Golgi α-mannosidase: opposing structures of Drosophila melanogaster and novel human model using molecular dynamics simulations and docking at different pHs.

The search for Golgi α-mannosidase II (GMII) potent and specific inhibitors has been a focus of many studies for the past three decades since this enzyme is a key target for cancer treatment. α-Mannosidases, such as those from Drosophila melanogaster or Jack bean, have been used as functional models of the human Golgi α-mannosidase II (hGMII) because mammalian mannosidases are difficult to purify and characterize experimentally. Meanwhile, computational studies have been seen as privileged tools able to explore assertive solutions to specific enzymes, providing molecular details of these macromolecules, their protonation states and their interactions. Thus, modelling techniques can successfully predict hGMII 3D structure with high confidence, speeding up the development of new hits. In this study, Drosophila melanogaster Golgi mannosidase II (dGMII) and a novel human model, developed in silico and equilibrated via molecular dynamics simulations, were both opposed for docking. Our findings highlight that the design of novel inhibitors should be carried out considering the human model's characteristics and the enzyme operating pH. A reliable model is evidenced, showing a good correlation between Ki/IC50 experimental data and theoretical ΔGbinding estimations in GMII, opening the possibility of optimizing the rational drug design of new derivatives.Communicated by Ramaswamy H. Sarma.

Effects of the Reactive Moiety of Phenolipids on Their Antioxidant Efficiency in Model Emulsified Systems.

Our previous research was focused on the effects of hydrophobicity on the antioxidant (AO) efficiency of series of homologous antioxidants with the same reactive moieties. In this work we evaluate the antioxidant efficiency of hydrophobic phenolipids in 4:6 olive oil-in-water emulsions, with different phenolic moieties (derived from caffeic, 4-hydroxycinnamic, dihydrocaffeic acids, tyrosol and hydroxytyrosol), with alkyl chains of 8 and 16 carbons, and compare the antioxidant efficiency with that of the parent compounds. All catecholic phenolipids, in particular the C8 derivatives, have proven to be better antioxidants for the oxidative protection of emulsions than their parental compounds with octyl dihydrocafffeate being the most efficient (16-fold increase in relation to the control). To understand the importance of some factors on the antioxidant efficiency of compounds in emulsions, Pearson's correlation analysis was carried out between antioxidant activity and the first anodic potential (Epa), reducing capacity (FRAP value), DPPH radical scavenging activity (EC50) and the concentration of antioxidants in each region of the emulsified system. Results confirm the importance of the effective concentration of AOs in the interfacial region (AOI) (ρ = 0.820) and of the Epa (ρ = -0.677) in predicting their antioxidant efficiency in olive oil-in-water emulsions.

Interfacial Concentrations of Hydroxytyrosol Derivatives in Fish Oil-in-Water Emulsions and Nanoemulsions and Its Influence on Their Lipid Oxidation: Droplet Size Effects.

Reports on the effect of droplet size on the oxidative stability of emulsions and nanoemulsions are scarce in the literature and frequently contradictory. Here, we have employed a set of hydroxytyrosol (HT) esters of different hydrophobicity and fish oil-in-water emulsified systems containing droplets of different sizes to evaluate the effect of the droplet size, surfactant, (ΦI) and oil (ΦO) volume fractions on their oxidative stability. To quantitatively unravel the observed findings, we employed a well-established pseudophase kinetic model to determine the distribution and interfacial concentrations of the antioxidants (AOs) in the intact emulsions and nanoemulsions. Results show that there is a direct correlation between antioxidant efficiency and the concentration of the AOs in the interfacial region, which is much higher (20-200 fold) than the stoichiometric one. In both emulsified systems, the highest interfacial concentration and the highest antioxidant efficiency was found for hydroxytyrosol octanoate. Results clearly show that the principal parameter controlling the partitioning of antioxidants is the surfactant volume fraction, ΦI, followed by the O/W ratio; meanwhile, the droplet size has no influence on their interfacial concentrations and, therefore, on their antioxidant efficiency. Moreover, no correlation was seen between droplet size and oxidative stability of both emulsions and nanoemulsions.

Influence of AO chain length, droplet size and oil to water ratio on the distribution and on the activity of gallates in fish oil-in-water emulsified systems: Emulsion and nanoemulsion comparison.

The distribution of a homologous series of polyphenol derivatives of increasing lipophilicity has been determined in fish oil-in-water emulsions and nanoemulsions by the pseudophase model. One of the hypotheses on which the pseudophase model is based, is that its application is independent of the size of emulsion droplets. In agreement with our hypothesis, results showed that the smaller droplet size found in nanoemulsions does not affect partition constants of gallic acid (GA) and its esters. The antioxidant efficiency of GA and gallates in the emulsified systems used, correlated positively with the concentration of antioxidant at the interfacial region. The increase in the oil/water ratio increased the overall oxidative stability of emulsions but decreased the antioxidant efficiency of the more lipophilic derivatives. This can be assigned to the fact that, increasing the oil phase volume, the interfacial concentration decreased for the more lipophilic antioxidants.

An efficient one-pot synthesis of polyphenolic amino acids and evaluation of their radical-scavenging activity.

A simple and efficient procedure for the synthesis of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy phenylglycine amides by a strategy based on the multicomponent Ugi reaction is proposed. Hydroxybenzaldehyde derivatives were reacted with 4-methoxybenzylamine, cyclohexyl isocyanide and benzoic acid or 2-naphthylacetic acid to give Ugi adducts that were treated with trifluoroacetic acid yielding N-acyl hydroxyphenylglycine amides in good yields. The same procedure using as acid component protocatechuic acid or hydrocaffeic acid gave N-catechoyl 3,4-dihydroxyphenylglycine amides. The use of N-benzyloxycarbonylglycine as acid component allowed the preparation of a 3,4-dihydroxyphenylglycyl dipeptide derivative. Radical-scavenging activity studies of the polyphenolic amino acid derivatives showed a sharp increase in activity with the increase in number of hydroxyl or catechol groups present. Cyclic voltammetry experiments established a correlation between oxidation peak potentials and the radical-scavenging activity.

Control of antioxidant efficiency of chlorogenates in emulsions: modulation of antioxidant interfacial concentrations.

BACKGROUND: Controlling the interfacial concentrations of antioxidants (AOs) in oil-in-water emulsions can be regarded as a unique approach for increasing the efficiency of AOs in inhibiting the oxidation of lipids. Classical methods to determine the AO distribution in binary systems cannot be employed and their distribution needs to be assessed in the intact emulsion. RESULTS: We have employed a well-established kinetic method to determine the distribution of a homologous series of AOs derived of chlorogenic acid in olive oil-in-water emulsions and analyse the effects of AO hydrophobicity on their distributions and their efficiencies. Results indicate that variations in the efficiency of chlorogenates in emulsions are due to differences in their interfacial concentrations. Their interfacial concentrations AOI were much higher (20- to 150-fold) than their stoichiometric concentrations. On the other hand, their concentrations in the oil region were 1.5- to 0.1-fold. Results also show the complex effect of the oil-to-water ratio employed in the preparation of the emulsions on the (AOI ) values. CONCLUSION: Results highlight the key role of the interfacial region and of its composition (interfacial AO molarity, emulsifier concentration, oil-to-water ratio) in interpreting the efficiency of AOs in inhibiting lipid oxidation in emulsions. Thus, a careful modulation of these parameters is necessary to ensure optimum AO efficiency. © 2019 Society of Chemical Industry.

Toxicity and structure-activity relationship (SAR) of α,ß-dehydroamino acids against human cancer cell lines.

A library of N-protected dehydroamino acids, namely dehydroalanine, dehydroaminobutyric acid and dehydrophenylalanine derivatives, was screened in three human cancer cell lines [(lung (A549), gastric (AGS) and neuroblastoma (SH-SY5Y)] in order to characterize their toxicological profile and identify new molecules with potential anticancer activity. Results showed N-protected dehydrophenylalanine and dehydroaminobutyric acid derivatives have no or low toxicity for all tested cell lines. The N-protected dehydroalanines exhibit significant toxic effects and the AGS and SH-SY5Y cells were significantly more vulnerable than A549 cells. Four α,ß-dehydroalanine derivatives, with IC50<62.5µM, were selected to investigate the pathways by which these compounds promote cell death. All compounds, at their IC50 concentrations, were able to induce apoptosis in both AGS and SH-SY5Y cell lines. In both cell lines, loss of mitochondrial membrane potential (ΔΨm) was found and caspase activity was increased, namely endoplasmic reticulum-resident caspase-4 in AGS cells and caspase-3/7 in SH-SY5Y cells. When evaluated in a non-cancer cell line, the molecules displayed no to low toxicity, thus suggesting some degree of selectivity for cancer cells. The results indicate that α,ß-dehydroalanine derivatives can be considered a future resource of compounds able to work as anticancer drugs.

A comparative analysis of oral and maxillofacial pathology over a 16-year period, in the north of Portugal.

OBJECTIVES: To determine the frequency and spectrum of oral and maxillofacial lesions biopsied in a hospital population in the northern region of Portugal. METHODS: We conducted descriptive analyses of pathology reports from biopsies of oral and maxillofacial lesions performed between 1990 and 2006, in Oporto Hospital Center. Information on gender and age of patient, location of the lesions and the histopathological diagnosis were analysed. RESULTS: The analyses revealed that 1,520 (47.7%) patients were male and 1,666 (52.3%) were female. They had a mean age ± standard deviation of 47.8 ± 18.6 years. The site most frequently biopsied was the labial mucosa (17.5%). A non-neoplastic diagnosis was established in 2,162 (63.3%) cases, potentially malignant disorders in 163 (5.1%) and neoplasms in 886 (27.6%) (403 benign and 483 malignant). The most commonly reported diagnosis was fibroepithelial polyp (n = 186; 15.9%), followed by squamous cell carcinoma (SCC) (n = 158; 13.6%). SCC was the lesion most commonly found in male patients (n = 279; 18.4%) whilst fibroepithelial polyp was the lesion most commonly found in female patients (n = 268; 16.1%). The most common lesion in patients 0-17 years of age was a follicular cyst (n = 25; 12.8%), whereas in patients 18-64 years of age it was a fibroepithelial polyp (n = 299; 13%). SCC was the most common type of lesion found in patients ≥ 65 years of age (n = 160; 24.6%). CONCLUSION: This large sample provides useful information about the incidence and distribution of oral biopsies over a period of 16 years, allowing valuable comparison with other countries. Non-neoplastic lesions were the types of lesion most commonly reported, with fibroepithelial polyp being most frequent. SCC was the second most common diagnosis.

High CDC20 expression is associated with poor prognosis in oral squamous cell carcinoma.

OBJECTIVES: Human Cell Division Cycle 20 (CDC20) homolog is a crucial target of the spindle assembly checkpoint. It is an activator of the Anaphase-Promoting Complex/Cyclosome (APC/C) which promotes anaphase onset and mitotic exit through the ubiquitination of securin and cyclin B1. Overexpression of CDC20 was previously reported in oral squamous cell carcinoma (OSCC). Here, we propose to explore the clinicopathological significance of CDC20 overexpression and its potential use as a prognostic marker in OSCC. METHODS: Using tissue microarray technology, we analyzed CDC20 expression in 65 primary OSCC tissues by immunohistochemistry. Statistical analysis was performed to evaluate the clinicopathological and prognostic significance of CDC20 expression in OSCC. RESULTS: Of the 65 cases of patients with OSCC studied, 37 (56.9%) showed high CDC20 protein expression. No clinicopathological features were correlated with CDC20 expression. Importantly, in univariable analysis, OSCC patients with higher CDC20 protein expression showed significantly shorter cancer-specific survival rate (P = 0.018). Multivariable analysis identified high CDC20 expression as an independent prognostic factor (P = 0.032). CONCLUSION: High CDC20 expression is associated with poor prognosis in OSCC and may be used to identify high-risk OSCC patients and may serve as a therapeutic target.

Incidence rates and trends of lip, oral and oro-pharyngeal cancers in Portugal.

OBJECTIVES: To analyse the trends and patterns of lip, oral and oro-pharyngeal cancer incidence in Portugal between 1998 and 2007. PATIENTS AND METHODS: Data on lip, oral and oro-pharyngeal cancers was collected from the databases maintained at the three main Regional Cancer Registries of Portugal (1998-2007). The data were analysed by gender, age and by site. Incidence rates were age standardized by the direct method, and joinpoint regression was used to estimate trends in incidence. RESULTS: During this 10-year period, a total of 9623 cases of lip, oral and oropharynx cancers were reported, 7565 (78.6%) in males and 2058 (21.4%) in females. There was an increase in the age-standardized incidence of oral cancers by 1.96% per year for both sexes grouped together and an increase of 4.34% per year for the female group. Oro-pharyngeal cancer showed an increase incidence trend of 3.49% per year for both sexes grouped together and an increase of 3.49% per year for male group among the sites analysed. Lip cancer showed a decrease in its incidence rate. CONCLUSION: In view of rising trends, it is necessary to implement policies on oral cancer control by initiating campaigns on oral cancer awareness and screening and to harness political measures on tobacco and alcohol control for the Portuguese population.

High yielding synthesis of N-ethyl dehydroamino acids.

