Association between maternal diet, smoking, and the placenta MTHFR 677C/T genotype and global placental DNA methylation.

INTRODUCTION: The placenta provides nutrients to the fetus, and it has protective effects against harmful substances. Unhealthy maternal diets and toxic agents might increase free radical (FR) production. Elevated FR levels are associated with a high risk of oxidative stress, which may cause DNA damage. DNA might be oxidized in the placenta, occasionally affecting its methylation profile due to 8-hidroxy-2'-deoxyguanosine formation. METHODS: This study assessed 130 mothers and their children. The maternal's nutritional patterns were determined using the Food Frequency Questionnaire. Information on smoking and alcohol consumption was collected during the medical examination. Data on placental DNA were obtained to determine the MTHFR 677C/T genotype and the proportion of placental DNA methylation (pDNAm). RESULTS: Consumption of vitamins and folic acid was above 85%. The pDNAm was found to be correlated with gestational age and coffee intake. Mothers with a smoking history had a low pDNAm. Placentas with the TT genotype had a higher but not significant pDNAm. In the placentas with the CC/CT genotype, the pDNAm was positively associated with carbohydrate and biotin intake. However, the TT genotype was negatively associated with folate and vegetable intake. DISCUSSION: The pDNAm was positively associated with coffee intake, but not with macro-, and micronutrient intake. However, it was negatively associated with cigarette smoking. The placentas with the CC/CT genotype had a lower pDNAm than those with the TT genotype. In the placentas with the CC/CT or TT genotype, methylation was positively, and negatively associated with micro- or macronutrients, respectively.

A novel and easy protocol to obtain 6-alkoxy-Δ4,6-diene-3-one derivatives from available sterols.

Herein we report an unprecedented and efficient methodology for accessing 6-alkoxy-Δ4,6-diene-3-one derivatives. Such scaffolds were serendipitously obtained in the course of the study of the reaction of Δ4-3-keto steroids with catalytic amounts of iodine in refluxing methanol. A series of 6-methoxy and 6-ethoxy- Δ4,6-diene-3-ones were prepared from easily-available sterols in a two-step sequence; first, oxidation of sterols furnished the Δ4-3-keto steroids, which were then refluxed with ethanol or methanol with I2 as catalyst to obtain a series of ten derivatives. Furthermore, this protocol was also effective for the introduction of a larger carbon chain at C-6. Druglikeliness properties of synthesized compounds were predicted using the SwissADME tool.

The sodium borate relieves the hypertrophic damage induced during pregnancy, it improves contractibility, reduces oxidative stress and stimulates cell proliferation.

INTRODUCTION: Fetal and postnatal hypertrophy develop in response to such different exposures or illnesses the mother suffers during gestation as anti-infectious and physical agents, obesity, hypertension, diabetes, and even advanced maternal age. This gives rise to high comorbidities in the newborn; therefore, looking for alternatives that contribute to cardiac homeostasis is quite necessary to inhibit the overgrowth of myocytes. Boron-derivative compounds could play a key role in exerting a repairing effect on chronic cardiac damage induced during gestation. METHODOLOGY: The cardiotoxic effect of 6.4, 12 and 100 mg/kg of sodium tetraborate administered by oral delivery route to healthy pregnant mice was assessed. After that, the use of the chemical compound was tested in the treatment of pregnant mice previously subjected to isoproterenol (fetal hypertrophy model) on the fifth day post coitus. Prior to the sacrifice of the pups of mice an electrocardiography (ECG) was done. Morphological and histological changes of heart were assessed in newborn pups. As a damage marker, the concentration of p38 nitrogen-activated protein kinases were evaluated by using Western Blot and the levels of malondialdehyde (MDA) as well as glutathione antioxidants (GSH) and glutathione peroxidase (GPx) were tested by spectrometry. Moreover, the mRNA expression for early response genes (c-jun, c-fos y c-myc), late response (GATA-4, Mef2c, NFAT) and heart damage (ANP and BNP) was measured by qPCR real time. RESULTS: The supply of 6,4 and 12 mg/kg-sodium tetraborate favored ventricular remodeling with histological alterations. By comparison, 100 mg/kg of sodium tetraborate administered during the fetal stage did not alter neither the cardiac morphology of six-week old pups nor the p38/P-p38MAPK ratio remained the same and no oxidative stress was observed. When pregnant females treated with isoproterenol were treated with 100 mg/kg sodium tetraborate during the fetal stage, an improvement in contractility was detected in the pups with an actual reduction in myocardial fibrosis and oxidative stress, but cardiac mass increased. In addition, the expression levels of c-jun, c-myc, GATA-4, MEF2c and ANP mRNA declined in comparison with CTR. However, the hypertrophic damage mechanism was sustained by c-fos, NFAT and BNP expressions. CONCLUSIONS: The set of results achieved suggests that high concentrations of sodium tetraborate have no cardiotoxic effects. Furthermore, sodium tetraborate mitigates hypertrophy induced during pregnancy, thereby improving contractibility, reducing oxidative stress and stimulating cell proliferation. Therefore, sodium tetraborate could be an excellent prophylactic treatment administered by delivery oral route during pregnancy when there is a risk of developing fetal left ventricular hypertrophy (LVH).

Fused Pyrroles in Cholestane and Norcholestane Side Chains: Acaricidal and Plant Growth-Promoting Effects.

Herein, we describe the synthesis and characterization of fused pyrroles in cholestane and norcholestane side chains derived from kryptogenin and diosgenin, respectively. Both conventional and microwave heating techniques were used to synthesize the steroidal pyrroles from primary amines, with the microwave method producing the highest yields. In particular, the norcholestane pyrroles were tested as acaricides against the two-spotted spider mite (Tetranychus urticae Koch) under laboratory conditions and as plant growth promoters on habanero pepper (Capsicum chinense Jacq) under greenhouse conditions.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus.

