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We provide an extensive review of 14 studies (11 independent and three industryfunded) on emissions generated by Electronic Cigarettes (ECs), specifically focusing on the evaluation of carbonyls present in these emissions and emphasizing a meticulous evaluation of their analytical methods and experimental procedures. Since the presence of carbonyl by-products in EC aerosol is concerning, it is important to evaluate the reliability of emission studies quantifying these compounds by verifying their compliance with the following criteria of experimental quality: authors must 1) supply sufficient information on the devices and experimental procedures to allow for potentially reproducing or replicating the experiments, 2) use of appropriate puffing protocols that approach consumer usage as best as possible, 3) use of appropriate analytical methods and 4) usage of blank samples to avoid false positive detection. Outcomes were classified in terms of the fulfilment of these conditions as reliable in seven studies, partially reliable in five studies, and unreliable in two studies. However, only five studies used blank samples and six studies failed the reproducibility criterion. Carbonyl yields were far below their yields in tobacco smoke in all reproducible studies, even in the partially reliable ones, thus supporting the role of ECs (when properly tested and operated) as harm reduction products. This review highlights the necessity to evaluate the quality of laboratory standards in testing EC emissions to achieve an objective assessment of the risk profile of ECs. [ABSTRACT FROM AUTHOR]

Diabetic neuropathy (DN) is a long-term complication of diabetes mellitus, affecting different periphery nerve systems including sensory and motor neurons. Hyperglycemia is the major cause of DN with symptoms such as weakness of balance or coordination, insensitivity to sensation, weakness of the muscles as well as numbness and pain in limbs Analgesic drug such as opioids can be effective to relief neuropathy pain but there is no effective treatment. Adiponectin is an anti-diabetic adipokine, which possesses insulin-sensitizing and neuroprotective effects. In this project, we aim to identify an agent which is dual acting to opioid and adiponectin receptors. Within a virtual screening repositioning campaign, a large collection of compounds with different structures comprehensive of adipoRon- like piperidine derivatives was screened by docking. Recently developed opioid receptor benzomorphanic agonists finally emerged as good ligands to adiponectin receptors showing some 2D and 3D structural similarities with AdipoRon. Particularly, we have identified (+)-MML1017, which has high affinity to the same binding domain of AdipoR1 and AdipoR2 as AdipoRon. Our western blot results indicate (+)-MML1017 activates AMPK phosphorylation through both adipoR1 and adipoR2 in neuronal cell line. Moreover, pretreatment of (+)-MML1017 can improve the cell viability with motor neurons under hyperglycermic conditions. The (+)-MML1017 also activates μ-opioid receptor cells in a concentration-dependent manner. Our study identified a novel compound having dual activity on opioid receptors and adiponectin receptors that may have analgesic effects and neuroprotective effects to treat diabetic neuropathy. [Display omitted] [ABSTRACT FROM AUTHOR]

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an ‘allosteric’ site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5 , bearing a diphenylic N -substituent on the benzomorphan nucleus, showed an affinity ( K i μ = 0.5 ± 0.2 nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with ‘orthosteric’ and ‘allosteric’ binding sites. [ABSTRACT FROM AUTHOR]

Although the epidemic caused by SARS-CoV-2 callings for international attention to develop new effective therapeutics, no specific protocol is yet available, leaving patients to rely on general and supportive therapies. A range of respiratory diseases, including pulmonary fibrosis, have been associated with higher iron levels that may promote the course of viral infection. Recent studies have demonstrated that some natural components could act as the first barrier against viral injury by affecting iron metabolism. Moreover, a few recent studies have proposed the combination of protease inhibitors for therapeutic use against SARS-CoV-2 infection, highlighting the role of viral protease in virus infectivity. In this regard, this review focuses on the analysis, through literature and docking studies, of a number of natural products able to counteract SARS-CoV-2 infection, acting both as iron chelators and protease inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: GNAO1 encephalopathy is characterized by severe hypotonia, psychomotor retardation, epilepsy, and movement disorders. Genetic variations in GNAO1 have been linked to neurological symptoms including movement disorders like dystonia. The correlation between the E246K mutation in the Gα subunit and aberrant signal transduction of G proteins has been established but no data are reported regarding the efficacy of medical treatment with tetrabenazine. Methods: Molecular modeling studies were performed to elucidate the molecular mechanisms underlying this mutation. We developed drug efficacy models using molecular dynamic simulations that replicated the behavior of wild-type and mutated proteins in the presence or absence of ligands. Results and discussion: We demonstrated that the absence of the mutation leads to normal signal transduction upon receptor activation by the endogenous ligand, but not in the presence of tetrabenazine. In contrast, the presence of the mutation resulted in abnormal signal transduction in the presence of the endogenous ligand, which was corrected by the drug tetrabenazine. Tetrabenazine was identified as a promising therapeutic option for pediatric patients suffering from encephalopathy due to an E246K mutation in the GNAO1 gene validated through molecular dynamics. This is a potential first example of the use of this technique in a rare neurological pediatric disease. [ABSTRACT FROM AUTHOR]