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain N-ethyl-α, ß-dehydroamino acid derivatives. The application of this N-alkylation procedure to several methyl esters of ß,ß-dibromo and ß-bromo, ß-substituted dehydroamino acids protected with standard amine protecting groups was subsequently reported. The corresponding N-ethyl, ß-bromo dehydroamino acid derivatives were obtained in fair to high yields and some were used as substrates in Suzuki cross-coupling reactions to give N-ethyl, ß,ß-disubstituted dehydroalanine derivatives. Herein, we further explore N-ethylation of ß-halo dehydroamino acid derivatives using triethyloxonium tetrafluoroborate as alkylating agent, but substituting N,N-diisopropylethylamine for potassium tert-butoxide as auxiliary base. In these conditions, for all ß-halo dehydroamino acid derivatives, reactions were complete and the N-ethylated derivative could be isolated in high yield. This method was also applied for N-ethylation of non-halogenated dehydroamino acids. Again, with all compounds the reactions were complete and the N-ethyl dehydroamino acid derivatives could be isolated in high yields. Some of these N-ethyl dehydroamino acid methyl ester derivatives were converted in high yields to their corresponding acids and coupled to an amino acid methyl ester to give N-ethyl dehydrodipeptide derivatives in good yields. Thus, this method constitutes a general procedure for high yielding synthesis of N-ethylated dehydroamino acids, which can be further applied in peptide synthesis.

Comparative effect of N-substituted dehydroamino acids and alpha-tocopherol on rat liver lipid peroxidation activities.

Free radical damage has been associated with a growing number of diseases and conditions, such as autoimmune diseases, neurodegenerative disorders and multiple types of cancer. Some dehydroamino acids and corresponding peptides can function as radical scavengers. In this study the in vitro effects on rat liver lipid peroxidation levels of fourteen N-substituted dehydroamino acid derivatives and alpha-tocopherol were investigated. alpha-Tocopherol is a powerful antioxidant that is beneficial in the treatment of many free radical related diseases. The results indicated that all the compounds showed very good inhibitory effect on the lipid peroxidation compound with alpha-tocopherol at 1 mM concentrations and the inhibition rate was in the range of 70-79 % with the exception of compound 5. At 0.1 mM concentrations compounds 1, 2 and 9 were found more active than alpha-tocopherol. The results confirmed that molecules such as dehydroamino acids which have reactive double bonds can act as a guard in vitro against oxidants.

Yellow eel (Anguilla anguilla) development in NW Portuguese estuaries with different contamination levels.

The aims of the present study were to compare the health status of yellow eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different levels of pollution and their physiological responses to combined effects of environmental variation and pollution. For this, a field study was performed using a multi-parameter approach, including eels condition indexes and biomarkers, water quality variables and other environmental factors. Sixteen biological parameters were assessed, namely: hepatosomatic index (LSI), Fulton's condition index (K), lipid peroxidation (LPO), total glutathione (TG), reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG, and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), sodium-potassium ATPase (Na(+)/K(+)-ATPase), ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR). Ten environmental factors were also measured in water: temperature, salinity, pH, phosphates, nitrates, nitrites, ammonium, silica, phenol and hardness. Globally, the biomarkers indicate exposure and toxic effects of pollutants on eels living in contaminated estuaries. The relationships between biological and environmental variables were assessed through redundancy analysis. K and LSI indexes, AChE and Na(+)/K(+)-ATPase, total glutathione levels and the antioxidant enzymes CAT, GR, and SOD where the factors most discriminating reference (Minho River estuary) from contaminated estuaries (Lima and Douro Rivers estuaries). Moreover, the most striking outcomes of pollutants exposure on biological responses were observed during winter, probably due to a joint effect of cold weather and pollution stress. Altogether, the results indicate that the development of eels in the polluted estuaries of Lima and Douro rivers is interfering with physiological functions determinant for their survival and performance. This may increase the mortality rates during the continental life-phase of the species and decrease the percentage of animals able to successfully complete their oceanic migration and, thus, reduce the contribution of each generation to the next one.

Exposure to retinoic acid at the onset of hindbrain segmentation induces episodic breathing in mice.

Hyperpnoeic episodic breathing (HEB), a cyclic waxing and waning of breathing, has been widely reported in pre-term neonates, patients with Joubert syndrome and adults (Cheyne-Stokes respiration) with congestive heart failure and brainstem infarction. We now provide a developmental mouse model of neonatal HEB. We used retinoic acid (RA) (0.5-10 mg/kg of maternal weight) to alter embryonic development of the respiratory neuronal network at the onset of hindbrain segmentation (7.5 days post-coitum). HEB was observed in vivo after RA treatment during post-natal days 1-7 but not in control animals. HEB persisted after reduction of the chemoafferent input by hypocapnic hyperoxia (100% O(2)). A large increase and decrease of the rhythm resembling an HEB episode was induced in vitro by stimulating the parafacial respiratory oscillator in treated but not in control neonates. Post-natal localization of the superior cerebellar peduncle and adjacent dorsal tegmentum was found to be abnormal in the pons of RA-treated juvenile mice. Thus, early developmental specifications in the rostral hindbrain are required for the development of neurones that stabilize the function of the respiratory rhythm generator, thereby preventing HEB during post-natal maturation.