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Screening of SERT and p11 mRNA Levels in Airline Pilots: A Translational Approach.

Airline pilots are frequently exposed to numerous flights per week, changes in their circadian rhythms, and extended periods away from home. All these stressors make pilots susceptible to developing psychiatric disorders. Recently, emphasis has been placed on the need for molecular tests that help in the diagnosis of depression. The genes SLC6A4 and S100A10 encode serotonin transporter (SERT) and p11 protein, respectively. Their expression has been frequently associated with stress and depression. In this work, we quantified, by quantitative PCR, the expression of SERT and p11 in peripheral mononuclear cells of airline pilots compared to patients with depression and healthy volunteers. Moreover, by mass spectrometry, we quantified the serum serotonin levels in the same three groups. We found that SERT and p11 were overexpressed in the mononuclear cells of airline pilots and depressed patients compared to healthy volunteers. Although serum serotonin was not different between healthy volunteers and airline pilots, a decreasing trend was observed in the latter. As expected, serum serotonin in the patients was significantly lower. Alterations in SERT and p11 in airline pilots could be related to professional stress, a condition that could potentially affect their long-term mental health.

The Heck reaction of allylic alcohols catalysed by an N-heterocyclic carbene-Pd(ii) complex and toxicity of the ligand precursor for the marine benthic copepod Amphiascoides atopus.

The palladium-catalysed reaction of aryl halides and allylic alcohols is an attractive method for obtaining α,ß-unsaturated aldehydes and ketones, which represent key intermediates in organic synthesis. In this context, a 1,2,3-triazol-5-ylidene (aNHC)-based palladium(ii) complex formed in situ has been found to be a selective catalyst for the syntheses of building blocks from the corresponding aryl halides and allylic alcohols, with yields ranging from 50% to 90%. The lack of toxic effects of the ligand precursor (1,2,3-triazolium salt) of the palladium(ii) complex for the harpacticoid copepod Amphiascoides atopus allowed us to contrast the efficiency of the catalytic system with the potential impact of the principal waste chemical in global aquatic ecosystems, which has not been previously addressed.

Synthesis, antiinflammatory activity, and molecular docking studies of bisphosphonic esters as potential MMP-8 and MMP-9 inhibitors.

Bisphosphonic acids (or bisphosphonates) have been successfully used in the clinic treatment of bone diseases for over decades. Additionally, the antiinflammatory activity of these compounds has been gaining attention. In our previous work, we synthesized and in vivo evaluated the bisphosphonic esters 1 and 2, finding a moderate edema inhibition upon oral and topical administration on BALB/c mice. Thus, in this work, the bioisosteric replacement of an amide functional group for an ester afforded the new bisphosphonates 3-6, which had a moderate oral edema inhibition (25 mg/kg dose) and a significant topical antiinflammatory activity (2 mg/ear) on BALB/c mice, with 6 being the most active hit (55.9% edema inhibition), comparable to the positive control (55.5% edema inhibition) on a TPA topical model. Next, to assess the acute toxicity of the synthesized derivatives, test animals were administered with 50-100 mg/kg of 3-6, respectively, by an oral route, and after 14 days, neither lethality nor a significative weight loss were observed. Finally, a structure-activity relationship (SAR) and a molecular docking analysis of 3-6 helped us to explain the trend observed in biological tests. Considering all these aspects, we propose the inhibition of MMP-8 and MMP-9 as a possible action mechanism of the synthesized derivatives.

Biochemical and Behavioral Characterization of IN14, a New Inhibitor of HDACs with Antidepressant-Like Properties.

Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using A.salina toxicity test, in vitro fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

LC-MS/MS proteomic analysis of starved Bacillus subtilis cells overexpressing ribonucleotide reductase (nrdEF): implications in stress-associated mutagenesis.

The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain B. subtilis YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the hisC952 (amber), metB5 (ochre) and leuC425 (missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC-MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆nrdR strain. Overall, our results revealed that amino acid-starved cells of strain B. subtilis ∆nrdR that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments.

Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations.

Invertebrate immune priming is a growing field in immunology. This phenomenon refers to the ability of invertebrates to generate a more vigorous immune response to a second encounter with a specific pathogen and can occur within and across generations. Although the precise mechanism has not been elucidated, it has been suggested that methylation of DNA is a cornerstone for this phenomenon. Here, using a novel method of analytical chemistry (a reversed-phase liquid chromatography procedure) and the beetle Tenebrio molitor as a model system, we did not find evidence to support this hypothesis taking into account the percentage of methylated cytosine entities in DNA (5mdC) within or across generations. However, we found a lower percentage of methylated cytosine entities in RNA (5mC) within but not across generations in immune priming experiments with adults against the bacteria Micrococcus lysodeikticus and larvae against the fungus Metarhizium anisopliae. To our knowledge, this is the first report suggesting a role of differential methylation on RNA during immune priming within generations.

Cr(VI) reduction by gluconolactone and hydrogen peroxide, the reaction products of fungal glucose oxidase: Cooperative interaction with organic acids in the biotransformation of Cr(VI).

The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI).

New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY cotyledon1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-related homeobox4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd(II) and Se(IV).

In the present work, application of the previously established reversed-phase liquid chromatography procedure based on fluorescent labeling of cytosine and methylcytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modulation of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation percentage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2'-deoxycytidine/5-methyl-2'-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mg L(-1) Cd and 1 mg L(-1) Se in the growth medium. For higher stressors' concentration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r = -0.6788, p = 0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context.

KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp.

BACKGROUND: The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors. RESULTS: To gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2. CONCLUSIONS: Agave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants.