The choice of endogenous controls in exosomal microRNA assessments from biofluids.

The assessment of differentially expressed microRNAs in patients and healthy controls is important to identify potential tumor biomarkers. Recently, it has been shown that the microRNA levels in exosomes are more correlated with the clinical-pathological variables than vesicle-free microRNAs (miRNAs) in biofluids; therefore, there is an increasing interest in these specific evaluations. However, these measurements can be affected by experimental problems that not always are evaluated and/or by inadequate procedural choices. In particular, exosome isolation and miRNA extraction procedures are crucial to avoid contaminations, and even the choice of the most suitable purity controls is important. Moreover, a stable endogenous RNA should be used for normalization of miRNA expression obtained by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in order to make these measures comparable among different samples. A rushed choice of the endogenous control can bias study conclusions without revealing inconsistencies. Unfortunately, a few studies systematically identified the best normalizer for their specific experimental context. Instead, sometimes, the normalization procedures were performed in a disputable way or the normalizer choices simply based on the previous literature. Here, we reviewed the studies where the exosomal miRNA profiling was assessed in human biofluids to point out the adopted procedures and the specific endogenous controls chosen for normalization.

To accelerate the Zika beat: Candidate design for RNA interference-based therapy.

Zika virus infection is associated with the development of severe neurological disorders in adults and newborns. Although at the moment Zika virus outbreak is not threatening to become again an emergency, infection cases are still being sporadically reported and there is still no effective therapy available. A possible treatment to suppress Zika replication is represented by short interfering RNAs (siRNAs), since they have been successfully used even against Ebola, H5N1 and SARS viruses and clinical trials of siRNA-based drugs are ongoing. In order to speed up the time consuming experimental validation of effective siRNAs, we have performed a comprehensive bioinformatic analysis to design only a few promising siRNAs against Zika virus. Besides siRNA efficacy, we paid attention to broad-spectrum antiviral activity, obtained by analysing all known Zika genomes, and siRNA safety, by excluding siRNAs that could potentially provoke an immune response or interfere with host mRNAs, lncRNAs, circRNAs and RNA binding proteins. In Zika genome we identified several highly conserved regions targetable by only 20 siRNAs. In particular, only a few siRNAs survived highly stringent criteria for siRNA safety. Notably, two of our candidate siRNAs have been successfully used against other flaviviruses like Zika, both in in vitro and in vivo models. Since they were effective against two different flaviviruses, by targeting a highly conserved region, it is reasonable to hypothesize that they could be active also against Zika. Therefore, we encourage researchers to experimentally validate these promising siRNAs.

Identification of candidate miRNA biomarkers for pancreatic ductal adenocarcinoma by weighted gene co-expression network analysis.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal prognosis which is, among others, due to a lack of suitable biomarkers and therapeutic targets. Previously, basic gene expression analysis methods have been used for their identification, but recently new algorithms have been developed allowing more comprehensive data analyses. Among them, weighted gene co-expression network analysis (WGCNA) has already been applied to several cancer types with promising results. METHODS: We applied WGCNA to miRNA expression data from PDAC patients. Specifically, we processed microarray-based expression data of 2555 miRNAs in serum from 100 PDAC patients and 150 healthy subjects. We identified network modules of co-expressed miRNAs in the healthy subject dataset and verified their preservation in the PDAC dataset. In the non-preserved modules, we selected key miRNAs and carried out functional enrichment analyses of their experimentally known target genes. Finally, we tested their prognostic significance using overall survival analyses. RESULTS: Through WGCNA we identified several miRNAs that discriminate healthy subjects from PDAC patients and that, therefore, may play critical roles in PDAC development. At a functional level, we found that they regulate p53, FoxO and ErbB associated cellular signalling pathways, as well as cell cycle progression and various genes known to be involved in PDAC development. Some miRNAs were also found to serve as novel prognostic biomarkers, whereas others have previously already been proposed as such, thereby validating the WGCNA approach. In addition, we found that these novel data may explain at least some of our previous PDAC gene expression analysis results. CONCLUSIONS: We identified several miRNAs critical for PDAC development using WGCNA. These miRNAs may serve as biomarkers for PDAC diagnosis/prognosis and patient stratification, and as putative novel therapeutic targets.

Purification and characterization of two forms of glyoxalase II from the liver and brain of Wistar rats.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes.

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.

Dietary restriction affects antioxidant levels in rat liver mitochondria during ageing.

