MiR-186-5p inhibition restores synaptic transmission and neuronal network activity in a model of chronic stress.

Chronic stress exerts profound negative effects on cognitive and emotional behaviours and is a major risk factor for the development of neuropsychiatric disorders. However, the molecular links between chronic stress and its deleterious effects on neuronal and synaptic function remain elusive. Here, using a combination of in vitro and in vivo approaches, we demonstrate that the upregulation of miR-186-5p triggered by chronic stress may be a key mediator of such changes, leading to synaptic dysfunction. Our results show that the expression levels of miR-186-5p are increased both in the prefrontal cortex (PFC) of mice exposed to chronic stress and in cortical neurons chronically exposed to dexamethasone. Additionally, viral overexpression of miR-186-5p in the PFC of naïve mice induces anxiety- and depressive-like behaviours. The upregulation of miR-186-5p through prolonged glucocorticoid receptor activation in vitro, or in a mouse model of chronic stress, differentially affects glutamatergic and GABAergic synaptic transmission, causing an imbalance in excitation/inhibition that leads to altered neuronal network activity. At glutamatergic synapses, we observed both a reduction in synaptic AMPARs and synaptic transmission, whereas GABAergic synaptic transmission was strengthened. These changes could be rescued in vitro by a miR-186-5p inhibitor. Overall, our results establish a novel molecular link between chronic glucocorticoid receptor activation, the upregulation of miR-186-5p and the synaptic changes induced by chronic stress, that may be amenable to therapeutic intervention.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination.

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV. HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e. This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

Changes in melanocyte RNA and DNA methylation favour pheomelanin synthesis and may avoid systemic oxidative stress after dietary cysteine supplementation in birds.

Cysteine plays essential biological roles, but excessive amounts produce cellular oxidative stress. Cysteine metabolism is mainly mediated by the enzymes cysteine dioxygenase and γ-glutamylcysteine synthetase, respectively coded by the genes CDO1 and GCLC. Here we test a new hypothesis posing that the synthesis of the pigment pheomelanin also contributes to cysteine homeostasis in melanocytes, where cysteine can enter the pheomelanogenesis pathway. We conducted an experiment with the Eurasian nuthatch Sitta europaea, a bird producing large amounts of pheomelanin for feather pigmentation, to investigate if melanocytes show epigenetic lability under exposure to excess cysteine. We increased systemic cysteine levels in nuthatches by supplementing them with dietary cysteine during growth. In feather melanocytes this led to the downregulation of genes involved in intracellular cysteine metabolism (GCLC), cysteine transport to the cytosol from the extracellular medium (Slc7a11) and from melanosomes (CTNS), and regulation of tyrosinase activity (MC1R and ASIP). These changes were mediated by increases in DNA m5 C in all genes except Slc7a11, which experienced RNA m6 A depletion. Birds supplemented with cysteine synthesized more pheomelanin than controls, but did not suffer higher systemic oxidative stress. These results suggest that excess cysteine activates an epigenetic mechanism that favours pheomelanin synthesis and may protect against oxidative stress.

Interaction between HFE and haptoglobin polymorphisms and its relation with plasma glutathione levels in obese children.

Obesity among children has emerged as a serious public health problem. The growing prevalence of childhood obesity has led to the appearance of serious complications, including a chronic systemic inflammation associated with oxidative stress.  In the present study, we analysed the interaction between two genes related with iron metabolism - HFE and haptoglobin - and the plasmatic concentration of glutathione, as a way to evaluate the antioxidant response capacity in obesity. To achieve this, 118 obese children and 89 eutrophic children were recruited for the study. Results showed that although obese children present a significantly decreased tGSH levels, once we analysed separately children based on their haptoglobin phenotype, the decreased tGSH levels is significant only for the Hp 2 allele. Additionally, Hp 2.2 obese children carrying H63D polymorphism show significantly lower tGSH/GSSG values. Our results found an association of haptoglobin and HFE with oxidative stress in childhood obesity.

Bis-Indolyl Benzenoids, Hydroxypyrrolidine Derivatives and Other Constituents from Cultures of the Marine Sponge-Associated Fungus Aspergillus candidus KUFA0062.

