Low Serum Tryptophan Levels as an Indicator of Global Cognitive Performance in Nondemented Women over 50 Years of Age.

Aging is a physiological decline process. The number of older adults is growing around the world; therefore, the incidence of cognitive impairment, dementia, and other diseases related to aging increases. The main cellular factors that converge in the aging process are mitochondrial dysfunction, antioxidant impairment, inflammation, and immune response decline, among others. In this context, these cellular changes have an influence on the kynurenine pathway (KP), the main route of tryptophan (Trp) catabolism. KP metabolites have been involved in the aging process and neurodegenerative diseases. Although there are changes in the metabolite levels with age, at this time, there is no study that has evaluated cognitive decline as a consequence of Trp catabolism fluctuation in aging. The aim of this study was to evaluate the relation between the changes in Trp catabolism and cognitive impairment associated with age through KP metabolites level alterations in women over 50 years of age. Seventy-seven nondemented women over 50 years old were examined with a standardized cognitive screening evaluation in Spanish language (Neuropsi), Beck anxiety inventory (BAI), and the geriatric depression scale (GDS). Also, serum levels of Trp, kynurenine (Kyn), kynurenic acid (KYNA), and 3-hydroykynurenine (3-HK) and the glutathione ratio (GSH/GSSG) were measured. Results showed a negative correlation between age and Trp levels and a positive correlation between age and KYNA/Trp and 3-HK/Trp ratios. The level of cognitive impairment showed a significant positive association with age and with kynurenine pathway activation and a significant negative correlation with Trp levels. The GSH/GSSG ratio correlated positively with Trp levels and negatively with Kyn/Trp and 3-HK/Trp ratios. The depression score correlated negatively with Trp and positively with the 3-HK/Trp ratio. We concluded that KP activation increases with age and it is strongly associated with the level of cognition performance in nondemented women over 50 years of age.

Novel giardicidal compounds bearing proton pump inhibitor scaffold proceeding through triosephosphate isomerase inactivation.

Giardiasis is a worldwide parasitic disease that affects mainly children and immunosuppressed people. Side effects and the emergence of resistance over current used drugs make imperative looking for new antiparasitics through discovering of new biological targets and designing of novel drugs. Recently, it has determined that gastric proton-pump inhibitors (PPI) have anti-giardiasic activity. The glycolytic enzyme, triosephosphate isomerase (GlTIM), is one of its potential targets. Therefore, we employed the scaffold of PPI to design new compounds aimed to increase their antigiardial capacity by inactivating GlTIM. Here we demonstrated that two novel PPI-derivatives (BHO2 and BHO3), have better anti-giardiasic activity than omeprazole in concentrations around 120-130 µM, without cytotoxic effect on mammal cell cultures. The derivatives inactivated GlTIM through the chemical modification of Cys222 promoting local structural changes in the enzyme. Furthermore, derivatives forms adducts linked to Cys residues through a C-S bond. We demonstrated that PPI can be used as scaffolds to design better antiparasitic molecules; we also are proposing a molecular mechanism of reaction for these novel derivatives.

Effects of PVP/PEI coated and uncoated silver NPs and PVP/PEI coating agent on three species of marine microalgae.

In the last years, applications for silver nanoparticles (Ag NPs) continue to increase together with the concerns about their potential input and hazards in aquatic ecosystems, where microalgae are key organisms. The aim of the present study was to assess the relative sensitivity of three marine microalgae species with differences in cell wall composition/structure exposed to Poly N-vinyl-2-pirrolidone/Polyethyleneimine (PVP/PEI) coated 5nm Ag NPs and uncoated 47nm Ag NP. As limited attention has been paid to the role of coating agents in NP toxicity, the effect of PVP/PEI alone was also evaluated. After 72h in artificial seawater, 47nm Ag NPs formed around 1400nm size aggregates while PVP/PEI coated 5nm Ag NPs reached around 90nm. Ag+ release in seawater was around 3% for 47nm Ag NPs and 30% for PVP/PEI coated 5nm Ag NPs. PVP/PEI coated 5nm Ag NP aggregates entrapped the algal cells in a network of heteroaggregates, while uncoated 47nm Ag NPs interacted to a lesser extent with algae. The concentration of PVP/PEI coated 5nm Ag NPs that exerted the median effect (EC50) on algae growth pointed out differences in algae sensitivity: T. suecica was about 10 times more sensitive than I. galbana and P. tricornutum. Further, the coating agent alone was as toxic to algae as PVP/PEI coated 5nm Ag NPs, suggesting that presence of the coating agent was the main driver of toxicity of coated NPs. Uncoated 47nm Ag NPs instead, showed similar toxicity towards algae although P. tricornutum was slightly less sensitive than T. suecica and I. galbana, which agrees with the presence of a resistant silicified cell wall in the diatom. The present work demonstrates differences in sensitivity of three marine microalgae, possibly related to their cell surface and size characteristics.

