Discovery and structural optimization of a new series of N-acyl-2-aminobenzothiazole as inhibitors of Zika virus.

Zika virus infection is associated to severe diseases such as congenital microcephaly and Zika fever causing serious harm to humans and special concern to health systems in low-income countries. Currently, there are no approved drugs against the virus, and the development of anti-Zika virus drugs is thus urgent. The present investigation describes the discovery and hit expansion of a N-acyl-2-aminobenzothiazole series of compounds against Zika virus replication. A structure-activity relationship study was obtained with the synthesis and evaluation of anti-Zika virus activity and cytotoxicity on Vero cells of nineteen derivatives. The three optimized compounds were 2.2-fold more potent than the initial hit and 20.9, 7.7 and 6.4-fold more selective. Subsequent phenotypic and biochemical assays were performed to evidence whether non-structural proteins, such as the complex NS2B-NS3pro, are related to the mechanism of action of the most active compounds.

Molecular network-guided chemical profile and mass spectrometry, volatile compounds, and antimicrobial activity of Scaptotrigona depilis propolis.

RATIONALE: Propolis has a great diversity in its composition due to numerous factors; therefore, each study is an important contribution to the knoFwledge of its composition and biological action. The objective of this study was to determine the chemical profile and biological activity of propolis produced by Scaptotrigona depilis. METHODS: Extracts with 70% ethanol (EPE70) and with cereal alcohol (CAPE) were elaborated, and then characterized using UHPLC-ESI(+)-MS/MS. Volatile compounds were extracted and then characterized using gas chromatography mass spectrometry (GC-MS). In addition, antimicrobial activities were verified against resistant strains. RESULTS: The volatile compounds of propolis predominantly consist of sesquiterpenes. Using the exploratory metabolomic approach, compounds of different classes were putatively identified in the ethanolic extracts, of which the most representative were terpenes, and some of the sesquiterpenes identified among the volatiles were also detected. The extracts were shown to be active against Escherichia coli and Staphylococcus aureus bacteria with a minimum inhibitory concentration (MIC) of 0.5 and 1.0 mg mL-1 , respectively. CONCLUSIONS: The molecular network approach proved to be determining the chemical profile of S. depilis propolis rapidly and accurately, and led to the identification of lipophilic compounds. The identification of compounds using GC-MS and UHPLC-ESI(+)-MS/MS is complementary and useful for the characterization of propolis.

Ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry and molecular networking analysis to investigate the chemodiversity of bioactive extracts of Annona jahnii Saff. fungi from the Brazilian Amazon.

RATIONALE: Annona species are of interest for the isolation of bioactive molecules; however, studies of Annona jahnii Saff. are limited. The exploration of bioactive metabolites of endophytes isolated from this species is unprecedented and allows the preservation of the host plant, in addition to enabling the discovery of compounds with promising biological activities. METHODS: Ethyl acetate extracts from the cultured media of five fungi were obtained. The antioxidant capacity of the extracts was measured using the 1,1-diphenyl-2-picrylhydrazyl free radical method. Antimicrobial activity was determined using the microdilution method in broth in 96-well plates. The exploration of the metabolic profile of the extracts and dereplication of the compounds were performed using ultra-high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (UHPLC/ESI-MS/MS) combined with analysis using molecular networking (MN). RESULTS: A total of 1818 MS features were detected in the five selected extracts, of which 39 compounds were putatively identified. The secondary metabolites with the highest abundance were alkaloids, naphthopyrons, and cytochalasins. Other secondary metabolites include fumonisins, coumarin, and a meroterpenoid. Most of these compounds are related to specific biological properties such as antioxidant, anti-inflammatory, antimicrobial, antiviral, and antitumor activities. Extracts F398 and F403 showed inhibitory activity of the four pathogens tested. Extracts F475 and F506 did not inhibit the growth of Staphylococcus aureus, and F407 did not inhibit the growth of Escherichia coli in addition to having potent antioxidant activity, with IC50 values of 10 µg/mL or less. CONCLUSIONS: The use of UHPLC/ESI-MS/MS data combined with MN proved useful in the dereplication of bioactive molecules of complex extracts that are still unexplored. These initial investigations should significantly assist in further research and increase the efficiency and speed in the discovery of new sources of secondary metabolites and new natural products.

Effects of feeding a live yeast on rumen fermentation and fiber degradability of tropical and subtropical forages.