A previously unreported bis-indolyl benzenoid, candidusin D (2e) and a new hydroxypyrrolidine alkaloid, preussin C (5b) were isolated together with fourteen previously described compounds: palmitic acid, clionasterol, ergosterol 5,8-endoperoxides, chrysophanic acid (1a), emodin (1b), six bis-indolyl benzenoids including asterriquinol D dimethyl ether (2a), petromurin C (2b), kumbicin B (2c), kumbicin A (2d), 2″-oxoasterriquinol D methyl ether (3), kumbicin D (4), the hydroxypyrrolidine alkaloid preussin (5a), (3S, 6S)-3,6-dibenzylpiperazine-2,5-dione (6) and 4-(acetylamino) benzoic acid (7), from the cultures of the marine sponge-associated fungus Aspergillus candidus KUFA 0062. Compounds 1a, 2a-e, 3, 4, 5a-b, and 6 were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. Only 5a exhibited an inhibitory effect against S. aureus ATCC 29213 and E. faecalis ATCC29212 as well as both methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE) strains. Both 1a and 5a also reduced significant biofilm formation in E. coli ATCC 25922. Moreover, 2b and 5a revealed a synergistic effect with oxacillin against MRSA S. aureus 66/1 while 5a exhibited a strong synergistic effect with the antibiotic colistin against E. coli 1410/1. Compound 1a, 2a-e, 3, 4, 5a-b, and 6 were also tested, together with the crude extract, for cytotoxic effect against eight cancer cell lines: HepG2, HT29, HCT116, A549, A 375, MCF-7, U-251, and T98G. Except for 1a, 2a, 2d, 4, and 6, all the compounds showed cytotoxicity against all the cancer cell lines tested.

Lichen Xanthones as Models for New Antifungal Agents.

Due to the emergence of multidrug-resistant pathogenic microorganisms, the search for new antimicrobial compounds plays an important role in current medicinal chemistry research. Inspired by lichen antimicrobial xanthones, a series of novel chlorinated xanthones was prepared using five chlorination methods (Methods A⁻E) to obtain different patterns of substitution in the xanthone scaffold. All the synthesized compounds were evaluated for their antimicrobial activity. Among them, 3-chloro-4,6-dimethoxy-1-methyl-9H-xanthen-9-one 15 showed promising antibacterial activity against E. faecalis (ATCC 29212 and 29213) and S. aureus ATCC 29213. 2,7-Dichloro-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one 18 revealed a potent fungistatic and fungicidal activity against dermatophytes clinical strains (T. rubrum, M. canis, and E. floccosum (MIC = 4⁻8 µg/mL)). Moreover, when evaluated for its synergistic effect for T. rubrum, compound 18 exhibited synergy with fluconazole (ΣFIC = 0.289). These results disclosed new hit xanthones for both antibacterial and antifungal activity.

Adaptive downregulation of pheomelanin-related Slc7a11 gene expression by environmentally induced oxidative stress.

Pheomelanin is a sulphur-containing yellow-to-reddish pigment whose synthesis consumes the main intracellular antioxidant (glutathione; GSH) and its precursor cysteine. Cysteine used for pheomelanogenesis cannot be used for antioxidant protection. We tested whether the expression of Slc7a11, the gene regulating the transport of cysteine to melanocytes for pheomelanogenesis, is environmentally influenced when cysteine/GSH are most required for antioxidant protection. We found that zebra finches Taeniopygia guttata developing pheomelanin-pigmented feathers during a 12-day exposure to the pro-oxidant diquat dibromide downregulated the expression of Slc7a11 in feather melanocytes, but not the expression of other genes that affect pheomelanogenesis by mechanisms different from cysteine transport such as MC1R and Slc45a2. Accordingly, diquat-treated birds did not suffer increased oxidative stress. This indicates that some animals have evolved an adaptive epigenetic lability that avoids damage derived from pheomelanogenesis. This mechanism should be explored in human Slc7a11 to help combat some cancer types related to cysteine consumption.