Genotoxic and cytotoxic effects of ZnO nanoparticles for Dunaliella tertiolecta and comparison with SiO2 and TiO2 effects at population growth inhibition levels.

The increasing use of oxide nanoparticles (NPs) in commercial products has intensified the potential release into the aquatic environment where algae represent the basis of the trophic chain. NP effects upon algae population growth were indeed already reported in literature, but the concurrent effects at cellular and genomic levels are still largely unexplored. Our work investigates the genotoxic (by COMET assay) and cytotoxic effects (by qualitative ROS production and cell viability) of ZnO nanoparticles toward marine microalgae Dunaliella tertiolecta. A comparison at defined population growth inhibition levels (i.e. 50% Effect Concentration, EC50, and No Observed Effect Concentration, NOEC) with SiO2 and TiO2 genotoxic effects and previously investigated cytotoxic effects (Manzo et al., 2015) was performed in order to elucidate the possible diverse mechanisms leading to algae growth inhibition. After 72h exposure, ZnO particles act firstly at the level of cell division inhibition (EC50: 2mg Zn/L) while the genotoxic action is evident only starting from 5mg Zn/L. This outcome could be ascribable mainly to the release of toxic ions from the aggregate of ZnO particle in the proximity of cell membrane. In the main, at EC50 and NOEC values for ZnO NPs showed the lowest cytotoxic and genotoxic effect with respect to TiO2 and SiO2. Based on Mutagenic Index (MI) the rank of toxicity is actually: TiO2>SiO2>ZnO with TiO2 and SiO2 that showed similar MI values at both NOEC and EC50 concentrations. The results presented herein suggest that up to TiO2 NOEC (7.5mg/L), the algae DNA repair mechanism is efficient and the DNA damage does not result in an evident algae population growth inhibition. A similar trend for SiO2, although at lower effect level with respect to TiO2, is observable. The comparison among all the tested nanomaterial toxicity patterns highlighted that the algae population growth inhibition occurred through pathways specific for each NP also related to their different physicochemical behaviors in seawater.

The diverse toxic effect of SiO2 and TiO2 nanoparticles toward the marine microalgae Dunaliella tertiolecta.

Nanoparticles (NPs) are widely used in many industrial applications. NP fate and behavior in seawater are a very important issue for the assessment of their environmental impact and potential toxicity. In this study, the toxic effects of two nanomaterials, silicon dioxide (SiO2) and titanium dioxide (TiO2) NPs with similar primary size (~20 nm), on marine microalgae Dunaliella tertiolecta were investigated and compared. The dispersion behavior of SiO2 and TiO2 NPs in seawater matrix was investigated together with the relative trend of the exposed algal population growth. SiO2 aggregates rapidly reached a constant size (600 nm) irrespective of the concentration while TiO2 NP aggregates grew up to 4 ± 5 µm. The dose-response curve and population growth rate alteration of marine alga D. tertiolecta were evaluated showing that the algal population was clearly affected by the presence of TiO2 NPs. These particles showed effects on 50 % of the population at 24.10 [19.38-25.43] mg L(-1) (EC50) and a no observed effect concentration (NOEC) at 7.5 mg L(-1). The 1 % effect concentration (EC1) value was nearly above the actual estimated environmental concentration in the aquatic environment. SiO2 NPs were less toxic than TiO2 for D. tertiolecta, with EC50 and NOEC values one order of magnitude higher. The overall toxic action seemed due to the contact between aggregates and cell surfaces, but while for SiO2 a direct action upon membrane integrity could be observed after the third day of exposure, TiO2 seemed to exert its toxic action in the first hours of exposure, mostly via cell entrapment and agglomeration.

