Intramolecular Nucleophilic Vinylic Substitution (SNV) by Carbon Nucleophiles: Conformationally Directed Formation of Dienes from N,N'-Diallyl Ureas.

Nucleophilic vinylic substitution (SNV) by carbon nucleophiles allows the formation of vinylic C-C bonds without transition metal catalysts. In this paper, we show that tethering two alkenes together through a urea linkage can lead to the formation of a diene by an intramolecular SNV reaction. The starting materials are fully substituted N,N'-diallyl ureas; the reaction proceeds in the presence of base, and entails a cascade of deprotonations, reprotonations, and an SNV reaction of an allylic carbanion on a rare electrophile: a vinylic urea. As a result, two allylic substituents couple to form a diene, despite the fact that neither is activated towards electrophilic attack. The reaction is tolerant of significant steric bulk, and exhibits regioselectivity with unsymmetrical diallyl ureas: ß-substituted allyl groups invariably behave as nucleophiles, while electrophilic behavior may be enforced by the use of an E-vinylic urea substituent that cannot be deprotonated under the reaction conditions.

Synthesis of Complex Tetracyclic Fused Scaffolds Enabled by (3 + 2) Cycloaddition.

We describe the single-step formation of complex tetracyclic fused scaffolds enabled by (3 + 2) cycloaddition of azomethine ylides. Various indoles, N-protecting groups, and amino acids are well tolerated. The products are obtained in a catalyst-free manner with moderate to excellent yield and high diastereoselectivity. Representing a new scaffold that is not yet found in nature, the construction of pyrrolidine-fused cyclohepta-, azepino-, or oxepinoindoles could be found valuable in the synthesis of new pseudo-natural products.

N-Methyl Allylic Amines from Allylic Alcohols by Mitsunobu Substitution Using N-Boc Ethyl Oxamate.

We report the practical, scalable synthesis of a range of N-methyl allylic amines. Primary and secondary allylic alcohols underwent a regioselective Mitsunobu reaction with readily accessible N-Boc ethyl oxamate to deliver the corresponding N-Boc allylic amines, including in enantiopure form via stereospecific substitution. Subsequent N-methylation and Boc deprotection without chromatography yielded the amine products as hydrochloride salts. This method solves the problem of converting commercially available alcohols into often volatile N-methyl allylic amines, many of which have limited commercial availability.

Synthetic (N,N-Dimethyl)doxorubicin Glycosyl Diastereomers to Dissect Modes of Action of Anthracycline Anticancer Drugs.

Anthracyclines are effective drugs in the treatment of various cancers, but their use comes with severe side effects. The archetypal anthracycline drug, doxorubicin, displays two molecular modes of action: DNA double-strand break formation (through topoisomerase IIα poisoning) and chromatin damage (via eviction of histones). These biological activities can be modulated and toxic side effects can be reduced by separating these two modes of action through alteration of the aminoglycoside moiety of doxorubicin. We herein report on the design, synthesis, and evaluation of a coherent set of configurational doxorubicin analogues featuring all possible stereoisomers of the 1,2-amino-alcohol characteristic for the doxorubicin 3-amino-2,3-dideoxyfucoside, each in nonsubstituted and N,N-dimethylated forms. The set of doxorubicin analogues was synthesized using appropriately protected 2,3,6-dideoxy-3-amino glycosyl donors, equipped with an alkynylbenzoate anomeric leaving group, and the doxorubicin aglycon acceptor. The majority of these glycosylations proceeded in a highly stereoselective manner to provide the desired axial α-linkage. We show that both stereochemistry of the 3-amine carbon and N-substitution state are critical for anthracycline cytotoxicity and generally improve cellular uptake. N,N-Dimethylepirubicin is identified as the most potent anthracycline that does not induce DNA damage while remaining cytotoxic.

Microplastics occurrence in the Tyrrhenian waters and in the gastrointestinal tract of two congener species of seabreams.

In this work it is reported for the first time the characterization of microplastics from sea water samples and in two congener species of seabreams: Pagellus erythrinus and P. bogaraveo, Mediterranean fish species of great commercial importance. An experimental survey was conducted on May-June 2017 in the southernmost part of the Tyrrhenian Sea. Microplastics found in the sea water and in the grastrointestinal tract of two teleosts were characterized by Raman and IR spectroscopies. Microplastics found in sea water samples appeared in the form of fragments made of plastics of low and high density (PVC and LPDE). All the microplastics found in fish belonged to Nylon 66, typical fibers used in industry and in fisheries. Our findings highlighted the importance of further studies along the food web chain for a better understanding of the diffusion and possible consequences of this terrible threat.

p62 improves AD-like pathology by increasing autophagy.

