Activation of NOP receptor increases vulnerability to stress: role of glucocorticoids and CRF signaling.

RATIONALE: Recently, we demonstrated that the activation of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) signaling facilitates depressive-like behaviors. Additionally, literature findings support the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal (HPA) axis. OBJECTIVES: Considering that dysfunctional HPA axis is strictly related to stress-induced psychopathologies, we aimed to study the role of the HPA axis in the pro-depressant effects of NOP agonists. METHODS: Mice were treated prior to stress with the NOP agonist Ro 65-6570, and immobility time in the forced swimming task and corticosterone levels were measured. Additionally, the role of endogenous glucocorticoids and CRF was investigated using the glucocorticoid receptor antagonist mifepristone and the CRF1 antagonist antalarmin in the mediation of the effects of Ro 65-6570. RESULTS: The NOP agonist in a dose-dependent manner further increased the immobility of mice in the second swimming session compared to vehicle. By contrast, under the same conditions, the administration of the NOP antagonist SB-612111 before stress reduced immobility, while the antidepressant nortriptyline was inactive. Concerning in-serum corticosterone in mice treated with vehicle, nortriptyline, or SB-612111, a significant decrease was observed after re-exposition to stress, but no differences were detected in Ro 65-6570-treated mice. Administration of mifepristone or antalarmin blocked the Ro 65-6570-induced increase in the immobility time in the second swimming session. CONCLUSIONS: Present findings suggest that NOP agonists increase vulnerability to depression by hyperactivating the HPA axis and then increasing stress circulating hormones and CRF1 receptor signaling.

Ferutinin induces osteoblast differentiation of DPSCs via induction of KLF2 and autophagy/mitophagy.

Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding.

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds' reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.

Assessment of Ferula hermonis Boiss fertility effects in immature female rats supported by quantification of ferutinin via HPLC and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferula hermonis is a small shrub renowned for its aphrodisiac abilities. Middle East herbalists have utilized Ferula hermonis seed and root as an aphrodisiac folk medicine to treat women's frigidity and male erectile and sexual dysfunction. AIM OF THE STUDY: Assessment of follicle-stimulating hormone-like (FSH), luteinizing hormone-like (LH), and estrogenic activities of the methanolic extract (ME) of the roots of Ferula hermonis on female reproductive function. MATERIALS AND METHODS: The methanolic extract was prepared from the root of F. hermonis and studied at dose level 6 mg/kg in immature female rats for FSH-like, LH-like, and estrogenic activities. These activities were determined by analyzing gross anatomical features, relative organ weight, and serum level of FSH, LH, progesterone and estrogen hormones, and histopathological characteristics. Quantification of the main phytoestrogenic component ferutinin carried out by HPLC. In addition, molecular docking for the binding affinity of ferutinin inside active sites of both estrogen receptor alpha (ERα) and FSH receptor (FSHR) was performed to predict the potential role of ferutinin in regulating the female reproductive process. RESULTS: Ferula hermonis (ME) showed potent FSH-like, LH-like activities and moderate estrogenic effect at the dose of 6 mg/kg. The content of ferutinin in F. hermonis was estimated to be 92 ± 1.33 mg/g of the methanolic extract. Molecular docking of ferutinin with ERα and FSHR displayed strong interaction with target proteins. CONCLUSIONS: Based on results, it can be concluded that Ferula hermonis can be considered as a suitable female fertility improving agent.

Screening of natural compounds identifies ferutinin as an antibacterial and anti-biofilm compound.

Antibacterial screenings are most commonly targeted at planktonic bacteria but less effort is dedicated to the exploration of agents acting on biofilms. Here, a natural compounds library was screened against Staphylococcus aureus using a 384-well plate platform to identify compounds preventing biofilm formation. Five structurally diverse hits were selected for follow-up studies: honokiol, tschimganidin, ferutinin, oridonin and deoxyshikonin. The compounds were evaluated against different bacterial species for their capacity to prevent and disrupt biofilms. The development of resistance and cytotoxicity were also investigated. Ferutinin displayed the best antibacterial activity, with a minimum inhibitory, bactericidal and biofilm preventive concentration of 25 µM against S. aureus. It efficiently disrupted pre-formed biofilms (over 5-log reduction of viable cells) and reduced biofilm formation on a catheter in the presence of neutrophils. This work provides new information on the antibacterial activity of five natural compounds and identified ferutinin as a promising candidate against S. aureus biofilms.

The Effect of Ferula communis Extract in Escherichia coli Lipopolysaccharide-Induced Neuroinflammation in Cultured Neurons and Oligodendrocytes.

In recent decades, interest in natural compounds has increased exponentially due to their numerous beneficial properties in the treatment of various acute and chronic diseases. A group of plant derivatives with great scientific interest is terpenic compounds. Among the plants richest in terpenes, the genus Ferula L. is one of the most representative, and ferutinin, the most common sesquiterpene, is extracted from the leaves, rhizome, and roots of this plant. As reported in the scientific literature, ferutinin possesses antioxidant and anti-inflammatory properties, as well as valuable estrogenic properties. Neurodegenerative and demyelinating diseases are devastating conditions for which a definite cure has not yet been established. The mechanisms involved in these diseases are still poorly understood, and oxidative stress is considered to be both a key modulator and a common denominator. In the proposed experimental system, co-cultured human neurons (SH-SY5Y) and human oligodendrocytes (MO3.13) were treated with the pro-inflammatory agent lipopolysaccharide at a concentration of 1 µg/mL for 24 h or pretreated with ferutinin (33 nM) for 24 h and subsequently exposed to lipopolysaccharide 1 µg/mL for 24 h. Further studies would, however, be needed to establish whether this natural compound can be used as a support strategy in pathologies characterized by progressive inflammation and oxidative stress phenomena.

Synthesis, activity and mechanism for double-ring conjugated enones.

The relationship between TLR4 and inflammation-related diseases has been paid more and more attention. The studies have shown that TLR4/NF-κB signaling pathway plays an important role in the transmission of inflammatory signals. A large number of pro-inflammatory factors, chemokines, adhesion factors, TLR4 and its ligands interact with each other, and jointly promote the development of diseases. In this work, 8 target compounds were synthesized to screen the inhibitory activity of TLR4 in vitro. The results of TLR4 inhibition test in vitro showed that the double-ring conjugated enones had a good inhibitory activity, and the IC50 value of compound 4f was 0.56 ± 0.10 µM, and it was superior to the positive control methotrexate. To further study the anti-inflammatory effect and mechanism of double-ring conjugated enones by using LPS induced rat synovial cell inflammation model. The results of the mechanism test showed that compound 4f could effectively promote the apoptosis of rat synovial cells, and the mechanism might be related to the up-regulation of the expression of apoptosis-related protein Caspase-3. In addition, compound 4f could significantly inhibit the increase of inflammatory factors TNF-α, IL-1ß and IL-6 in rat synovial cells induced by LPS, showing a good anti-inflammatory activity. In the TLR4/NF-κB signaling pathway test of rat synovial cells, compound 4f can effectively regulate the expression levels of TLR4, MyD88, NF-κB and IκB related proteins in TLR4/NF-κB signaling pathway, which may be due to its inhibition of LPS-induced inflammation in rat synovial cells. At the same time, it inhibits the abnormal proliferation of cells and its important mechanism promoted of apoptosis.

Antinociceptive Efficacy of the µ-Opioid/Nociceptin Peptide-Based Hybrid KGNOP1 in Inflammatory Pain without Rewarding Effects in Mice: An Experimental Assessment and Molecular Docking.

Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund's adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

Topical azole treatments for otomycosis.

BACKGROUND: Otomycosis is a fungal infection of the outer ear, which may be treated with topical antifungal medications. There are many types, with compounds belonging to the azole group ('azoles') being among the most widely used. OBJECTIVES: To evaluate the benefits and harms of topical azole treatments for otomycosis. SEARCH METHODS: The Cochrane ENT Information Specialist searched the Cochrane ENT Register; Central Register of Controlled Trials (CENTRAL); Ovid MEDLINE; Ovid Embase; CINAHL; Web of Science; ClinicalTrials.gov; ICTRP and additional sources for published and unpublished trials. The search date was 11 November 2020. SELECTION CRITERIA: We included randomised controlled trials (RCTs) in adults and children with otomycosis comparing any topical azole antifungal with: placebo, no treatment, another type of topical azole or the same type of azole but applied in different forms. A minimum follow-up of two weeks was required. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were: 1) clinical resolution as measured by the proportion of participants with complete resolution at between two and four weeks after treatment (however defined by the authors of the studies) and 2) significant adverse events. Secondary outcomes were 3) mycological resolution and 4) other less serious adverse effects. We used GRADE to assess the certainty of evidence for each outcome. MAIN RESULTS: We included four studies with 559 participants from Spain, Mexico and India. Three studies included children and adults; one included only adults. The duration of symptoms was not always explicitly stated. Mycological resolution results were only reported in one study. The studies assessed two comparisons: one type of topical azole versus another and the same azole but administered in different forms (cream versus solution). A. Topical azoles versus placebo None of the studies assessed this comparison. B. Topical azoles versus no treatment None of the studies assessed this comparison. C. One type of topical azole versus another type of topical azole i) Clotrimazole versus other types of azoles (eberconazole, fluconazole, miconazole) Three studies examined clotrimazole versus other types of azoles. The evidence is very uncertain about the difference between clotrimazole and other types of azole in achieving complete clinical resolution at four weeks (risk ratio (RR) 0.80, 95% confidence interval (CI) 0.59 to 1.07; 3 studies; 439 participants; very low-certainty evidence). The anticipated absolute effects are 668 per 1000 for clotrimazole versus 835 per 1000 for other azoles. One study planned a safety analysis and reported no significant adverse events in either group. The evidence is therefore very uncertain about any differences between clotrimazole and other types of azole (no events in either group; 1 study; 174 participants; very low-certainty evidence). Clotrimazole may result in little or no difference in mycological resolution at two weeks follow-up (RR 1.01, 95% CI 0.96 to 1.06; 1 study; 174 participants; low-certainty evidence) or in other (less serious) adverse events at two weeks follow-up (36 per 1000, compared to 45 per 1000, RR 0.79, 95% CI 0.18 to 3.41; 1 study; 174 participants; very low-certainty evidence). ii) Bifonazole cream versus bifonazole solution One study compared bifonazole 1% cream with solution. Bifonazole cream may have little or no effect on clinical resolution at two weeks follow-up when compared to solution, but the evidence is very uncertain (RR 1.07, 95% CI 0.73 to 1.57; 1 study; 40 ears; very low-certainty evidence). Bifonazole cream may achieve less mycological resolution compared to solution at two weeks after the end of therapy, but the evidence for this is also very uncertain (RR 0.53, 95% CI 0.29 to 0.96; 1 study; 40 ears; very low-certainty evidence). Five out of 35 patients sustained severe itching and burning from the bifonazole solution but none with the bifonazole cream (very low-certainty evidence). AUTHORS' CONCLUSIONS: We found no studies that evaluated topical azoles compared to placebo or no treatment. The evidence is very uncertain about the effect of clotrimazole on clinical resolution of otomycosis, on significant adverse events or other (non-serious) adverse events when compared with other topical azoles (eberconazole, fluconazole, miconazole). There may be little or no difference between clotrimazole and other azoles in terms of mycological resolution. It may be difficult to generalise these results because the range of ethnic backgrounds of the participants in the studies is limited.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells.

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5-15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-ß-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

Ferutinin: A phytoestrogen from ferula and its anticancer, antioxidant, and toxicity properties.

This study was performed to evaluate the antioxidant, anticancer, and toxicity properties of ferutinin, a phytoestrogen derived from Ferula species. The human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line and normal human fibroblast (HDF) were cultured and treated with different ferutinin concentrations. The cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell death-defining tests (a comparative real-time polymerase chain reaction [for Bax and Bcl-2 genes], flow cytometry, and acridine orange/propidium iodide cell staining). Moreover, 15 white male balb/c mice were divided into three groups of five (one untreated control group and two groups), which received different doses of ferutinin-supplemented water (500 and 1000 µg/kg mice weight) to check the mice liver and kidney pathomorphological alterations and to determine the antioxidant enzymes' expression profile (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase) in the mentioned tissues. Finally, the liver lipid peroxidation of mice was analyzed. The results of MTT and cell death-defining tests indicate the significant reduction in cell viability and induction of apoptotic death in MCF-7 cells (enhanced sub-G1 peaks, Bax overexpression, Bcl-2 downregulation, and increased apoptotic cells). The antioxidant enzymes (SOD and CAT) in the mice liver and kidney cells were found to be upregulated (p < .05) in response to the increasing doses of ferutinin. Besides, the lipid peroxidation of the liver tissue of mice was significantly reduced. According to the results, we suggest that ferutinin has the potential to be served as a selective anticancer compound for breast cancer treatment.

Ferula L. Plant Extracts and Dose-Dependent Activity of Natural Sesquiterpene Ferutinin: From Antioxidant Potential to Cytotoxic Effects.

The employment studies of natural extracts in the prevention and treatment of several diseases highlighted the role of different species of genus Ferula L., belonging to the Apiaceae family, dicotyledonous plants present in many temperate zones of our planet. Ferula communis L. is the main source of sesquiterpene ferutinin, a bioactive compound studied both in vitro and in vivo, because of different effects, such as phytoestrogenic, antioxidant, anti-inflammatory, but also antiproliferative and cytotoxic activity, performed in a dose-dependent and cell-dependent way. The present review will focus on the molecular mechanisms involved in the different activities of Ferutinin, starting from its antioxidant potential at low doses until its ionophoric property and the subsequent mitochondrial dysfunction induced through administration of high doses, which represent the key point of its anticancer action. Furthermore, we will summarize the data acquired from some experimental studies on different cell types and on several diseases. The results obtained showed an important antioxidant and phytoestrogenic regulation with lack of typical side effects related to estrogenic therapy. The preferential cell death induction for tumor cell lines suggests that ferutinin may have anti-neoplastic properties, and may be used as an antiproliferative and cytotoxic agent in an estrogen dependent and independent manner. Nevertheless, more data are needed to clearly understand the effect of ferutinin in animals before using it as a phytoestrogen or anticancer drug.

Fear expression is reduced after acute and repeated nociceptin/orphanin FQ (NOP) receptor antagonism in rats: therapeutic implications for traumatic stress exposure.

RATIONALE: Evaluation of pharmacotherapies for acute stress disorder (ASD) or post-traumatic stress disorder (PTSD) is challenging due to robust heterogeneity of trauma histories and limited efficacy of any single candidate to reduce all stress-induced effects. Pursuing novel mechanisms, such as the nociceptin/orphanin FQ (NOP) system, may be a viable path for therapeutic development and of interest as it is involved in regulation of relevant behaviors and recently implicated in PTSD and ASD. OBJECTIVES: First, we evaluated NOP receptor antagonism on general behavioral performance and again following a three-species predator exposure model (Experiment 1). Then, we evaluated effects of NOP antagonism on fear memory expression (Experiment 2). METHODS: Adult, male rats underwent daily administration of NOP antagonists (J-113397 or SB-612,111; 0-20 mg/kg, i.p.) and testing in acoustic startle, elevated plus maze, tail-flick, and open field tests. Effects of acute NOP antagonism on behavioral performance following predator exposure were then assessed. Separately, rats underwent fear conditioning and were later administered SB-612,111 (0-3 mg/kg, i.p.) prior to fear memory expression tests. RESULTS: J-113397 and SB-612,111 did not significantly alter most general behavioral performance measures alone, suggesting minimal off-target behavioral effects of NOP antagonism. J-113397 and SB-612,111 restored performance in measures of exploratory behavior (basic movements on the elevated plus maze and total distance in the open field) following predator exposure. Additionally, SB-612,111 significantly reduced freezing behavior relative to control groups across repeated fear memory expression tests, suggesting NOP antagonism may be useful in dampening fear responses. Other measures of general behavioral performance were not significantly altered following predator exposure. CONCLUSIONS: NOP antagonists may be useful as pharmacotherapeutics for dampening fear responses to trauma reminders, and the present results provide supporting evidence for the implication of the NOP system in the neuropathophysiology of dysregulations in fear learning and memory processes observed in trauma- and stress-related disorders.

Silyl Tosylate Precursors to Cyclohexyne, 1,2-Cyclohexadiene, and 1,2-Cycloheptadiene.

Transient strained cyclic intermediates have become valuable intermediates in modern synthetic chemistry. Although silyl triflate precursors to strained intermediates are most often employed, the instability of some silyl triflates warrants the development of alternative precursors. We report the syntheses of silyl tosylate precursors to cyclohexyne, 1,2-cyclohexadiene, and 1,2-cycloheptadiene. The resultant strained intermediates undergo trapping in situ to give cycloaddition products. Additionally, the results of competition experiments between silyl triflates and silyl tosylates are reported.

Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation.

Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.

Blockade of NOP receptor modulates anxiety-related behaviors in mice exposed to inescapable stress.

RATIONALE: Depression and anxiety frequently co-occur, and this has important clinical implications. Previous studies showed that activation of the nociceptin/orphanin FQ receptor (NOP) elicits anxiolytic effects, while its blockade promotes consistent antidepressant actions. NOP antagonists are effective in reversing footshock-induced depressive-like behaviors, but their effects on stress-induced anxiety are still unclear. OBJECTIVE: This study aimed to investigate the effects of the NOP antagonist SB-612111 on footshock stress-induced anxiety behaviors. METHODS: Male Swiss mice were exposed to inescapable electric footshock stress, and behavioral phenotype was screened based on the ability to escape from footshock (i.e., helpless or non-helpless). Animals were then treated with diazepam (1 mg/kg) and SB-612111 (0.1-10 mg/kg), and their behavior was assessed in the elevated plus-maze (EPM) and open field test. RESULTS: When compared with non-stressed mice, helpless, but not non-helpless, animals displayed significant reductions in the time spent in and entries into open arms in the EPM. Diazepam significantly increased open arms exploration in helpless, non-helpless, and non-stressed mice. However, treatment with the NOP antagonist SB-612111 was inactive in naive mice, while it reversed anxiogenic-related behaviors in helpless mice and increased anxiety states in non-helpless mice. No effects on locomotion were observed. CONCLUSION: Helpless mice displayed increased anxiety compared to non-stressed and non-helpless animals, thus supporting use of this approach as an animal model to investigate anxiety/depression comorbidity. Additionally, SB-612111 modulated anxiety-like behaviors in male mice depending on individual stress susceptibility. Ultimately, NOP antagonists could be useful for treating anxiety in depressed patients.

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake.

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

Potent and selective NOP receptor activation reduces cocaine self-administration in rats by lowering hedonic set point.

Developing new medications for the treatment of cocaine dependence continues to be a research priority. Compelling evidence indicates that mixed opioid receptor agonists, particularly bifunctional compounds that target nociceptin/orphanin FQ peptide (NOP) and mu opioid receptors, may be useful for the treatment of cocaine addiction. Here, we verify that potent and selective pharmacological activation of NOP receptors is sufficient to reduce relevant facets of cocaine addiction in animal models. Accordingly, we determined whether systemic injections of the small molecule AT-312 (0, 1, 3 mg/kg) could reduce operant cocaine self-administration, motivation for cocaine, and vulnerability to cocaine relapse in rats. Results indicate that a potent and selective NOP receptor agonist was equally efficacious in reducing the number of cocaine infusions in short (1-hour), as well as long (6-hour) access sessions. When tested on an economic-demand reinforcement schedule, AT-312 reduced Q0 , the parameter that describes the amount of drug consumed at zero price, while leaving the parameter α, a measure of motivation for drug consumption, unaltered. Furthermore, AT-312 successfully reduced conditioned reinstatement of cocaine seeking. In contrast, the NOP receptor agonist did not modify food self-administration. Blockade of the NOP receptor with the antagonist SB-612111 prevented the effect of AT-312 in decreasing cocaine-reinforced responding under a 2-hour fixed ratio 1 schedule, suggesting a NOP receptor-mediated mechanism. This work demonstrates that potent and selective activation of NOP receptors is sufficient to decrease cocaine taking and seeking behaviors in rats.

Ferula hermonis: A Review of Current Use and Pharmacological Studies of its Sesquiterpene Ester Ferutinin.

Ferula hermonis Boiss, is an endemic plant of Lebanon, locally known as "shilsh Elzallouh". It has been extensively used in the traditional medicine as an aphrodisiac and for the treatment of sexual impotence. Crude extracts and isolated compounds of ferula hermonis contain phytoestrogenic substances having a wide spectrum of in vitro and in vivo pharmacological properties including anti-osteoporosis, anti-inflammatory, anti-microbial and anti-fungal, anti-cancer and as sexual activity enhancer. The aim of this mini-review is to highlight the traditional and novel applications of this plant's extracts and its major sesquiterpene ester, ferutinin. The phytochemical constituents and the pharmacological uses of ferula hermonis crude extract and ferutinin specifically will be discussed.

Ferutinin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling.

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/ß-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.