Molecular and catalytic properties of the aldehyde dehydrogenase of Gluconacetobacter diazotrophicus, a quinoheme protein containing pyrroloquinoline quinone, cytochrome b, and cytochrome c.

Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus: molecular and catalytic properties.

Gluconacetobacter xylinus possesses a constitutive membrane-bound oxidase system for the use of ethanol. Its alcohol dehydrogenase complex (ADH) was purified to homogeneity and characterized. It is a 119-kDa heterodimer (68 and 41 kDa subunits). The peroxidase reaction confirmed the presence of haem C in both subunits. Four cytochromes c per enzyme were determined by pyridine hemochrome spectroscopy. Redox titrations of the purified ADH revealed the presence of four haem c redox centers, with apparent mid-point potential values (Em(7)) of -33, +55, +132 and +310 mV, respectively. The ADH complex contains one mol of pyrroloquinoline quinone as determined by HPLC. The enzyme was purified in full reduced state; oxidation was induced by potassium ferricyanide and substrate restores full reduction. Activity responses to pH were sharp, showing two distinct optimal pH values (i.e. pH 5.5 and 6.5) depending on the electron acceptor used. Purified ADH oxidizes primary alcohols (C2-C6) but not methanol. Noteworthy, aliphatic aldehydes (C1-C4) were also good substrates. Myxothiazol and antymicin A were powerful inhibitors of the purified ADH complex, most likely acting at the ubiquinone acceptor site in subunit II.

The membrane-bound quinohemoprotein alcohol dehydrogenase from Gluconacetobacter diazotrophicus PAL5 carries a [2Fe-2S] cluster.

Gluconacetobacter diazotrophicus stands out among the acetic acid bacteria as it fixes dinitrogen and is a true endophyte. It has a set of constitutive enzymes to oxidize ethanol and acetaldehyde which is upregulated during N(2)-dependent growth. The membrane-bound alcohol dehydrogenase (ADH) is a heterodimer (subunit I approximately 72 kDa, subunit II approximately 44 kDa) and constitutes an important component of this organism. ADH of Ga. diazotrophicus is a typical quinohemoprotein with one pyrroloquinoline quinone (PQQ) and four c-type cytochromes. For the first time, a [2Fe-2S] cluster has been identified by EPR spectroscopy in this type of enzyme. This finding is supported by quantitative chemical analysis, revealing 5.90 +/- 0.15 Fe and 2.06 +/- 0.10 acid-labile sulfurs per ADH heterodimer. The X-band EPR spectrum of ADH (as isolated in the presence of dioxygen, 20 K) showed three broad resonances at g 2.007, 1.941, and 1.920 (g(av) 1.956), as well as an intense narrow line centered at g = 2.0034. The latter signal, which was still detected at 100 K, was attributed to the PQQ semiquinone radical (PQQ(sq)). The broad resonances observed at lower temperature were assigned to the [2Fe-2S] cluster in the one-electron reduced state. The oxidation-reduction potentials E(m) (pH 6.0 vs SHE) of the four c-type cytochromes were estimated to E(m1) = -64 (+/-2) mV, E(m2) = -8 (+/-2) mV, E(m3) = +185 (+/-15) mV, and E(m4) = +210 (+/-10) mV (spectroelectrochemistry), E(mFeS) = -250 (+/-5) mV for the [2Fe-2S] cluster, and E(mPQQ) = -210 (+/-5) mV for the PQQ/PQQH(2) couple (EPR spectroscopy). We propose a model for the membrane-bound ADH of Ga. diazotrophicus showing hypothetical intra- and intermolecular electron pathways. Subunit I binds the PQQ cofactor, the [2Fe-2S] cluster, and one c-type cytochrome. Subunit II harbors three c-type cytochromes, thus providing an efficient electron transfer route to quinones located in the cytoplasmic membrane.