The multifunctional protein p62 is associated with neuropathological inclusions in several neurodegenerative disorders, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and Alzheimer's disease (AD). Strong evidence shows that in AD, p62 immunoreactivity is associated with neurofibrillary tangles and is involved in tau degradation. However, it remains to be determined whether p62 also plays a role in regulating amyloid-ß (Aß) aggregation and degradation. Using a gene therapy approach, here we show that increasing brain p62 expression rescues cognitive deficits in APP/PS1 mice, a widely used animal model of AD. The cognitive improvement was associated with a decrease in Aß levels and plaque load. Using complementary genetic and pharmacologic approaches, we found that the p62-mediated changes in Aß were due to an increase in autophagy. To this end, we showed that removing the LC3-interacting region of p62, which facilitates p62-mediated selective autophagy, or blocking autophagy with a pharmacological inhibitor, was sufficient to prevent the decrease in Aß. Overall, we believe these data provide the first direct in vivo evidence showing that p62 regulates Aß turnover.

CHF5074 (CSP-1103) induces microglia alternative activation in plaque-free Tg2576 mice and primary glial cultures exposed to beta-amyloid.

Activation of microglia associated with neuroinflammation and loss of phagocytic activity is considered to play a prominent role in the pathogenesis of Alzheimer's disease (AD). CHF5074 (CSP-1103) has been shown to improve cognition and reduce brain inflammation in patients with mild cognitive impairment (MCI). CHF5074 was also found to reverse impairments in recognition memory and improve hippocampal long-term potentiation when administered to plaque-free Tg2576 mice (5-month-old) for 4 weeks. Though, no investigation has focused on the consequence of CHF5074 treatment on microglia polarization yet. In this study we evaluated the effect of CHF5074 administration (375 ppm in the diet) to 5-month-old Tg2576 mice on the expression of pro-inflammatory (M1) genes, Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor alpha (TNFα) and inducible Nitric Oxide Synthase (iNOS), and anti-inflammatory/phagocytic (M2) markers Mannose Receptor type C 1 (MRC1/CD206), Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Chitinase 3-like 3 (Ym1). No changes of pro-inflammatory gene transcription but a reduced expression of MRC1/CD206, TREM2 and Ym1 were detected in the hippocampus of young Tg2576 mice receiving normal diet, when compared to wild-type littermates. CHF5074 did not affect the pro-inflammatory transcription but significantly increased the expression of MRC1/CD206 and Ym1. CHF5074 effects appeared to be hippocampus-specific, as the M2 transcripts were only slightly modified in the cerebral cortex. In primary cultures of mouse astrocyte-microglia, CHF5074 totally suppressed the expression of TNF-α, IL-1ß and iNOS induced by 10 µM ß-amyloid1-42 (Aß42). Moreover, CHF5074 significantly increased the expression of anti-inflammatory/phagocytic markers MRC1/CD206 and TREM2, reduced by the Aß42 application alone. The effect of CHF5074 was not reproduced by ibuprofen (3 µM or 500 µM) or R-flurbiprofen (3 µM or 100 µM), as both compounds limited the pro-inflammatory gene expression but did not modify the anti-inflammatory/phagocytic transcription. These data show that CHF5074 specifically drives the expression of microglia M2 markers either in young Tg2576 hippocampus or in primary astrocyte-microglia cultures, suggesting its potential therapeutic efficacy as microglial modulator in the early phase of AD.

Multi-component modeling of quasielastic neutron scattering from phospholipid membranes.

