Preliminary studies on environmental pollutants in chamois and wild boar from Eastern Piedmont, Italy.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) are synthetic chlorinated compounds classified as POPs whereas only the penta e tetra-brominated polybromodiphenyl ethers (PBDEs) are so defined by the Stockolm Convention (Stockholm Convention, 2005) in order to elimitate or restrict the use of POPs. Organophosphorus insecticides (OCPs) represent important environmental and food contamination sources, widely used in agriculture. Among polyciclic aromatic hydrocarbons (PAHs), benzo[a]pirene is classified by IARC in Group 1, as cancerogen  and Benzo[a]fluoranthene as a Group 2B, as possible cancerogen (IARC, 2012; IARC, 2010).  EFSA (European Food Safety Authority) has released a scientific opinion on the risks to public health related to the presence of brominated flame retardants in food (EFSA, 2011) and in 2014 European commission has asked Member States to monitor the presence of brominated flame retardants (BFRs) in food over the next two years (EC, 2014). Due to their heir n-octanol/water partition coefficient (log Kow), they accumulate in fat tissue, bioconcentrate and biomagnify in the animals at the higher trophic levels, possibly causing, through chronic exposure, endocrine disruption and cancer (Wania et al., 1995; Vallack et al., 1998). The aim of this study is to evaluate the presence of OCPs, PCBs, PBDEs and PAHs in chamois and wild boar from Eastern Piedmont, Italy. A total of 20 chamois and 20 wild boar muscle samples were collected during the hunting season 2017, from Verbania Cusio Ossola (VCO) (Fig 1). The chemical analysis for the detection of OCPs, PCBs, PBDEs, and PAHs   was performed by GC-MS/MS on muscle samples purified and extracted using a QuEChERS technique, validated according to SANTE 2017 (SANTE/11183/2017). These preliminary results show the ubiquitary presence of the studied contaminants. PCBs have been found more in chamois (45%) than in wild boar (35%). No PBDEs were detected in wild boar but in chamois were found with a prevalence of 35%  and  concentration 0.25-1.52 ng g-1. About OCPs, phorate and demeton were found in wild boar (55%-15%) and chamois (32%- 35%) with range concentrations 0.21-20.1 ng g-1. No PAHs were detected, expect antharacene for one samples in wild boar (0.53 ng g-1). Further studies are in progress in order to correlate environmental contamination and game animals

Metals in mussels from Italian mollusc culture plants

The beneficial effects on human health of seafood are well known. However, seafood is a major source of exposition for consumers of most of the contaminants due to human activities such as breeding, industries, mining and agriculture: the overall level in biota, therefore seafood and particularly molluscs, dramatically increased over this last two centuries. This study evaluates the presence of Lead, Mercury, Cadmium, Arsenic, Nickel and Chromium in mussels from the Italian mussel culture plants, and estimate the risk that Italian consumer undergoes eating these molluscs. Mussels where collected at the wholesale fish market of Milan, the most important wholesale Italian fish market. The molluscs belonged to the   37 FAO marine area (corresponding to Mediterranean Sea), particularly from FAO 37.2.1 Ligury, 37.2.2. North Adriatic, middle Adriatic, Puglia, 37.2.3 Lazio and Sardinia, and were collected from July 2016 to February 2017. (FIG1). Analyses were carried out through inductively coupled plasma-mass spectrometry (ICP-MS) according to the Environmental Protection Agency (EPA) 3050B method. The sample concentrations were below the Maximum Levels (ML) given by Commission Regulation (EC) No 1881/2006 for Cadmium, Lead and Mercury, except one sample from south Adriatic sea, that showed Mercury concentration of 0.528 mg kg-1. Arsenic, Nickel and Chromium ML are not stated by EU. Arsenic concentration ranged from 2.05 to 13.35 mg kg-1, with the highest values found in Italian molluscs, Nickel concentration ranged from 0.00 to 3.98 mg kg-1. Chromium was found only in 5 of 30 sample analysed with a maximum concentration of 0.590 g kg-1. The tolerable intakes recommended by EFSA and on EU maximum levels, indicate that Italian mussels do not pose a risk consumers

Listeria monocytogenes sensitivity to antimicrobial treatments depends on cell origin

In this study we investigated how cell origin could affect the efficacy of an antimicrobial treatment (mild heating combined with terpenoids) in Listeria monocytogenes Scott A, considering cells from: 1. single colony, 2. glycerol stock, 3. cold adapted culture, and 4. fresh culture in stationary phase. After treatment, culturability on BHI medium and viability assessed by flow cytometry were evaluated. Our results showed that the cell origin significantly impacted viability and culturability of L. monocytogenes towards antimicrobial treatment. The mild heat treatment combined or not with terpenoids mainly affected culturability rather than viability, although the culturability of cells from single colony was less impacted. Therefore, to mimic the worst scenario, these latter were selected to contaminate Gorgonzola rind and roast beef slices and we evaluated the ability of L. monocytogenes cells to recover their culturability (on ALOA agar medium) and to growth on the food matrix stored at 4 °C for 7 days. Our results suggest that only Gorgonzola rind allowed a partial recovery of the culturability of cells previously heated in presence or not of terpens. In conclusion, we found a connection between the cell history and sensitivity toward an antimicrobial treatment, underlying the importance to standardize the experimental procedures (starting from the cells to be used in the assay) in the assessment of cell sensitivity to a specific treatment. Finally, our study clearly indicated that VBNC cells can resuscitate under favorable conditions on a food matrix, becoming a threat for consumer’s health