Predictability of copper, irgarol, and diuron combined effects on sea urchin Paracentrotus lividus.

The aim of this work was to investigate the mixture toxicity of Irgarol (2-methylthio-4-t-butylamino-6-cyclopropylamino-s-triazine), Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and copper upon the sea urchin Paracentrotus lividus and to compare the observed data with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA). Copper spermiotoxicity was more sensitive (EC50 = 0.018 mg/L) than embryotoxicity (EC50 = 0.046 mg/L). The offspring malformations were mainly P1 type (skeletal alterations) in both cases, probably because copper competes to fix Ca2+. Irgarol and Diuron toxicity has been previously investigated. EC50 mixture embryotoxicity showed an EC50 of 1.79 mg/L, whereas spermiotoxicity mixture effects were lower than 11%. Both CA and IA modeling approaches failed to predict accurately mixture toxicity. For embryotoxicity, the IA model overestimated the mixture toxicity at effect levels of <80%. CA does not represent the worst-case approach showing values lower than IA (embryotoxicity) or similar (spermiotoxicity).

Toxic effects of irgarol and diuron on sea urchin Paracentrotus lividus early development, fertilization, and offspring quality.

Irgarol and Diuron are the most representative "organic booster biocides" that replaced organotin compounds in antifouling paints. It cannot be assumed beforehand that their use will have no environmental impact: more ecotoxicological data and a significant environmental monitoring are required. Spermio and embryotoxicities of the biocides Irgarol and Diuron were investigated on Paracentrotus lividus, the dominant echinoid species of the Mediterranean Sea. Spermiotoxicity was studied by assessing the effects of sperm exposure on fertilization rate as well as on the induction of transmissible damages to the offspring. Embryotoxicity was studied by assessing the developmental defects in the exposed larvae. The experimental results show a Diuron EC50 of 2.39 (+/- 0.21) mg/L with a NOEL of 0.25 mg/L for embryos, and of 5.09 (+/- 0.45) mg/L with a NOEL of 0.5 mg/L for sperms, respectively. Data obtained from the embryotoxicity test on Irgarol [EC50 0.99 (+/- 0.69) mg/L] are of the same order of magnitude as the literature data about Japanese urchins. Spermiotoxicity tests show an Irgarol EC50 of 9.04 (+/- 0.45) mg/L with a NOEL of 0.1 mg/L. These data show the different sensitivities of the two tests: embryos are more sensitive than sperms for both the tested chemicals and Diuron seems to be the less toxic one. Moreover, as a major output of the experimental work, tested herbicides exert transmissible damage to spermatozoa evidenced by larval malformations in the offspring, mainly P1 type (skeletal alterations). The comparison of the endpoints results offers an interesting indication of a probable different mode of action (Irgarol seems to interact with calcium homeostasis) of the two biocides.

Partial bioenergetic characterization of Gluconacetobacter xylinum cells released from cellulose pellicles by a novel methodology.

AIMS: Gluconacetobacter xylinum is well known for its ability to produce large amounts of cellulose, however, little is known about its cell physiology. Our goal was to study the respiratory metabolism and components of the respiratory system of this bacterium in static cultures. To reach our goal, a medium formulation had to be designed to improve cell growth and cellulose production together with a novel method for the recovery of cells from cellulose pellicles. METHODS AND RESULTS: Successive modifications of a nutrient medium improved G. xylinum cell growth 4.5-fold under static culture conditions. A blender homogenization procedure for the releasing of cells from the cellulose matrix gave a high yield of cells recovered. Respiratory activities of purified cells were greatly stimulated by exogenous substrates and showed to be resistant to KCN. Unexpectedly, exogenous NADH was oxidized at high rates. Cytochromes a, b, c and d were identified after spectral analyses. CONCLUSIONS: Partial bioenergetic characterization of G. xylinum cells allowed us to propose a scheme for its respiratory system. In addition, the growth medium for biomass production and the procedure for the efficient recovery of cells from cellulose pellicles were significantly improved. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first-ever bioenergetic characterization of G. xylinum grown in static cultures. In addition, a novel methodology to obtain purified cells in suitable quantities for biochemical research is described.