Genetic favouring of pheomelanin-based pigmentation limits physiological benefits of coloniality in lesser kestrels Falco naumanni.

Pheomelanin contributes to the pigmentation phenotype of animals by producing orange and light brown colours in the integument. However, pheomelanin synthesis in melanocytes requires consumption of glutathione (GSH), the most important intracellular antioxidant. Therefore, a genetic control favouring the production of large amounts of pheomelanin for pigmentation may lead to physiological costs under environmental conditions that promote oxidative stress. We investigated this possibility in the context of breeding coloniality, a reproductive strategy that may affect oxidative stress. We found in lesser kestrel Falco naumanni nestlings that the GSH:GSSG ratio, which decreases with systemic oxidative stress, increased with the size of the colony where they were reared, but the expression in feather melanocytes of five genes involved in pheomelanin synthesis (Slc7a11, Slc45a2, CTNS, MC1R and AGRP) did not vary with colony size. The antioxidant capacity (TEAC) of lesser kestrel nestlings also increased with colony size, but in a manner that depended on Slc7a11 expression and not on the expression of the other genes. Thus, antioxidant capacity increased with colony size only in nestlings least expressing Slc7a11, a gene with a known role in mediating cysteine (a constituent amino acid of GSH) consumption for pheomelanin production. The main predictor of the intensity of pheomelanin-based feather colour was Slc45a2 expression followed in importance by Slc7a11 expression, hence suggesting that the genetic regulation of the pigmentation phenotype mediated by Slc7a11 and a lack of epigenetic lability in this gene limits birds from benefiting from the physiological benefits of coloniality.

Gyrfalcons Falco rusticolus adjust CTNS expression to food abundance: a possible contribution to cysteine homeostasis.

Melanins form the basis of animal pigmentation. When the sulphurated form of melanin, termed pheomelanin, is synthesized, the sulfhydryl group of cysteine is incorporated to the pigment structure. This may constrain physiological performance because it consumes the most important intracellular antioxidant (i.e., glutathione, GSH), of which cysteine is a constitutive amino acid. However, this may also help avoid excess cysteine, which is toxic. Pheomelanin synthesis is regulated by several genes, some of them exerting this regulation by controlling the transport of cysteine in melanocytes. We investigated the possibility that these genes are epigenetically labile regarding protein intake and thus contribute to cysteine homeostasis. We found in the Icelandic population of gyrfalcon Falco rusticolus, a species that pigments its plumage with pheomelanin, that the expression of a gene regulating the export of cystine out of melanosomes (CTNS) in feather melanocytes of developing nestlings increases with food abundance in the breeding territories where they were reared. The expression of other genes regulating pheomelanin synthesis by different mechanisms of influence on cysteine availability (Slc7a11 and Slc45a2) or by other processes (MC1R and AGRP) was not affected by food abundance. As the gyrfalcon is a strict carnivore and variation in food abundance mainly reflects variation in protein intake, we suggest that epigenetic lability in CTNS has evolved in some species because of its potential benefits contributing to cysteine homeostasis. Potential applications of our results should now be investigated in the context of renal failure and other disorders associated with cystinosis caused by CTNS dysfunction.

A New Dihydrochromone Dimer and Other Secondary Metabolites from Cultures of the Marine Sponge-Associated Fungi Neosartorya fennelliae KUFA 0811 and Neosartorya tsunodae KUFC 9213.

A previously unreported dihydrochromone dimer, paecilin E (1), was isolated, together with eleven known compounds: ß-sitostenone, ergosta-4,6,8 (14), 22-tetraen-3-one, cyathisterone, byssochlamic acid, dehydromevalonic acid lactone, chevalone B, aszonalenin, dankasterone A (2), helvolic acid, secalonic acid A and fellutanine A, from the culture filtrate extract of the marine sponge-associated fungus Neosartorya fennelliae KUFA 0811. Nine previously reported metabolites, including a chromanol derivative (3), (3ß, 5α, 22E), 3,5-dihydroxyergosta-7,22-dien-6-one (4), byssochlamic acid, hopan-3ß,22-diol, chevalone C, sartorypyrone B, helvolic acid, lumichrome and the alkaloid harmane were isolated from the culture of the marine-sponge associated fungus Neosartorya tsunodae KUFC 9213. Paecilin E (1), dankasterone A (2), a chromanol derivative (3), (3ß, 5α, 22E)-3,5-dihydroxyergosta-7,22-dien-6-one (4), hopan-3ß,22-diol (5), lumichrome (6), and harmane (7) were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. While paecilin E (1) was active against S. aureus ATCC 29213 and E. faecalis ATCC 29212, dankastetrone A (2) was only effective against E. faecalis ATCC 29212 and the multidrug-resistant VRE E. faecalis A5/102. Both compounds neither inhibit biofilm mass production in any of the strains at the concentrations tested nor exhibit synergistic association with antibiotics.