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4–7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of β-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline. [ABSTRACT FROM AUTHOR]

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer. [ABSTRACT FROM AUTHOR]

Over the last few years, the study of the SARS-CoV-2 spike protein and its mutations has become essential in understanding how it interacts with human host receptors. Since the crystallized structure of the spike protein bound to the angiotensin-converting enzyme 2 (ACE2) receptor was released (PDB code 6M0J), in silico studies have been performed to understand the interactions between these two proteins. Specifically, in this study, heterocyclic compounds with different chemical characteristics were examined to highlight the possibility of interaction with the spike protein and the disruption of the interaction between ACE2 and the spike protein. Our results showed that these compounds interacted with the spike protein and interposed in the interaction zone with ACE2. Although further studies are needed, this work points to these heterocyclic push–pull compounds as possible agents capable of interacting with the spike protein, with the potential for the inhibition of spike protein–ACE2 binding. [ABSTRACT FROM AUTHOR]

Brassica incana subsp. raimondoi is an endemic taxon present in a restricted area located on steep limestone cliffs at an altitude of about 500 m a.s.l. in eastern Sicily. In this research, for the first time, studies on the phytochemical profile, the antioxidant properties in cell-free and cell-based systems, the cytotoxicity on normal and cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, and on Artemia salina Leach, were performed. The total phenolic, flavonoid, and condensed tannin contents of the leaf hydroalcoholic extract were spectrophotometrically determined. Ultra-performance liquid chromatography—tandem mass spectrometer (UPLC-MS/MS) analysis highlighted the presence of several phenolic acids, flavonoids, and carotenoids, while High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) identified various kaempferol and isorhamnetin derivatives. The extract exhibited different antioxidant properties according to the five in vitro methods used. Cytotoxicity by MTT assay evidenced no impact on normal human fibroblasts (HFF-1) and prostate cancer cells (DU145), and cytotoxicity accompanied by necrotic cell death for colon cancer cells (CaCo-2) and hepatoma cells (HepG2), starting from 100 μg/mL and 500 μg/mL, respectively. No cytotoxic effects were detected by the A. salina lethality bioassay. In the H2O2-induced oxidative stress cell model, the extract counteracted cellular reactive oxygen species (ROS) production and preserved non-protein thiol groups (RSH) affected by H2O2 exposure in HepG2 cells. Results suggest the potential of B. incana subsp. raimondoi as a source of bioactive molecules. [ABSTRACT FROM AUTHOR]

The growing interest in natural bioactive molecules, as an approach to many pathological contexts, is widely justified by the necessity to overcome the disadvantageous benefit–risk ratio related to traditional therapies. Among them, mangiferin (MGF) shows promising beneficial properties such as antioxidant, anti-inflammatory, and immunomodulatory effects. In this study, we aimed to investigate the antioxidant and anti-inflammatory properties of MGF on lipopolysaccharide (LPS)-induced lung NCI-H292 cells, focusing on its role against COVID-19 adsorption. In order to obtain this information, cells treated with LPS, with or without MGF, were analyzed performing wound healing, gene expression of inflammatory cytokines, GSH quantification, and JC-1 staining. Moreover, the inhibition of viral adsorption was evaluated microbiologically and the results were further confirmed by molecular docking analysis. In this regard, MGF downregulates the expression of several inflammatory factors, enhances GSH levels, promotes the wound healing rate, and restores the mitochondrial dysfunction caused by LPS. In addition, MGF significantly inhibits SARS-CoV-2 adsorption as shown by the gene expression of ACE2 and TMPRSS-2, and furtherly confirmed by microbiological and molecular modeling evaluation. Although more investigations are still needed, all data obtained constitute a solid background, demonstrating the cytoprotective role of MGF in inflammatory mechanisms including COVID-19 infection. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is the leading cause of death worldwide. The severity of BC strictly depends on the molecular subtype. The less aggressive hormone-positive subtype is treated with adjuvant endocrine therapy (AET), which causes both physical and psychological side effects. This condition strongly impacts the adherence and persistence of AET among oncologic patients. Moreover, viral infections also constitute a serious problem for public health. Despite their efficacy, antiviral agents present several therapeutic limits. Accordingly, in the present work, we investigated the antitumor and antiviral activities of Orobanche crenata Forssk. (O. crenata), a parasitic plant, endemic to the Mediterranean basin, traditionally known for its beneficial properties for human health. Methods: The MTT assay was carried out to evaluate the cytotoxic effect of O. crenata leaf extract (OCLE) on human breast cancer cells (MCF-7 and MDA-MB-231) and the primary HFF-1 cell line. The lactic dehydrogenase (LDH) assay was performed on MCF-7 cells to analyze necrotic cell death. The antioxidant effect of OCLE was evaluated by intracellular determination of the reactive oxygen species and thiol groups, by DPPH and ABTS assays. The antiviral activity of OCLE was determined against Poliovirus 1, Echovirus 9, Human respiratory syncytial virus, Adenovirus type 2 and type 5, Coxsackievirus B1 (CoxB1) and B3 (CoxB3), Herpes simplex type 1 (HSV-1) and type 2 (HSV-2), and β-Coronavirus by the plaque reduction assay. Results: The extract, after 24 h of incubation, did not affect MDA-MB-231 and HFF-1 cell viability. However, at the same time point, it showed a dose-dependent inhibitory effect on MCF-7 cells, with an increase in LDH release. OCLE exhibited free radical scavenging activity and significantly increased non-protein thiol levels in MCF-7 cells. OCLE effectively inhibited HSV-1, HSV-2, CoxB1, and CoxB3 replication. Conclusions: The overall results showed an interesting inhibitory effect of OCLE on both MCF-7 cell survival and viral replication. [ABSTRACT FROM AUTHOR]

Abstract: Nocistatin (NST) and Nociceptin/Orphanin FQ (N/OFQ) are derived from the same precursor protein, pre-proN/OFQ, and exert opposite effects on the modulation of pain signals. However, the role of the peripheral N/OFQ and the NOP receptor, which is located at the endings of sensory nerves, in inflammatory pain was not ascertained. NST administered intrathecally (i.t.) prevented the nociceptive effects induced by i.t. N/OFQ and PGE2. Moreover an up regulation of N/OFQ was shown in the rat in response to peripheral inflammation. Here, we investigated the effects of intraplantar (i.pl.) administration of functional N/OFQ and NOP receptor antagonists in a rat model of inflammatory pain. Our findings showed that i.pl. injection of (±)-J 113397, a selective antagonist of the NOP receptor, and NST, the functional N/OFQ antagonist, prior to carrageenan significantly reduced the paw allodynic and thermal hyperalgesic threshold induced by the inflammatory agent. The resulting antiallodynic and antihyperalgesic effects by co-administering NST and (±)-J 113397 prior to carrageenan were markedly enhanced, and the basal latencies were restored. Thus, it is likely that the peripheral N/OFQ/NOP receptor system contributes to the abnormal pain sensitivity in an inflammatory state. [Copyright &y& Elsevier]

Abstract: Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG. [Copyright &y& Elsevier]

Abstract: The effects of a novel N-methyladamantan-1-amine derivative [(−)-MR22] with high σ1 receptor affinity were investigated on retinal degeneration using a rat model of ischemia–reperfusion injury. The animals were anaesthetized and retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 45 min. The drug was injected intraperitoneally before the ischemic damage. Retinal biochemical changes, i.e. increase of lactate content and decrease of glucose and ATP were significantly inhibited by the new and selective σ1 receptor ligand compared to the ischemic control group. The effect of (−)-MR22 was antagonized by pre-treatment with the σ1 site antagonist. The protective effect of (−)-MR22 on ischemic retina was confirmed by the histological analysis. These findings suggest that (−)-MR22 serves as a retinal neuroprotective agent and acts as a σ1 receptor agonist. [Copyright &y& Elsevier]

The development of Tyrosinase inhibitors (TYRIs) could represent an efficacious strategy for pharmacological intervention on skin pathologies related to aberrant production of melanin. Based on in silico studies we designed and tested a library of twenty-four compounds bearing the 4-(4-fluorobenzyl)piperazin-1-yl]-fragment. As result, we identified several compounds with excellent inhibit effects at low micromolar concentration against TYR from Agaricus bisporus (TyM). Among them, compound 25 (IC 50 = 0.96 μM) proved to be ∼20-fold more potent than the reference compound kojic acid (IC 50 = 17.76 μM) having wide applications in the cosmetics and pharmaceutical industries. The mode of interaction of active inhibitor 25 was deciphered by means of crystallography as well as molecular docking and these results were consistent with kinetic experiments. Moreover, the identified compound 25 exhibited no considerable cytotoxicity and showed anti-melanogenic effects on B16F10 melanoma cells. Therefore, a combination of computational and biochemical approaches could represent a rational guidelines for further structural modification of this class of compounds as future anti-melanogenic agents. Image 1 • Computational studies suggested the rational design of new tyrosinase inhibitors. • Twenty-four 4-(4-fluorobenzyl)piperazine derivatives were synthesized and screened. • All compounds inhibited tyrosinase (TyM) at low micromolar concentration. • Compound 25 demonstrated antimelanogenic effects and low cytotoxicity. • Crystal complex with TyBm demonstrated the interaction within enzymatic cavity. [ABSTRACT FROM AUTHOR]

A series of compounds generated by ring expansion/opening and molecular elongation/simplification of the 1,3-dioxolane scaffold were prepared and tested for binding affinity at 5-HT 1A R and α 1 adrenoceptors. The compounds with greater affinity were selected for further functional studies. N-((2,2-diphenyl-1,3-dioxan-5-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-ammonium hydrogen oxalate (12) emerged as highly potent full agonist at the 5-HT 1A R (pKi 5-HT 1A = 8.8; pD 2 = 9.22, %E max = 92). The pharmacokinetic data in rats showed that the orally administered 12 has a high biodistribution in the brain compartment. Thus, 12 was further investigated in-vivo, showing an anxiolytic and antidepressant effect. Moreover, in the formalin test, 12 was able to decrease the late response to the noxious stimulus, indicating a potential use in the treatment of chronic pain. Image 1 • A new series of 1,3-dioxolane analogues were prepared and tested in vitro for binding affinity, potency, efficacy at 5-HT 1A R and α 1 adrenoceptors. • Compound 12 emerged as a potent and selective 5-HT 1A R agonist with an high biodistribution in the brain compartment as assessed by pharmacokinetic data in rats. • Compound 12 exhibited anxiolytic (Elevated Plus Maze and Open Field test) and antidepressant (Forced Swim test) effect. • Compound 12 showed anti-nociceptive activity in the formalin test. [ABSTRACT FROM AUTHOR]

Recently, 1-(1,4-dioxaspiro[4,5]dec-2-ylmethyl)-4-(2-methoxyphenyl)piperazine ( 1 ) was reported as a potent 5-HT 1A R agonist with a moderate 5-HT 1A R selectivity. In an extension of this work a series of derivatives of 1, obtained by combining different heterocyclic rings with a more flexible amine chain, was synthesized and tested for binding affinity and activity at 5-HT 1A R and α 1 adrenoceptors. The results led to the identification of 14 and 15 as novel 5-HT 1A R partial agonists, the first being outstanding for selectivity (5-HT 1A /α 1d = 80), the latter for potency (pD 2 = 9.58) and efficacy (E max = 74%). Theoretical studies of ADME properties shows a good profile for the entire series and MDCKII-MDR1 cells permeability data predict a good BBB permeability of compound 15 , which possess a promising neuroprotective activity. Furthermore, in mouse formalin test, compound 15 shows a potent antinociceptive activity suggesting a new strategy for pain control. [ABSTRACT FROM AUTHOR]

The benzomorphan scaffold has great potential as lead structure and the nature of the N -substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N -substituent ( 9 – 15 ). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity ( K i MOR = 38 ± 4 nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA 2 ) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD 50 = 2.0 mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N -substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function. [ABSTRACT FROM AUTHOR]

Herein we report the synthesis and biological activity of new sigma receptor (σR) ligands obtained by combining different substituted five-membered heterocyclic rings with appropriate σR pharmacophoric amines. Radioligand binding assay, performed on guinea pig brain membranes, identified 25b (1-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)-4-benzylpiperazine) as the most interesting compound of the series, displaying high affinity and selectivity for σ 1 R (pK i σ 1 = 9.13; σ 1 /σ 2 = 47). The ability of 25b to modulate the analgesic effect of the κ agonist (−)-U-50,488H and μ agonist morphine was evaluated in vivo by radiant heat tail-flick test. It exhibited anti-opioid effects on both κ and μ receptor-mediated analgesia, suggesting an agonistic behavior at σ 1 R. Docking studies were performed on the theoretical σ 1 R homology model. The present work represents a new starting point for the design of more potent and selective σ 1 R ligands. [ABSTRACT FROM AUTHOR]

Germinated brown rice is considered a functional food in relation to the presence of beneficial nutrients and bioactive compounds in considerable amounts. The present study, evaluated, for the first time, the suitability of germinated brown rice for the production of value-added fermented products functionalized by the addition of probiotics. Four different fermented products with (EYGG, EYBB536, EYBB12, and EYM) and without (EY) probiotics addition were formulated. Microbiological and chemical profiles, presence of bioactive compounds, antioxidant activity, sensory attributes, and shelf-life during refrigerated storage were evaluated. Results showed that the fermentation process determined the improvement of both bioactive compound profile and anti - inflammatory properties of germinated brown rice. All the fermented products were microbiologically stable during refrigerated storage at +4 °C for 28 days. In addition, a high count of both lactobacilli and bifidobacteria was achieved during the shelf-life, indicating the suitability of the germinated brown rice as a probiotic carrier. Based on the sensory profile, high acceptability scores were attributed by panelists to the germinated brown rice experimental products. Based on the aforementioned results, germinated brown rice can be processed as a new fermented formulation with potential health benefits. • Germinated brown rice is suitable for the formulation of fermented products. • The fermentation improved both bioactive compounds and anti - inflammatory properties. • The fermentation increased both GABA and oryzanol neutralizing the phytic acid. • The fermented products downregulated the genes expression of IL-1β, IL-6 and TNF α. • Probiotics survived in fermented products during refrigerated storage for 28 days. [ABSTRACT FROM AUTHOR]