Sea urchin embryotoxicity test: proposal for a simplified bioassay.

Sea urchin embryotoxicity tests are widely used for evaluating the biological effects of contaminants in marine environments. The currently used traditional and standardized protocols are quite slow and laborious. The present work shows a modified bioassay (new embryotoxicity test; NET) in an attempt to speed up laboratory work using a limited number of fertilized eggs. Several experiments have been conducted both with a traditional bioassay and with the NET, using the same test conditions, in order to evaluate the reliability of the proposed simplified bioassay. Adult Paracentrotus lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples) and embryos, reared in filtered seawater, were exposed to increasing potassium dichromate and copper sulfate concentrations. Then the EC(50) was calculated. The analysis of the results evidenced good repeatability. The confidence limits in all tests overlapped; moreover, data correlation analysis between the results of both tests showed a high significant accordance (chromium, R2 = 0.93, P < 0.01; copper, R2 = 0.86, P < 0/05). In conclusion, the NET seems to be a good alternative to the traditional tests; it could be a first step toward a new routine ecotoxicological kit for seawater.

A novel one-shot anastomotic stapler prototype for coronary bypass grafting on the beating heart: feasibility in the pig.

OBJECTIVE: The nonpenetrating, arcuate-legged clip has proved its ability to provide a high-quality microvascular anastomosis. This study assessed the feasibility of constructing a coronary end-to-side anastomosis on the beating heart with a novel mechanical, sutureless anastomotic device that applies 12 circumferential clips simultaneously. METHODS: In 14 consecutive pigs (70-90 kg), the left internal thoracic artery (diameter, 3 mm) was grafted to the left anterior descending coronary artery (diameter, 3 mm) by means of a one-shot anastomotic stapler prototype. Endothelial denudation, medial necrosis, and intimal hyperplasia were analyzed quantitatively and compared with those seen in conventionally sutured anastomoses (n = 4). RESULTS: In 8 of 14 anastomoses, the one-shot anastomotic stapler successfully applied all 12 clips circumferentially across the everted arteriotomy edges. In the remaining, either 1 (n = 4) or 3 and 4 adjoining malaligned clips had to be replaced manually with a single-clip applicator. Coronary occlusion was limited to approximately 3 minutes. At follow-up, all anastomoses were patent angiographically. At 2 days, in 2 of 7 cases, a local coronary dissection was observed, and there was a considerable loss of endothelial cells and medial damage. At 28 days, however, minimal intimal hyperplasia was seen at the anastomotic lining, although more pronounced when compared with conventionally sutured anastomoses. CONCLUSIONS: The one-shot anastomotic stapler prototype enabled short-occlusive (3 minutes), sutureless end-to-side grafting on the beating porcine heart. In spite of early endothelial and medial damage and 2 local dissections, all anastomoses remained patent with minimal intimal hyperplasia at 4 weeks.

Facilitated vascular anastomoses: the one-shot device.

BACKGROUND: A mechanical system for facilitating vascular anastomosis (end-to-side, end-to-end) is described that enables the rapid construction of nonpenetrated, compliant junctions. The instrument (United States Surgical One-Shot system) simultaneously applies either 10 or 12 nonpenetrating, arcuate-legged titanium clips to everted vessel or prosthetic conduit edges. METHODS AND RESULTS: The instrument has been tested in animals (jugular and femoral vein jump grafts in carotid and femoral arteries, interpositional grafts, 20 pigs) and human cadaveric constructs (saphenous veins to left anterior descending coronary arteries, 20 cases, 5 brachiocephalic access fistulas) as end-to-side constructs. Clipped constructs have equivalent or superior physical properties to control sutured constructs (6-0 polypropylene) as gauged by burst and tensile strength. All studies were performed under Food and Drug Administration Good Laboratory Practice standards, and the device has been approved for marketing by the Food and Drug Administration. CONCLUSIONS: The device enables rapid and reproducible vascular anastomotic constructs with vessels as small as 1.8 mm outer diameter. The constructs are flanged, interrupted, and nonpenetrated.