We investigated molecular motions in the 0.3-350 ps time range of D2O-hydrated bilayers of 1-palmitoyl-oleoyl-sn-glycero-phosphocholine and 1,2-dimyristoyl-sn-glycero-phosphocholine in the liquid phase by quasielastic neutron scattering. Model analysis of sets of spectra covering scale lengths from 4.8 to 30 Å revealed the presence of three types of motion taking place on well-separated time scales: (i) slow diffusion of the whole phospholipid molecules in a confined cylindrical region; (ii) conformational motion of the phospholipid chains; and (iii) fast uniaxial rotation of the hydrogen atoms around their carbon atoms. Based on theoretical models for the hydrogen dynamics in phospholipids, the spatial extent of these motions was analysed in detail and the results were compared with existing literature data. The complex dynamics of protons was described in terms of elemental dynamical processes involving different parts of the phospholipid chain on whose motions the hydrogen atoms ride.

Status of the circle of Willis and intolerance to carotid cross-clamping during carotid endarterectomy.

PURPOSE: During carotid endarterectomy (CEA), an intolerance to the cross-clamping (CC) can occur. The purpose of this study was to evaluate whether preoperative magnetic resonance angiography (MRA) can predict CC intolerance. MATERIAL AND METHODS: Seventy-one patients (57 males, 14 females, mean age 71.8 years, age range 46-86 years) underwent 71 CEA procedures under local anaesthesia. Before CEA, patients underwent an MRA of the Circle of Willis (CoW) and were then classified into three groups: group A consisted of patients with a complete CoW, group B included patients with one agenesia/obstruction in the CoW and group C comprised patients with two or more agenesiae/obstructions in the CoW. The association between the number of anatomical variants in the CoW, corrected for the status of the contralateral carotid artery, and the onset of CC intolerance was evaluated. RESULTS: The prevalence of intolerance to CC was 15.5% (11/71). The Fisher test and logistic regression analysis showed a statistically significant association between the intolerance to CC and two or more agenesiae/obstructions in the CoW (p value < 0.00001 and p < 0.001, respectively). No neurological complications were observed. CONCLUSION: The results of our study showed that two or more agenesiae/obstructions of the CoW identified by MRA were associated with a high risk of intolerance to CC during CEA.

The acetylation of RelA in Lys310 dictates the NF-κB-dependent response in post-ischemic injury.

The activation of nuclear factor kappa B (NF-κB) p50/RelA is a key event in ischemic neuronal injury, as well as in brain ischemic tolerance. We tested whether epigenetic mechanisms affecting the acetylation state of RelA might discriminate between neuroprotective and neurotoxic activation of NF-κB during ischemia. NF-κB activation and RelA acetylation were investigated in cortices of mice subjected to preconditioning brain ischemia or lethal middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to preconditioning or lethal oxygen-glucose deprivation (OGD). In mice subjected to MCAO and in cortical neurons exposed to lethal OGD, activated RelA displayed a high level of Lys310 acetylation in spite of reduced total acetylation. Also, acetylated RelA on Lys310 interacted strongly with the CREB-binding protein (CBP). Conversely, RelA activated during preconditioning ischemia appeared deacetylated on Lys310. Overexpressing RelA increased Bim promoter activity and neuronal cell death both induced by lethal OGD, whereas overexpressing the acetylation-resistant RelA-K310R, carrying a mutation from Lys310 to arginine, prevented both responses. Pharmacological manipulation of Lys310 acetylation by the sirtuin 1 activator resveratrol repressed the activity of the Bim promoter and reduced the neuronal cell loss. We conclude that the acetylation of RelA in Lys310 dictates NF-κB-dependent pro-apoptotic responses and represents a suitable target to dissect pathological from neuroprotective NF-κB activation in brain ischemia.

NMR evidence of a sharp change in a measure of local order in deeply supercooled confined water.

Using NMR, we measure the proton chemical shift delta, of supercooled nanoconfined water in the temperature range 195 K < T < 350 K. Because delta is directly connected to the magnetic shielding tensor, we discuss the data in terms of the local hydrogen bond geometry and order. We argue that the derivative -( partial differential ln delta/ partial differentialT)(P) should behave roughly as the constant pressure specific heat C(P)(T), and we confirm this argument by detailed comparisons with literature values of C(P)(T) in the range 290-370 K. We find that -( partial differential ln delta/ partial differentialT)(P) displays a pronounced maximum upon crossing the locus of maximum correlation length at approximately 240 K, consistent with the liquid-liquid critical point hypothesis for water, which predicts that C(P)(T) displays a maximum on crossing the Widom line.

Structural investigation of the confinement of finite amounts of trehalose in water-containing sodium Bis(2-ethylhexyl)sulfosuccinate reversed micelles.

The structural effect of trehalose confined in water-containing sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles at water to AOT molar ratio W = 5 and 10 as a function of the trehalose to AOT molar ratio T (0 < T < 0.1) has been investigated by small-angle neutron scattering (SANS). SANS data analysis is consistent with the hypothesis that trehalose is encapsulated within the quite spherical hydrophilic micellar cores of water-containing reversed micelles, causing an increase of the aggregate size and a decrease of the polydispersion. Moreover, SANS results suggest that the trehalose confinement in water-containing reversed micelles involves marked changes on the molecular packing of the water-containing micellar cores. In particular, according to the obtained findings, we can hypothesize the intercalation of the trehalose molecules between the polar surfactant headgroups. The preferential solubilization in this specific nanodomain could explain the trehalose capability to prevent, upon dehydration, the transition to a gel phase, hindering serious damage to biostructures.

Tetrahedral order in homologous disaccharide-water mixtures.

The present work aims at evidencing the "kosmotrope" nature of trehalose through the analysis of inelastic neutron scattering measurements on trehalose and sucrose water solutions at different temperatures. Neutron spectra were collected by using the spectrometer MARI at the ISIS pulsed neutron source of the Rutherford Appleton Laboratory (Chilton, UK). To study the structural modifications induced on the tetrahedral hydrogen-bond network of water by homologous disaccharides, as a first step, the vibrational properties of pure water at different temperatures have been investigated. In particular, the temperature behavior of the intramolecular OH stretching mode has been analyzed. Successively, the vibrational properties for pure water have been compared with those of the sugar water solutions focusing the attention on the tetrahedral network-forming tendency. Finally, the obtained findings have been compared with previous Raman scattering evidences, and the results interpreted in the frame of recent molecular dynamics simulation works.

Role of polyols in thermal inactivation of shark ornithine transcarbamoylase.

The ability of activity modulators of ornithine transcarbamoylase (OCT) from the liver of the thresher shark Alopias vulpinus to stabilize the enzyme against thermal denaturation was investigated in the tri-buffer at pH 7.8, at temperatures ranging from 60 to 70 (o)C, in the presence of polyhydroxylic molecules such as glycerol and sugars. The study indicated that in the presence of 0.5 M sugars and 1.6 M glycerol in the preincubation medium the OCT activity increases. When trehalose is introduced directly in the reaction mixture in a range of concentration of 0.25-0.5 M, the activity is lower than that with maltose, glycerol and buffer alone. Kinetic data for carbamoyl phosphate and ornithine with and without maltose and glycerol are similar, whereas trehalose increases the kinetic values. Arrhenius plots show an increase of activation energy due to trehalose, whereas values obtained with maltose and glycerol are similar to the control.

Cytokinin-like activity of N,N'-diphenylureas. N,N'-bis-(2,3-methylenedioxyphenyl)urea and N,N'-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root formation in apple rootstock M26 (Malus pumila Mill.).

Vegetative propagation of cuttings is a widespread method to multiplicate plants. Adventitious root formation is a key step in vegetative propagation and considerable progress has recently been made in understanding root formation. But, in spite of the efforts made, no new rooting treatments have been developed. Here, we report for the first time, that N,N'-bis-(2,3-methylenedioxyphenyl)urea and N,N'-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root formation in microcuttings of Malus pumila Mill. rootstock M26. Roots emerge without auxin supplementation in the darkness, transfer in hormone free medium, or callus formation. With the use of different bioassays, we also demonstrate that these two diphenylurea derivatives do not show cytokinin- or auxin-like activity.

Experimental study of the hydration properties of homologous disaccharides.

To get some insight into the hydration mechanisms of homologousdisaccharides, we report measurements on trehalose, maltose, and sucroseaqueous solutions. The interest on these systems is mainly due to theextraordinary properties of disaccharides and especially of trehalose, themost effective bio-protector against freezing and dehydration. To carry outthis study we have investigated the volumetric properties of the threedisaccharide solutions, by performing density and ultrasonic velocitymeasurements at different concentration and temperature values. Whatemerges from these studies is that trehalose shows, in comparison withmaltose and sucrose, the greatest structural sensitivity to temperaturechanges and the smallest values of the partial molar volume in all theinvestigated temperature range, this circumstance being indicative of a morepacked conformational arrangement.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum.

Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.