Six experimental groups of young (7-month-old) and aged (24-32-month-old) rats, underwent different dietary manipulations (i.e. dietary restriction and/or a vitamin E-depleted diet), and their liver mitochondria were assayed for several antioxidants and peroxidation markers. Glutathione levels were affected both by age and dietary treatment. Coenzyme Q9 and C0Q10 showed the highest levels in the oldest rats where ageing, as well as other oxidative stresses, could induce ubiquinone biosynthesis.

Relaxed thiol substrate specificity of glutathione transferase effected by a non-substrate glutathione derivative.

Rat glutathione transferase 4-4 catalyzed the conjugation of 2-mercaptoethanol with 1-chloro-2,4-dinitrobenzene in the presence of S-methyl-glutathione. The reaction was linearly dependent on enzyme concentration and saturation was seen with respect to both 2-mercaptoethanol and S-methyl-glutathione concentration. High concentrations of S-methyl-glutathione were inhibitory. The results suggest that the natural substrate glutathione has two distinct functions in the normal catalytic reaction, (i) induction of a catalytically competent conformation of the enzyme and (ii) provision of the substrate sulfhydryl group in the reaction catalyzed.

Studies on the life prolonging effect of food restriction: glutathione levels and glyoxalase enzymes in rat liver.

Cytosolic and mitochondrial levels of glutathione (GSH) as well as the activities of glyoxalase I (GI) and glyoxalase II (GII), GSH-dependent enzymes involved in the detoxification of 2-ketoaldehydes, were investigated in the liver of ad libitum (AL) fed and food restricted (FR) rat during aging. Both cytosolic and mitochondrial GSH level was lower in old than in adult AL fed rats. Food restriction did not prevent this decrease, but its extent was attenuated considering the cytosolic GSH. As regards the mitochondrial GSH, its content was higher in adult FR animals than in the age-matched AL fed ones. Thus, the subsequent age-dependent decrease of GSH, occurring also in FR animals, resulted in a thiol concentration not different from that observed in young and adult AL fed animals. Considering the enzymatic activities, cytosolic GI decreased in old rats irrespective of diet, whereas GII activity remained constant in all the experimental groups. The higher glutathione content found in both cellular compartments of old FR rats as compared to the old AL fed ones, could help to explain the life prolonging effect of FR treatment. Moreover, the observation that the activity of glyoxalases was not influenced by food restriction does not necessarily mean that the cells of diet-conditioned animals are scarcely protected against the toxic effect of methylglyoxal. Indeed, the production of this compound should be lower in FR animals as compared to AL fed ones, due to the lower level serum glucose concentration during the life span of the former with respect to the latter group.

Glyoxalase II and glutathione levels in rat liver mitochondria during cold storage in Euro-Collins and University of Wisconsin solutions.

Glutathione and glyoxalase II levels were evaluated in cytosolic and mitochondrial compartments of rat liver after 7 and 24 hr of cold storage in University of Wisconsin (UW) and Euro-Collins solutions. 1-4 A similar time-dependent depletion of cytosolic glutathione up to about 60% of control values was observed in both Euro-Collins and UW solutions. Cytosolic glyoxalase II showed activity oscillations in livers stored in Euro-Collins but not in UW. Mitochondrial glutathione and glyoxalase II were severely depleted soon after 7 hr of cold storage in Euro-Collins, whereas the same parameters did not change in liver stored in UW after 24 hr. UW is confirmed to be the most suitable solution for liver cold storage and we conclude that mitochondrial glutathione and glyoxalase II can be important parameters in assessing mitochondrial and cell function.

Separation of the four pairs of enantiomers of vincamine alkaloids by enantioselective high-performance liquid chromatography.

The four enantiomeric pairs of vincamine group alkaloids were separated by HPLC using Chiralpak AD as chiral stationary phase (CSP) and various n-hexane-2-propanol and n-hexane-ethanol mobile phases. (+)-cis-Vincamine, which is used in pharmaceutical preparations, is eluted much faster than its optical isomer, with separation factors of 2.4 and 3.5, respectively in these mobile phases. Other CSPs gave negative results. A chiral recognition mechanism is proposed and circular dichroism spectra of the individual enantiomers are presented.

Direct high-performance liquid chromatographic separation of the enantiomers of methoxy and hydroxy derivatives of 3,4-dihydro-3-(dipropylamino)-2H-1-benzopyrans with dopaminergic activity.