Preparation and Double Michael Addition Reactions of a Synthetic Equivalent of Nazarov Reagent

A synthetic equivalent of the Nazarov reagent, the silyl derivative 2, able to undergo base-catalyzed double Michael addition reactions with a,b-unsaturated carbonyl compounds, has been developed. The new reagent satisfactorily reacts with unsaturated indolo[2,3-a]quinolizidine lactams to give pentacyclic yohimbinone-type derivatives

A Straightforward Synthesis of Functionalized cis-Perhydroisoquinolin-1-ones

Base-catalyzed annulation reactions of 5,6-dihydro-2(1H)-pyridones with Nazarov-type reagents are reported. The effect of the solvent polarity and the concentration of the reagents is studied. The process involves two successive Michael additions and stereoselectively provides functionalized cis-perhydroisoquinolin-1-ones

Stereocontrolled annulations of indolo[2,3-a]quinolizidine-derived lactams with a silylated Nazarov reagent. Access to allo and epiallo yohimbine-type derivatives

The facial selectivity of double Michael addition reactions of the silylated Nazarov reagent 4 to unsaturated indolo[2,3-a]quinolizidine lactams 3 has been studied. Pentacyclic 3-H/15-H trans adducts 5 are generated from Nind -unsubstituted lactams, but the corresponding cis isomers 6 are formed when the indole nitrogen has a tert-butyloxycarbonyl (Boc) substituent. This reversal in the facial selectivity of the annulation has been rationalized by means of theoretical calculations, which indicate that the initial nucleophilic attack under stereoelectronic control is hampered by the presence of the bulky Boc group. The synthetic usefulness of the pentacyclic Nazarov-derived adducts is demonstrated by their conversion into allo and epiallo yohimbine-type targets

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2

Síntesis estereocontrolada de sistemas tipo yohimbano. Nitraraína, un alcaloide de estructura no resuelta