In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents.

Quaternary ammonium compounds (QAC) are widely used, cheap, and chemically stable disinfectants and topical antiseptics with wide-spectrum antimicrobial activities. Within this group of compounds, we recently showed that there are significant differences between the pharmacodynamics of n-alkyl quaternary ammonium surfactants (QAS) with a short (C12) alkyl chain when in vitro toxicities toward bacterial and mammalian epithelial cells are compared. These differences result in an attractive therapeutic window that justifies studying short-chain QAS as prophylactics for sexually transmitted infections (STI) and perinatal vertically transmitted urogenital infections (UGI). We have evaluated the antimicrobial activities of short-chain (C12) n-alkyl QAS against several STI and UGI pathogens as well as against commensal Lactobacillus species. Inhibition of infection of HeLa cells by Neisseria gonorrhoeae and Chlamydia trachomatis was studied at concentrations that were not toxic to the HeLa cells. We show that the pathogenic bacteria are much more susceptible to QAS toxic effects than the commensal vaginal flora and that QAS significantly attenuate the infectivity of N. gonorrhoeae and C. trachomatis without affecting the viability of epithelial cells of the vaginal mucosa. N-Dodecylpyridinium bromide (C12PB) was found to be the most effective QAS. Our results strongly suggest that short-chain (C12) n-alkyl pyridinium bromides and structurally similar compounds are promising microbicide candidates for topical application in the prophylaxis of STI and perinatal vertical transmission of UGI.

Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis.

OBJECTIVES: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. METHODS: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. RESULTS: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. CONCLUSIONS: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

The role of CYBA (p22phox) and catalase genetic polymorphisms and their possible epistatic interaction in cervical cancer.

Human papillomavirus (HPV) infection is necessary but not a sufficient cause for the development of invasive cervical cancer (ICC). Epithelial tissues, target for HPV, are exposed to reactive oxygen species (ROS) associated with tumor initiation and progression. The NADPH oxidase (NOX) and catalase (CAT) are involved in hydrogen peroxide (H2O2) production and inactivation, respectively. P22phox is the NOX subunit encoded by the CYBA gene that has a functional polymorphism (C-242T). This protein is involved in the regulation of electron transfer to oxygen. CAT is a hemic enzyme that plays a role in regulating H2O2 concentration, with a functional polymorphism (C-262T) in its gene. We evaluated CYBA C-242T and CAT C262T genetic polymorphisms and their interaction in 132 women with ICC. We found that CYBA C-242T and CAT C262T genotype frequencies were significantly different between ICC and controls (χ (2) test, p = 0.017 and p = 0.009, respectively). Women with the C/T CYBA-242 genotype had a lower risk for ICC development (odds ratio (OR) = 0.515, 95% confidence interval (CI) 0.291-0.914, p = 0.023) whereas T/T CAT-262 genotype carriers present an increased risk for ICC (OR = 3.034, 95% CI 1.462-6.298, p = 0.003). Women with C/C genotype for CYBA and T/T genotype for CAT had an increased risk to develop ICC comparing with the interaction of the other possible genotypes of both genes (OR = 3.952, 95% CI 1.075-14.521, p = 0.032). The CYBA C-242T and CAT C-262T genetic polymorphisms and their epistatic interactions can be associated with ICC through mechanisms related with the role of ROS in cell proliferation and apoptosis.