The direct HPLC resolution of the enantiomers of methoxy and hydroxy derivatives of 3,4-dihydro-3-(dipropylamino)-2H-1-benzopyrans and of unsubstituted amino compounds was achieved using Chiralcel OD and/or Chiralpak AD stationary phases. The position of the substituent (methoxyl or hydroxyl) in the aromatic ring has a strong effect on the enantioselectivity. Circular dichroism spectra of the single enantiomers of one compound were measured.

Synthesis of brain DNA during acquisition of an active avoidance task.

The possible involvement of cerebral DNA synthesis in the learning process was examined in rats injected intracerebrally with 3H thymidine. During the period of incorporation (4.5 hr) one rat was trained to an active avoidance task while a second animal was kept in the same experimental room. In comparison with control rats paired to learning animals, the concentration of PCA-soluble radioactivity and of radioactive DNA of the cerebral cortex increased in all animal groups, i.e., control rats paired to non-learning animals, learning rats and non-learning rats. No change occurred in liver. In the cerebral cortex the slope of the regression line obtained by plotting the concentration of radioactive DNA versus the concentration of PCA-soluble radioactivity was lower in learning rats than in the group of pooled control animals. A comparable effect was noted in the hippocampus. In non-learning animals a similar decrease was present in the cerebral cortex and in cerebellum. In addition, it was found that in learning animals the percent incorporation was inversely related to the total number of avoidances only in the cerebral cortex. In non-learning rats a similar inverse relationship was present in the cerebral cortex and in cerebellum. In the former region the regression line of learning rats was shifted upwards in comparison with the regression line of non-learning animals. These results are interpreted to indicate that the incorporation of 3H-thymidine into cerebral DNA is directly related to the level of stress and is increased by learning.

Effect of methylglyoxal on Bufo bufo embryo development: morphological and biochemical aspects.

Methylglyoxal (2-oxopropanal) is a cytotoxic compound that can be formed endogenously as a by-product of glycolytic pathway; so its concentration is expected to increase when glycolysis activity increases such as during embryo development. In this work we study the effect of exogenous methylglyoxal on development and embryo viability during Bufo Bufo development and on the enzymes and cofactors involved in its detoxication process (glyoxalase I and II, reduced glutathione and glyceraldehyde 3-phosphate dehydrogenase). The results show that exogenous methylglyoxal does not affect the enzymatic pattern until stage 20, while it induces a significant activity decrease of the tested enzymes at stage 25. On the contrary methylglyoxal positively influences the reduced glutathione concentration at all the considered stages. At morphological and histological levels methylglyoxal causes a strong retardation of cell division in the early stages, that results in various abnormalities in the late development. In conclusion, methylglyoxal enters the embryo and is antiproliferative and teratogenic: the data further supports the hypothesis of the importance of the glyoxalase system in the process of cell growth and division.

Effect of ascorbic and dehydroascorbic acid on glyoxalase enzyme system.

The authors studied the effect of ascorbic and dehydroascorbic acid on glyoxalase enzyme system, in order to establish if these substances influence cell proliferation by interfering with glyoxalase activity. The results demonstrate that both ascorbic and dehydroascorbic acid do not alter glyoxalase activity in vitro, even if employed at high doses.

[Bio-metabolic changes produced by 2 commercial fluoride-containing compounds on human keratinocytes]. / Alterazioni bio-metaboliche indotte da due composti fluorati commerciali su cheratinociti umani.

BACKGROUND: Although fluoride has been used for decades either systemically or topically to prevent dental caries, the cellular and molecular mechanisms underlying its action are poorly understood. METHODS: An in vitro study of the human keratinocyte cell line NCTC 2544 was conducted in the presence of two different fluoride-containing commercial compounds (Zymafluor and Elmex) to investigate their toxicity threshold and the sequence of events involved in fluoride ion toxicity in this cell population. The toxicity threshold was determined by incubating cells with rising concentrations of Zymafluor and Elmex for 20 h. The study of the sequence of events involved in ion toxicity was performed through a time-effect study by exposing cells to 4 mM fluoride ions and testing them at 2, 6, and 20 h. Cell viability and ultrastructural parameters were assessed: degree of confluence, semiquantitative assessment of dead cells and debris in the supernatant, and morphology. RESULTS: Ultrastructural morphological analysis showed different cell behaviours with the two compounds; moreover, their toxic effect appeared to be both concentration- and time-dependent. CONCLUSIONS: These data underline the susceptibility of the intracellular communication system to fluoride and show that exceeding the therapeutic dose of fluoride involves substantial risk of toxicity.