects of NPS has yet to be studied and understood.

Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

A novel lecithin:cholesterol acyltransferase for soybean oil refining provides higher yields and extra nutritional value with a cleaner process.

The growing demand for food and biofuels urges the vegetable oil processing industry to adopt cleaner technologies to mitigate the environmental pollution caused by chemical refining processes. Over the past decade, several enzymatic methods have proven to be efficient at reducing the generated waste, but improving the benefit-cost ratio is still necessary for the widespread adoption of this technology. In this work, we show that lecithin:cholesterol acyltransferase from Aeromonas enteropelogenes (LCATAE) provides a higher extra-yield of soybean oil than a type A1 phospholipase (PLA) enzyme currently commercialized for soybean oil deep degumming. Our model indicates that crude soybean oil treated with the new enzyme generates 87% more neutral oil from phospholipids than the widely used PLA, with the corresponding reduction in waste and byproducts generation. The refined oil retains the phytosterols naturally present in crude oil, enriching its nutritional value. The results presented here position LCATAE as a promising candidate to provide the green solutions needed by the industrial oil processing sector. Key points • Selected LCAT gene candidates were expressed in E. coli. • Aeromonas enteropelogenes LCAT hydrolyzes all the phospholipids present in crude soybean oil. • The LCAT enzyme provides a higher yield of neutral oil than commercial PLA enzymes and generates less waste. • The degummed oil retains sterols with high nutritional value.