[spa] Los alcaloides indólicos monoterpénicos constituyen un grupo muy importante de productos naturales. Estos alcaloides presentan una gran variedad de estructuras químicas y muchos de los miembros de esta clase de alcaloides tienen importantes actividades farmacológicas, con un gran potencial para su utilización clínica, como los alcaloides antitumorales vinblastina y vincristina. A partir de la lactama 1, hemos descrito la formación estereocontrolada de los diasteroisómeros 12b-Ry 12b-S de las correspondientes indolo[2,3-a]quinolicidinas, utilizando HCl o BF3·Et2O, respectivamente, como ácidos promotores de la reacción de alfa-amidoalquilación intramolecular. Para la construcción del anillo carbocíclico E del núcleo yohimbánico a partir de las lactamas indoloquinolicidínicas insaturadas hemos decidido emplear un procedimiento basado en el uso del reactivo de Nazarov. El reactivo de Nazarov es un agente utilizado frecuentemente en reacciones de anulación de Robinson y ha sido empleado extensivamente en la síntesis de terpenos y alcaloides. Este reactivo tiene el inconveniente de ser muy inestable bajo las condiciones básicas por su gran tendencia a la polimerización. Numerosos derivados sustituidos con grupos alquilos en las posiciones alfa y beta han sido también empleados en la síntesis de decalinas y esteroides. Mientras la reacción de doble adición de Michael con nuestras lactamas insaturadas activadas con el derivado metilado en la posición beta tuvo lugar con excelente rendimiento y estereoselectividad, el reactivo de Nazarov original no condujo al producto de adición esperado. Recientemente hemos desarrollado el derivado sililado 5, un nuevo equivalente sintético del reactivo de Nazarov, más estable bajo las condiciones básicas requeridas para la reacción de doble adición de Michael con compuestos carbonílicos alfa, beta-insaturados. En esta transformación se forman dos enlaces carbono-carbono (C-15/C-16 y C-19/C-20), con generación de dos centros estereogénicos (C-15 y C-20) en una reacción one-pot. En el contexto de nuestros estudios sobre el uso de lactamas derivadas del triptofanol como scaffolds enantioméricos para la síntesis enantioselectiva de alcaloides indólicos, hemos desarrollado una metodología para el acceso diastereodivergente a sistemas de tipo yohimbano mediante una reacción de doble adición de Michael entre una lactama indoloquinolicidínica insaturada y el reactivo de Nazarov sililado 5. Gracias a la modulación de las condiciones de reacción podemos controlar la estereoselectividad de la ciclación: utilizando Cs2CO3 y DCM, el sistema pentacíclico H-3/H-15 trans (6-a) se obtiene con elevada diastereoselectividad, mientras que el uso de DBU en THF cambia la selectividad de la reacción, conduciendo exclusivamente al correspondiente isómero H-3/H-15 cis (6-b). Se han realizado cálculos teóricos que han permitido explicar y racionalizar la distinta estereoselectividad observada en la reacción de doble adición de Michael. El potencial sintético de la metodología estudiada se ha demostrado mediante su aplicación a la síntesis de derivados de los alcaloides de tipo yohimbina como la 17a-carbaakuamigina. Por otro lado, hemos sintetizado las estructuras propuestas para el alcaloide nitraraína, un producto natural cuya asignación estructural no está todavía confirmada, ya que los datos físicos y espectroscópicos de los compuestos sintetizados no coinciden con los descritos para el compuesto aislado. Originalmente se había propuesto una estructura tipo yohimbina mientras que posteriormente, Erwan Poupon ha propuesto una estructura basada en un esqueleto de tipo tangutorina. En este contexto, hemos sintetizado los compuestos 14-a/b y los derivados O-acetilnitraraína 15-a/b, dihidronitraraína16-a/bynitraraidina 17b, productos naturales relacionados con la nitraraína y aislados de las mismas plantas. Hemos sintetizado también la estructura 18, propuesta por Erwan Poupon para el alcaloide nitraraína mediante una secuencia que tiene como etapas claves: i) una ciclocondensación estereoselectiva entre el (S)-triptofano y un ceto-éster oportunamente funcionalizado; ii) una ciclación de Bischler-Napieralski; iii) la eliminación de la cadena de hidroximetilo. La comparación de los datos físicos y espectroscópicos de estos compuestos sintéticos con los descritos en la literatura para los productos naturales nos permitió concluir que ninguna de las estructuras sintetizadas parece ser la correcta asignación para la nitraraína y los alcaloides relacionados.[eng] Monoterpenoid indole alkaloids constitute an important group of natural products with a wide variety of complex chemical structures. A large number of members of this class of alkaloids display pharmacologically important activities, with considerable potential for clinical use, such as the antitumor alkaloids vinblastine and vincristine. Starting from lactams we have described the stereocontrolled formation of C‐12b diastereomeric indolo[2,3‐a]quinolizidines using either HCl or BF3∙Et2O, respectively, to promote the intramolecular alpha-amidoalkylation reaction. In order to assemble the carbocyclic E ring of the yohimbane nucleus from tetracyclic unsaturated lactams we have devised an expeditious procedure based on the use of Nazarov reagents. The Nazarov reagent is a well‐known annelating agent that has been extensively used in terpene and alkaloid syntheses. The silyl derivative, a new stable and practical synthetic equivalent of the Nazarov reagent recently developed in our laboratory, is able to undergo base‐promoted double Michael annulations with alpha, beta-unsaturated carbonyl compounds. In this transformation, two carbon‐carbon bonds would be formed (C‐15/C‐16 and C‐19/C‐20), with generation of two stereogenic centers (C‐15 and C‐20), in a one‐pot reaction. In the context of our studies on the use of tryptophanol‐derived lactams in the enantioselective synthesis of indole alkaloids, we have developed a methodology for the diastereodivergent access to yohimbinone‐type systems by a double Michael reaction between an unsaturated indolo[2,3‐a]quinolizidine lactam and the Nazarov reagent equivalent By simply modulating the reaction conditions we are able to control the stereochemical outcome of the cyclization: when Cs2CO3 and DCM are used, the H‐3/H‐15 trans pentacyclic system is obtained with high diastereoselectivity, whereas the use of DBU in THF reverses the selectivity, exclusively leading to the cis isomer. This methodology allows the straightforward construction of pentacyclic yohimbinonetype systems. Following the reported methodology, we prepared compounds and studied their transformation into the dihydro derivative, and the quaternary salt, structures originally proposed for the natural product nitraraine and related alkaloids. We also synthesized the structure proposed by Erwan Poupon for nitraraine. Comparison of the physical and spectroscopic data of these synthetic compounds with those reported for the natural products allowed us to conclude that none of the synthesized structures seem to be the correct assignment for the natural compounds nitraraine, dihydronitraraine, and nitraraidine

A Straightforward Synthesis of Functionalized <i>cis</i>-Perhydroisoquinolin-1-ones

Base-catalyzed annulation reactions of 5,6-dihydro-2(1H)-pyridones with Nazarov-type reagents are reported. The effect of the solvent polarity and the concentration of the reagents is studied. The process involves two successive Michael additions and stereoselectively provides functionalized cis-perhydroisoquinolin-1-ones