The new sigma ligand cis-(±)-methyl (1R,2S/1S,2R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl) methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(±)-PPCC] is a promising tool for the treatment of various diseases. With the aim to investigate the absorption of (±)-PPCC by the cell membranes, in this study we evaluated the influence on thermotropic behavior of membrane model exerted by PPCC both as free base or as oxalic salt. To fulfill this purpose differential scanning calorimetry was used. The findings highlight that PPCC affects the thermodynamic parameters of phospholipids in different manner depending on whether it is in the salt or base form as well as function of the amount of drugs dispersed in the lipid matrix. The salt form of PPCC was uptaken by the membrane model faster than the free base. In addition, preliminary information on the use of a lipophilic carrier for PPCC was obtained. [ABSTRACT FROM AUTHOR]

Abstract: An emerging approach in pain management is the use of multitarget opioid ligands, owing an improved analgesic effect coupled to a reduced incidence of side effects. With a mu opioid receptor agonist/delta opioid receptor antagonist profile, the benzomorphan-based compound LP1 belongs to multitarget ligands class. Previous in vivo investigations showed that LP1 – subcutaneously administered as oxalate salt – was an antinociceptive agent as potent as morphine with a low tolerance-inducing capability. Because the renal toxicity of oxalate is known, an alternative approach allowing the administration of LP1 freebase could be more biocompatible. In this study the interaction of LP1 freebase and LP1 oxalate salt with multilamellar vesicles, as membrane model, was evaluated using differential scanning calorimetry technique. Despite the good membrane interaction showed by LP1 freebase, it was not capable to diffuse in the aqueous medium and to be uptaken by multilamellar vesicles. On the other hand, LP1 freebase possessed a good transfer profile by a liposomal carrier to a biomembrane model. Considering our findings and the need of safe formulations, studies for the development of a suitable carrier for a systemic administration of LP1 freebase are in progress. [Copyright &y& Elsevier]

Objective and design: The sigma 1 (σ) receptor, which is widely distributed in the CNS in areas that are known to be important for pain control, may play a role in persistent pain characterized by the hypersensitivity of nociceptive transmission. Here, we investigated the effect of σ blockade in an inflammatory pain model. Treatment and methods: An intraplantar injection of carrageenan (2 %) was used to induce paw inflammation. The effects of the σ antagonist (+)-MR200, given subcutaneously at a dose of 0.1, 0.25, 0.5,1, 1.5, and 2 mg/kg prior to injection of carrageenan, on inflammatory pain and inflammation were assessed. Mechanical allodynia with von Frey filaments, thermal hyperalgesia with the plantar test and edema evaluation with a plethysmometer were measured. Intergroup comparisons were assessed by one- or two-way analysis of variance when appropriate, followed by post-hoc tests (Dunnett's test for one-way or Bonferroni for two-way ANOVA). Results: (+)-MR200 dose-dependently prevented allodynia and hyperalgesia induced by carrageenan. Furthermore, it reduced paw edema with a significant inhibition percentage of 37.82 % at 3 h after carrageenan treatment. Conclusions: The blockade of the σ receptor with the selective antagonist (+)-MR200 may contribute to the suppression of the typical symptoms of inflammatory pain. [ABSTRACT FROM AUTHOR]

The compound (−)-MRV3 [(−)-methyl (1S,2R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)-methyl]-1-phenylcyclopropanecarboxylate] has an assessed antagonist sigma 1 (σ) profile and showed improved σ/σ selectivity with respect to the parent compound (+)-MR200. The σ receptor is reported to play a role in both central sensitization and pain hypersensitivity, which suggests a potential use of σ antagonists for the treatment of persistent pain conditions. The present study was performed to assess the effects of the selective σ antagonist (−)-MRV3, in carrageenan-induced inflammatory hyperalgesia, allodynia, and edema. Mechanical allodynia with a series of calibrated von Frey's filaments, thermal hyperalgesia with plantar test, and edema evaluation with a plethysmometer were measured. Subcutaneous (s.c.) treatment with (−)-MRV3 (1, 2, 3, 4, 5 mg/kg) dose-dependently reduced allodynia and hyperalgesia induced by intraplantar carrageenan. Furthermore, treatment with (−)-MRV3 (3 mg/kg s.c.) also inhibited paw edema with a significant inhibition of 37.82 % 3 h after carrageenan treatment. These results provide a strong basis for the use of σ receptor antagonists in the treatment of inflammatory pain. [ABSTRACT FROM AUTHOR]

Abstract: Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA, a small series of 1-(1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives has been synthesized by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only the 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity. Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward σ2 receptor (K i values of 10nM and 20nM, respectively). Thus, in this case the discovery of new σ2 receptor selective ligands was an unexpected result emerging from the screening of cross-activity against other CNS receptors. [Copyright &y& Elsevier]

The revolutions in biotechnology and information technology have produced clinical data, which complement biological data. These data enable detailed descriptions of various healthy and diseased states and responses to therapies. For the investigation of the physiology and pathology of the immune responses, computer and mathematical models have been used in the last decades, enabling the representation of biological processes. In this modeling effort, a major issue is represented by the communication between models that work at cellular and molecular level, that is, multiscale representation. Here we sketch some attempts to model immune system dynamics at both levels. [ABSTRACT FROM AUTHOR]