Association of myeloperoxidase polymorphism (G463A) with cervix cancer.

Cervical cancer is the fourth most common cancer affecting women worldwide, according to the latest IARC release with 528 000 new cases every year. Infection by high-risk human papillomavirus (HPV) is necessary but not sufficient for progression to cancer. Epithelial tissues, the target of HPV infection, are heavily exposed to reactive oxygen species (ROS). Hypochlorous acid (HOCl) is a very potent ROS, and it is produced by myeloperoxidase (MPO). MPO, a lysosomal enzyme expressed in polymorphonuclear neutrophils (PMN), has the potential to kill HPV transformed cells, as a component of an intercellular induced-apoptosis pathway. This enzyme catalyzes the production of HOCl in the presence of hydrogen peroxide (H2O2). The H2O2 produced by the Doderlein's bacillus will interact with MPO, contributing to the intercellular induced-apoptosis pathway. We studied a functional polymorphism in the promoter region of MPO (G463A) and how it may affect the risk of developing cervix cancer. A sample of 100 patients with invasive cervical cancer and 122 control women were genotyped for MPO polymorphism by PCR-RFLP method. The statistical method used was χ(2). We found that women with the GG genotype had lower risk for cervical cancer than the women who displayed the heterozygous genotype GA (OR = 0.546, 95 % CI = 0.315-0.939, p = 0.028, OR = 2.210, 95 % CI = 1.257-3.886, p = 0.008, respectively). The genotype that leads to a higher concentration of ROS (GG) presents itself as a protection factor in comparison to the homozygous genotype (AA). This can be explained by the interaction of HOCl and superoxide of transformed cells that will generate apoptosis-inducing hydroxyl radicals.

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Analysis of Genes Involved in Oxidative Stress and Iron Metabolism in Heart Failure: A Step Forward in Risk Stratification.

INTRODUCTION: Heart failure (HF) is a clinical syndrome characterized by cardinal symptoms that may be accompanied by signs. It results from structural and/or functional abnormalities of the heart leading to elevated intracardiac pressures and/or inadequate cardiac output at rest and/or during exercise. The prevalence of iron deficiency and anemia justifies the current guidelines recommendation of screening. Genes HP, ACE, MTHFR, HFE, and CYBA are involved in oxidative mechanisms, iron metabolism, and hematologic homeostasis. This study investigates the contribution of variants Hp1/2 (HP), I/D (ACE), C677T (MTHFR), C282Y and H63D (HFE), and C242T (CYBA) to the development of HF, either independently or in epistasis. METHODS: We used a database of 389 individuals, 143 HF patients, and 246 healthy controls. Genotypes were characterized through PAGE electrophoresis, PCR, PCR-RFLP, and multiplex-ARMS. Data analysis was performed with the SPSS® 26.0 software (IBM Corp., Armonk, NY). RESULTS: We observed a significant association between the MTHFR gene and HF predisposition. The presence of allele T and genotype CT constituted risk, while genotype CC granted protection. Epistatic interactions revealed risk between genotype II of the ACE gene and genotypes CC (C282Y) or HH (H63D) of the HFE gene. Risk was also observed for interactions between genotype CC (CYBA)and genotypes 2-2 (HP), CT (MTHFR), or HH (HFE-H63D). CONCLUSION: We concluded that genes HP, ACE, MTHFR, HFE, and CYBA contribute to the susceptibility for HF, individually or in epistasis. This study contributes to the clarification of the role that genes involved in oxidative mechanisms and iron metabolism play in the physiopathology of HF. It is, therefore, a step forward in risk stratification and personalized medicine.

Leiomyoma and the importance of genetic variation on genes related to the vasculature system - CßS, MTHFR, NOS3, CYBA, and ACE1.

OBJECTIVE: The link between the systemic vasculature system and tumor biology is here investigated by studying the contribution of CßS (844ins68), MTHFR (677C > T), NOS3 (4a/4b), CYBA (C242T), and ACE1 (I/D) genes to leiomyoma onset, uterus and leiomyoma volumes. METHODS: DNA samples from 130 women with leiomyomas and 527 from healthy women were genotyped by PCR or PCR-RFLP. Qui-square (χ2) or Fisher's exact test were used to test associations. All the mentioned tests were performed in IBM® SPSS® Statistics Version 28. Statistical significance was defined as a p-value < 0.05. RESULTS: Results revealed that CßS (in the codominant and allelic models, p = 0.044 and, p = 0.015, OR = 1.791 [1.114-2.879], respectively), MTHFR (in the codominant, allelic and dominant models, p = 0.009, p = 0.002, OR = 0.585 [0.416-0.824] and p = 0.003, OR = 0.527 [0.346-0.802], respectively) and ACE1 (dominant model, p = 0.045, OR = 0.639 [0.411-0.992]) genes are associated with leiomyoma onset. NOS3 4a4a genotype is associated with a lower uterus volume (p = 0.004). This study also uncovers intriguing epistatic interactions among some genes that further accentuate their roles in disease modulation. Indeed, the epistatic interactions between the CC genotype (MTHFR) and (+/+) (CßS; p = 0.003), 4b4b (NOS3; p = 0.006, OR = 2.050 [1.223-3.439]) or DD (ACE1; p < 0.001, OR = 2.362 [1.438-3.880]) were shown to be associated with the disease, while 4a presence (NOS3) in epistasis with I presence (ACE1), increased the effect protection having just the I allele presence (p = 0.029, OR = 0.446 [0.214-0.930]). CONCLUSIONS: We conclude that variation in genes related to the systemic vascular system can play a role in the onset and development of leiomyoma.

Mitochondrial dysfunction is the focus of quaternary ammonium surfactant toxicity to mammalian epithelial cells.

Surfactants have long been known to have microbicidal action and have been extensively used as antiseptics and disinfectants for a variety of general hygiene and clinical purposes. Among surfactants, quaternary ammonium compounds (QAC) are known to be the most useful antiseptics and disinfectants. However, our previous toxicological studies showed that QAC are also the most toxic surfactants for mammalian cells. An understanding of the mechanisms that underlie QAC toxicity is a crucial first step in their rational use and in the design and development of more effective and safer molecules. We show that QAC-induced toxicity is mediated primarily through mitochondrial dysfunction in mammalian columnar epithelial cell cultures in vitro. Toxic effects begin at sublethal concentrations and are characterized by mitochondrial fragmentation accompanied by decreased cellular energy charge. At very low concentrations, several QAC act on mitochondrial bioenergetics through a common mechanism of action, primarily by inhibiting mitochondrial respiration initiated at complex I and, to a lesser extent, by slowing down coupled ADP phosphorylation. The result is a reduction of cellular energy charge which, when reduced below 50% of its original value, induces apoptosis. The lethal effects are shown to be primarily a result of this process. At higher doses (closer to the critical micellar concentration), QAC induce the complete breakdown of cellular energy charge and necrotic cell death.

Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase G pathway to the proliferation of neural stem cells stimulated by nitric oxide.

Nitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

Different levels of hsp70 and hsc70 mRNA expression in Iberian fish exposed to distinct river conditions.

Comprehension of the mechanisms by which ectotherms, such as fish, respond to thermal stress is paramount for understanding the threats that environmental changes may pose to wild populations. Heat shock proteins are molecular chaperones with an important role in several stress conditions such as high temperatures. In the Iberian Peninsula, particularly in Portugal, freshwater fish of the genus Squalius are subject to daily and seasonal temperature variations. To examine the extent to which different thermal regimes influence the expression patterns of hsp70 and hsc70 transcripts we exposed two species of Squalius (S. torgalensis and S. carolitertii) to different temperatures (20, 25, 30 and 35 °C). At 35 °C, there was a significant increase in the expression of hsp70 and hsc70 in the southern species, S. torgalensis, while the northern species, S. carolitertii, showed no increase in the expression of these genes; however, some individuals of the latter species died when exposed to 35 °C. These results suggest that S. torgalensis may cope better with harsher temperatures that are characteristic of this species natural environment; S. carolitertii, on the other hand, may be unable to deal with the extreme temperatures faced by the southern species.