BACKGROUND: The effect of live yeast Saccharomyces cerevisiae strain CNCM I-1077 (SC) on the ruminal degradability of different forages commonly found in dairy diets in South America was evaluated. We also assessed if SC supplementation interacts with forage group to affect ruminal fiber degradability. Four non-lactating rumen-cannulated Holstein cows were randomly assigned to two treatment sequences: Control-SC-Control or SC-Control-SC, in a switchback design, with three 30-day periods. Cows in the SC treatment were supplied with 1 × 1010 colony-forming units of yeast daily via rumen cannula. In situ degradability of dry matter (DM) and neutral detergent fiber (aNDF) was measured in 15 forages collected in South America. Forages were assigned to one of three groups: corn silages; tropical grasses (sugarcane silages and tropical grass silages); and temperate grasses and alfalfa (oat silages, ryegrass silages, alfalfa silage, and alfalfa hay). RESULTS: Cows supplemented with SC had higher (P = 0.05) counts of yeasts and lower (P = 0.03) concentration of lactate in rumen fluid. There was no interaction between forage group and yeast supplementation (P > 0.10) on in situ degradability. The SC increased DM (by 4.6%) and aNDF degradation (by 10.3%) at 24 h of incubation (P < 0.05). Metabolomics revealed that a chemical entity (C17 H29 N6 O3 , m/z 365.2284 [M + H]+ ) from the family of lipids and related molecules was suppressed in the rumen fluid of cows supplemented with SC. CONCLUSION: The SC supplementation improved DM and aNDF degradability regardless of the forage group. © 2021 Society of Chemical Industry.

Litsea cubeba essential oil: chemical profile, antioxidant activity, cytotoxicity, effect against Fusarium verticillioides and fumonisins production.

The purpose of this study was to determine the chemical profile of Litsea cubeba essential oil, carry out an in vitro evaluation of its antioxidant potential and its cytotoxicity, as well as its antifungal and antimicotoxigenic activities against Fusarium verticillioides. Most of the compounds observed in the EO were neral (32.75%) and geranial (37.67%). The radical scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 104.4 and 56.4 mmol Trolox mg-1, respectively, indicating good antioxidant activity. The EO studied by us revealed cytotoxic effect against HT-29 and HeLa cancer cells. The Minimum Inhibitory and Minimum Fungicidal Concentrations against F. verticillioides were both 125 µg mL-1. Morphological investigation, performed by fluorescence microscopy and scanning electron microscopy, showed that hyphae and microconidia structures underwent changes after treatment with the EO. Analyses performed with the EO strongly reduced the mycelial development of F. verticillioides and the synthesis of fumonisins B1 and B2 in dose-dependence effect compared (P < 0.01) with the fungal control (105 conidia mL-1) and positive control (fludioxonil + metalaxyl-M). Thus, the results obtained in vitro suggest that L. cubeba EO has excellent antioxidant, fungicidal, and antimycotoxigenic effects.

Use of alginate edible coating and basil (Ocimum spp) extracts on beef characteristics during storage.

The effect of alginate edible coating and the addition of two levels of basil (Ocimum spp) on polyphenols, DPPH and ABTS activities, meat characteristics (lipid oxidation, pH, weight loss, texture, and color) during storage and on consumer acceptability were evaluated. Four treatments were developed: CON (control-without coating), AEC (with alginate edible coating), B01 (with alginate edible coating + 1% of basil extract) and B02 (with alginate edible coating + 2% of basil extract). The addition of basil increased the polyphenols and antioxidant activity of meat and reduced the lipid oxidation. The pH values were not altered by the addition of basil and ageing time. The meat with coating was darker, more red and yellow. The inclusion of basil extract in the alginate-based edible coating improved meat acceptability. Thus, edible coatings with natural sources of antioxidants improve meat stability during storage and can be used in the food industry.

Metabolomics of soybean green stem and foliar retention (GSFR) disease using mass spectrometry and molecular networking.