Abstract: Persistent pain states, such as those caused by nerve injury or inflammation, are associated with altered sensations, allodynia and hyperalgesia, that are resistant to traditional analgesics. A contribution to development and maintenance in altered pain perception comes from nociceptive processing and descending modulation from supraspinal sites. A multitarget ligand seems to be useful for pain relief with a decreased risk of adverse events and a considerable analgesic efficacy. The multitarget MOR agonist–DOR antagonist LP1, (3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive agent with low potential to induce tolerance. LP1 was tested in models of neuropathic pain – induced by chronic constriction injury (CCI) of the left sciatic nerve – and inflammatory pain – produced by intraplantar injection of carrageenan. In CCI rats, subcutaneous (s.c.) LP1 (3 mg/kg) showed a significant antiallodynic effect, measured with von Frey filaments, and antihyperalgesic effect, evoked in response to a radiant heat stimulus with plantar test. Analogously, LP1 significantly reduced allodynic and hyperalgesic thresholds in a model of inflammatory pain induced by carrageenan. To evaluate the contribution of opioid receptor subtypes in LP1 antinociceptive effects, the multitarget LP1 profile was assessed using selective opioid antagonists. Moreover, functional electrophysiological in vitro assays, using primary cortical and spinal cord networks, allowed to define the “pharmacological fingerprint” of LP1. The EC50 values in this functional screening seem to confirm LP1 as a potent opioid ligand (EC50 = 0.35 fM and EC50 = 44 pM in spinal cord and frontal cortex, respectively). Using a NeuroProof data-base of well characterised reference compounds, a similarity profile of LP1 to opioid and non-opioid drugs involved in pain modulation was detected. Our studies seem to support that multitarget ligand approach should be useful for persistent pain conditions in which mechanical allodynia and thermal hyperalgesia are significant components of the nociceptive response. [Copyright &y& Elsevier]

Abstract: Aims: Opioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects. Main methods: We explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug''s DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist. Key findings: Data obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia. Significance: LP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain. [Copyright &y& Elsevier]

Abstract: Aims: Powerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine. Main methods: Here, we evaluated the pharmacological effects of LP1 administered at a dose of 4mg/kg subcutaneously (s.c.) twice per day for 9days to male Sprague–Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced [35S]GTPγS binding to the MOR and DOR. Key findings: Data obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced [35S]GTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR. Significance: LP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR. [Copyright &y& Elsevier]

Abstract: Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ1 receptor affinity (K i values <25nM) and good σ1/σ2 selectivity (K iσ2 >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ1 receptor ligands (K i =0.70 and 0.86nM, respectively) and to display significantly high selectivity over σ2, μ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ1 receptor antagonist BD-1063 totally counteracted this effect, confirming that σ1 receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ1 agonist profile. As a part of our work, a threedimensional σ1 pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ1 receptor subtype. [Copyright &y& Elsevier]

Novel enantiomers and diastereoisomers structurally related to σ ligand (+)-MR200 were synthesized to improve σ1/σ2 subtype selectivity. The selective σ1 ligand (–)-8 showed an antagonist profile determined by phenytoin differential modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The σ2 ligand (–)-9 displayed agonist properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines. [ABSTRACT FROM AUTHOR]

Sigma-1 receptor agonists have recently attracted much attention as potential therapeutic drugs for cognitive and affective disorders, however, it is still unclear whether they act via modulation of transmitter release or activation of sigma-1 receptors in memory-related brain regions. In the present study, we have investigated the anti-amnesic and neuroprotective actions of the compound (-)-methyl (1S,2R)-2-{[1-adamantyl(methyl)amino]methyl}-1-phenylcyclopropane-carboxylate) [(-)-MR22], a selective sigma-1 receptor agonist able to protect cultured cortical neurons from amyloid toxicity. To this aim, cognitive deficits, cholinergic loss, and amyloid peptide accumulation were obtained in the rat by simultaneous injections of a selective immunotoxin and pre-aggregated amyloid peptide into the basal forebrain and the hippocampus, respectively. At about five-six weeks post-lesion, the double-lesioned animals exhibited dramatic deficits in spatial learning and memory, whereas animals with single injections of either compound were not or only marginally affected, in spite of equally severe cholinergic loss or amyloid deposition. Administration of (-)-MR22 appeared to reverse cognitive impairments in double lesioned animals, whereas pre-treatment with the selective sigma-1 antagonist BD1047 abolished this effect. Moreover, (-)-MR22 normalized the levels of cell-associated amyloid-β protein precursor (AβPP) in the neocortex and hippocampus, thus sustaining a non-amyloidogenic AβPP processing. By contrast, treatment with (-)-MR22 produced no effects whatsoever in intact animals. Thus, sigma-1 receptor agonists such as (-)-MR22 may ameliorate perturbed cognitive abilities and exert a protective action onto target neurons, holding promises as viable tools for memory enhancement and neuroprotection. [ABSTRACT FROM AUTHOR]

Abstract: 2-Benzo[d]thiazolyl- and 2-benzo[d]isothiazolyl-imino-5-benzylidene-4-thiazolidinone derivatives were investigated as potential metalloproteinases (MMPs) inhibitors and evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1β, using an experimental model that reproduces the mechanisms involved in osteoarthritic (OA) diseases. Cell viability, the amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) were measured. The most potent compound, 5-(4-methoxy-benzylidene)-2-(benzo[d]isothiazol-3-ylimino)-thiazolidin-4-one (4b), a MMP-13 inhibitor at nanomolar concentration (IC50 =0.036μM), could be considered as a lead compound for the development of novel clinical agents, inhibitors of cartilage degradation, for the treatment of OA. [ABSTRACT FROM AUTHOR]

Abstract: Complex mechanisms of prostate cancer progression prompt to novel therapeutic strategies concerning a combination of drugs or of single molecules able to interact with more crucial targets. Histone deacetylase inhibitors and sigma ligands with mixed σ1 antagonist and σ2 agonist properties were proposed as new potential tools for treatment of prostate cancer. (±)-MRJF4 was synthesized as phenylbutyrate ester of haloperidol metabolite II, which is a molecule consisting of a histone deacetilase inhibitor (4-phenylbutyric acid) and a sigma ligand (haloperidol metabolite II). Antiproliferatives activities of 4-phenylbutyric acid, haloperidol metabolite II, equimolar mixture of both compounds and (±)-MRJF4 were evaluated in vitro on LNCaP and PC3 prostate cancer cells. Preliminary binding studies of (±)-MRJF4 for σ1, σ2, D2 and D3 receptors and inhibition HDAC activity were reported. MTT cell viability assays highlighted a notable increase of antiproliferative activity of (±)-MRJF4 (IC50 = 11 and 13 μM for LNCaP and PC3, respectively) compared to 4-phenylbutyric acid, haloperidol metabolite II and the respective equimolar pharmacological association. (±)-MRJF4 was also used in combination with σ1 agonist (+)-pentazocine and σ2 antagonist AC927 in order to evaluate the role of σ receptor subtypes in prostate cancer cell death. [Copyright &y& Elsevier]

Abstract: 6,7-Benzomorphan derivatives, exhibiting different μ, δ, and κ receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2′-hydroxy-6,7-benzomorphan derivatives (12–22). Data obtained by competition binding assays showed that the μ opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide. Highly stringent features are required for δ receptor interaction, while an N-acetamide spacer and/or bulkier amide could preferentially lead to κ receptor selectivity. In the propanamide series, compound 12 (named LP1) displayed high μ affinity (K i =0.83nM), good δ affinity (K i =29nM) and low affinity for the κ receptor (K i =110nM), with a selectivity ratio δ/μ and κ/μ of 35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a μ/δ agonist profile, with IC50 values of 4.8 and 12nM at the μ and δ receptors, respectively. The antinociceptive potency of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine (ED50 =2.03 and 2.7mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a μ/δ agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent in vivo analgesic activity and a reduced tendency to induce side effects. [Copyright &y& Elsevier]

Abstract: Aims: It has been well documented that ventrolateral periaqueductal gray (vlPAG) matter is a crucial component of the descending pain modulatory system in the chronic pain condition. The aim of the present study was to identify the role of the vlPAG Nociceptin/Orphanin FQ/NOP receptor system in allodynia, a nociceptive behavioral response associated with chronic pain. Main methods: We used two animal models of persistent pain: chronic constriction injury (CCI) and inflammation induced by carrageenan. In each, Nociceptin/Orphanin FQ transmission was abolished using UFP-101, a selective NOP receptor antagonist, which was injected into the vlPAG at a dose of 18 µg/1 µl/rat. Key findings: We found that treatment with the NOP antagonist reversed the decrease in allodynic threshold in CCI rats fourteen days after the ligature, which was the timepoint of the greatest reduction in threshold. Moreover, UFP-101 administered immediately prior to or 2 h after intra plantar (i.pl.) carrageenan injection prevented or reversed, respectively, allodynic behavior in rats with inflammation. Significance: Our findings support the hypothesis that the endogenous Nociceptin/Orphanin FQ/NOP receptor system is tonically active at the vlPAG level during neuropathic states or carrageenan inflammation. [Copyright &y& Elsevier]

Previous studies have reported that selective sigma-1 agonists may improve cognitive abilities in experimental animals possibly via a cholinergic mechanism. However, the issue of a direct action on to sigma-1 receptors in memory-related brain areas has been much less investigated. The newly synthetised compound methyl(1 R,2 S/1 S,2 R)-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(±)-PPCC] has recently been shown to possess high affinity for the sigma-1 receptor where it specifically acts as an agonist. Here, the functional effects of (±)-PPCC were investigated in rat models of mild or severe cognitive dysfunction based on a sub-total (≤ 70–80%) or complete (≥ 90–95%) central cholinergic depletion induced by different doses of the selective immunotoxin 192 IgG-saporin injected intraventricularly. At 5–6 weeks post-surgery, the lesioned animals exhibited dose-dependent deficits in reference memory, as assessed using the Morris water maze task, whereas working memory abilities, evaluated using the radial arm water maze task, appeared equally impaired in the two dose groups. Daily treatment with (±)-PPCC significantly improved both reference and working memory performance in all lesioned animals but it did not affect intact or sham-lesioned subjects. In a separate test, treatment with (±)-PPCC reversed the learning deficits induced by the muscarinic receptor antagonist atropine sulphate in both control and mild-lesioned rats. The effect was blocked in lesioned, but not normal animals by pre-treatment with the sigma-1 antagonist N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(dimethylamino)ethylamine. The results suggest that (±)-PPCC may efficiently ameliorate perturbed cognitive abilities, and that these anti-amnesic effects most probably occur via a direct interaction of the compound with sigma-1 receptors. [ABSTRACT FROM AUTHOR]

Abstract: The compound (1R,2S/1S,2R)-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(±)-PPCC] is a ligand with high affinity for sigma (σ) sites of which the selectivity towards several other receptor systems has been demonstrated. Given the existence of a relationship between the σ system and the kappa opioid (KOP)-mediated analgesia, to characterize the pharmacological properties of (±)-PPCC we analyzed its influence on the analgesic effect of the systemic injected kappa agonist (−)-U-50,488H comparing the effects with those shown by (+)-pentazocine and BD1047. The results demonstrate that the systemic administration of (±)-PPCC (1 mg/kg s.c.) does not modify basal tail-flick latency. Pre-treatment with (±)-PPCC, at the same dose, significantly decreased the antinociceptive effect of (−)-U-50,488H, analogously to the σ compounds used. This study confirms that (±)-PPCC plays the role of σ agonist in this model and strengthens the hypothesis of the σ receptor modulatory role on KOP-mediated analgesia. [Copyright &y& Elsevier]

Abstract: 5-Arylidene-3-hydroxyalkyl-2-phenylimino-4-thiazolidinones (7,8) were synthesized and evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1β. This in vitro model has proven to be a useful experimental model to reproduce the mechanisms involved in arthritic diseases. The cell viability, the amount of GAGs, the production of NO and PGE2 and the inhibition of MMP-3 were measured. Several thiazolidinones 7 and 8 exhibited the ability to block the production or action of the degenerative factors induced by IL-1β. [Copyright &y& Elsevier]

Abstract: The compound (+)-MR200 [(+)-methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate] is a sigma ligand with increased affinity and selectivity compared to the structurally related ligand haloperidol. From the results of a previous study on the modulation of a systemically injected KOP opioid agonist analgesia by (+)-MR200, we analysed the influence of this sigma ligand on the antinociceptive effect of centrally injected MOP, DOP, and KOP selective agonists using the tail-flick test in rats. The results obtained confirmed that systemic administration of (+)-MR200 (1mg/Kg s.c.) did not modify basal tail-flick latency. Pre-treatment with 1mg/Kg s.c. of (+)-MR200 provided a significant increase in the antinociceptive effect of DAMGO (100ng/rat i.c.v.) and DPDPE (20 μg/rat i.c.v.). Conversely to previous reports, pre-treatment with (+)-MR200 reversed, in these experimental conditions, U-50488H (100μg/rat i.c.v.) analgesia. The mechanism involved in these effects was not clear, but provided additional data on a diverging modulator role of selective sigma-1 antagonists on KOP analgesia. [Copyright &y& Elsevier]

Natural bioactive compounds may be used in obese patients because of their ability to impact on various key mechanisms involved in the complex pathophysiological mechanisms of such condition. The aim of this study was to investigate the effect of a Mangifera indica L. leaf extract (MLE) on adipogenic differentiation of murine preadipocyte cells. 3T3-L1 cells were treated during their differentiation with various concentrations of (Mangifera indica L.) leaves extract (MLE) (750, 380, 150, 75 and 35 μg) in order to assess their lipid content, adiponectin production, expression profile of genes involved in lipid metabolism, oxidative stress and inflammation. Our results showed that MLE was particularly enriched in polyphenols (46.30 ± 0.083 mg/g) and that pharmacological treatment of cells resulted in a significant increase of adiponectin levels and reduction of intracellular lipid content. Consistently with these results, MLE resulted in a significant decrease of the expression of genes involved in lipid metabolism (FAS, PPARG, DGAT1, DGAT2, and SCD-1). In conclusion, our results suggest that MLE may represent a possible pharmacological tool for obese or metabolic syndrome patients. [ABSTRACT FROM AUTHOR]