RATIONALE: The nematode Aphelenchoides besseyi is the causal agent of green stem and foliar retention, a soybean disease recently described in Brazil. This condition can reduce soybean yield by up to 100%. However, little is known about chemical interactions between the plant and pathogen. Therefore, this work aimed to investigate metabolites from healthy soybean roots and from soybean roots that were inoculated with A. besseyi. METHODS: A. besseyi were multiplied in vitro with Fusarium sp. colonies in Petri dishes for 25 days, and were axenically inoculated into hydroponics healthy soybean plants. The metabolites were extracted from the roots of healthy and A. besseyi-infected plants 16 days post-inoculation. These extracts were analyzed using an untargeted metabolomic method with an ultra-high-performance liquid chromatography/electrospray ionization /tandem mass spectrometry (UHPLC/ESI-MS/MS) and molecular networking approach. RESULTS: Roots from infected plants showed morphological alterations such as shrinkage, darkening, and arching. Similarly, they also showed an increased presence of flavonoids, compared with healthy roots. Compounds such as neobavaisoflavone, glycitin, genistin, and genistein were putatively identified and had greater intensity in inoculated roots. These compounds are linked to the defensive mechanisms in plants against nematodes. Moreover, coumaric acid, also exclusively putatively identified in inoculated roots, shows activity related to inhibition of root growth. CONCLUSIONS: Liquid chromatography, mass spectrometry, and molecular networking approaches proved to be a powerful tool for the metabolomic study of GSFR. This study showed metabolomics differences of protective substances in the roots, evidencing a quick response of the plant to the attack of A. besseyi.

Understanding mechanisms of oocyte development by follicular fluid lipidomics.

PURPOSE: The present study aimed to provide a non-invasive approach to studying mechanisms responsible for oocyte development. METHODS: To this end, follicular fluid (FF) from 62 patients undergoing in vitro fertilization (IVF) cycles was split into two groups depending on the pregnancy outcome: pregnant (n = 28) and non-pregnant (n = 34) groups. Data were acquired by the MALDI-TOF mass spectrometry. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to the data set. A ROC curve, to predict success rate, was constructed, and the lipids were attributed. RESULTS: Six ions were differentially represented in FF of pregnant and non-pregnant patients, with an area under the curve of 0.962. Phosphatidic acid, phosphatidylglycerol, and triacylglycerol were hyper-represented in the pregnant group, while glucosylceramide was hyper-represented in the non-pregnant group. Enriched functions related to these lipids are steroidogenesis, cellular response, signal transduction, cell cycle, and activation of protein kinase C for the pregnant group and apoptosis inhibition for the non-pregnant group. CONCLUSION: Human FF fingerprinting can both improve the understanding concerning mechanisms responsible for oocyte development and its effect on embryo implantation potential and assist in the management of IVF cycles.

Ovarian environment aging: follicular fluid lipidomic and related metabolic pathways.

PURPOSE: The decline in female fecundity with age may be caused by decreased oocyte quality, a factor that may be associated with the altered composition of follicular fluid (FF). METHODS: In an effort to better understand follicular aging and the role of lipids in a given biological system, we present a prospective study that compares lipid profiles of FF from women older than 35 years (aging group, n = 12) to women equal or younger than 35 years old (control group, n = 17). FF lipids were extracted, and mass spectra were generated using a Waters Synapt G1 Q-TOF in MS mode. MS data was evaluated for both multi- and univariate statistics. The lipids identified as potential biomarkers of follicle aging were attributed by the online databases Lipid Maps, followed by pathway network analysis using Cytoscape software. RESULTS: The in vitro fertilization (IVF) parameters showed significant differences in aging, number of follicles, total number of oocytes and oocytes in MII, and number of injected oocytes. Additionally, FF from the aging group revealed 11 lipids with higher abundance, while FF from the control group included 4 lipids with higher abundance. CONCLUSIONS: We suspect that aging may influence lipid metabolism in a downstream cascade leading, ultimately, to decreased oocyte quality. The discovery of target lipids may assist oocyte selection for IVF in the future. Furthermore, systems biology approach based on post-genomic medicine may help unravel a number of altered mechanisms not previously understood.

Fortification of the whey protein isolate antioxidant and antidiabetic activity with fraction rich in phenolic compounds obtained from Stevia rebaudiana (Bert.). Bertoni leaves.

A stevia fraction (ASF) free of steviol glycosides was extracted from Stevia rebaudiana leaves (Stevia UEM-13). ASF essentially constitutes phenolic compounds (52.42%), which were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) as caffeic acid, quercetin-3-o-glycoside, cyanidin-3-glucoside, kaempferol, quercetin, apigenin, rozmarinic acid, chlorogenic acid and dicaffeoylquinic acid. ASF was used as a multi-functional source of phenolic compounds to fortify the whey protein isolate (WPI) obtained by membrane separation. WPI fortified with 0.2% ASF showed an 80% increase in its antioxidant activity and more pronounced antidiabetic effects than the unfortified WPI, mainly in the glycemic control of diabetic animals induced by streptozotocin. The in vitro and in vivo antioxidant effects of ASF may enhance the effects of WPI. Indeed, this pioneering study revealed that ASF can be used to enrich the antioxidant and antidiabetic properties of WPI.

Metabolomic kinetics investigation of Camellia sinensis kombucha using mass spectrometry and bioinformatics approaches.

Kombucha is created through the fermentation of Camellia sinensis tea leaves, along with sucrose, utilizing a symbiotic consortium of bacteria and yeast cultures. Nonetheless, there exists a dearth of comprehensive information regarding the spectrum of metabolites that constitute this beverage. To explore this intricate system, metabolomics was used to investigate fermentation kinetics of Kombucha. For that, an experimental framework was devised to assess the impact of varying sucrose concentrations and fermentation temperatures over a ten-day period of kombucha fermentation. Following fermentation, samples were analyzed using an LC-QTOF-MS system and a distinctive metabolomic profile was observed. Principal component analysis was used to discriminate between metabolite profiles. Moreover, the identified compounds were subjected to classification using the GNPS platform. The findings underscore notable differences in compound class concentrations attributable to distinct fermentation conditions. Furthermore, distinct metabolic pathways were identified, specially some related to the biotransformation of flavonoids. This comprehensive investigation offers valuable insights into the pivotal role of SCOBY in driving metabolite production and underscores the potential bioactivity harbored within Kombucha.

Synthesis, design, and optimization of a potent and selective series of pyridylpiperazines as promising antimalarial agents.

An optimization of the pyridylpiperazine series against Plasmodium falciparum has been performed, exploring a structure-activity relationship carried out on the toluyl fragment of hit 1, a compound with low micromolar activity against Plasmodium falciparum discovered by high-throughput screening. After confirming the crucial role played by this aryl fragment in the antiplasmodial activity, the replacement of the ortho-methyl substituent of 1 by halogenated ones led to an improvement for four analogs, either in terms of potency, expected pharmacokinetics profile, or both. Further introduction of endocyclic nitrogens in this fragment identified two more optimized compounds, 20 and 23, which are expected to be much more metabolically stable than 1. Additional assessment of the cytotoxicity, Ligand Lipophilic Efficiency, potency against the chloroquine-resistant Dd2 strain and in silico ADMET predictions revealed a satisfactory profile for most compounds, ultimately identifying the four optimized compounds 7, 9, 20 and 23 as promising compounds for further lead optimization of this series against Plasmodium falciparum.

Evaluation and discovery of novel benzothiazole derivatives as promising hits against Leishmania infantum.

An early exploration of the benzothiazole class against two kinetoplastid parasites, Leishmania infantum and Trypanosoma cruzi, has been performed after the identification of a benzothiazole derivative as a suitable antileishmanial initial hit. The first series of derivatives focused on the acyl fragment of its class, evaluating diverse linear and cyclic, alkyl and aromatic substituents, and identified two other potent compounds, the phenyl and cyclohexyl derivatives. Subsequently, new compounds were designed to assess the impact of the presence of diverse substituents on the benzothiazole ring or the replacement of the endocyclic sulfur by other heteroatoms. All compounds showed relatively low cytotoxicity, resulting in decent selectivity indexes for the most active compounds. Ultimately, the in vitro ADME properties of these compounds were assessed, revealing a satisfying water solubility, gastrointestinal permeability, despite their low metabolic stability and high lipophilicity. Consequently, compounds 5 and 6 were identified as promising hits for further hit-to-lead exploration within this benzothiazole class against L. infantum, thus providing promising starting points for the development of antileishmanial candidates.

Proteomic analysis of follicular fluid from women with and without endometriosis: new therapeutic targets and biomarkers.

Endometriosis is a gynecological disease that affects women of reproductive age. The protein profiles of women with endometriosis who were able or unable to achieve pregnancy and women without endometriosis who did achieve pregnancy were compared in this study. The follicular fluid was collected from 21 patients undergoing in vitro-fertilization treatment, according to the following groups: nine women in the control group (Group C), four women with endometriosis who achieved pregnancy (Group E.P), and eight women with endometriosis who did not achieve pregnancy (Group E.NP). Follicular fluid proteins were separated using 2D-electrophoresis, and their spots were compared, excised, and submitted to LC-ESI-MS/MS for proteins identification. The analysis showed 29 differentially expressed spots among the groups, and from these, 21 proteins were identified. Analysis showed some functional enrichment in the E.P group, including response to oxidative stress and apoptosis, while the E.NP group showed functions related to response to reactive oxygen species and positive regulation of apoptosis. These data suggest that endometriosis leads to differential protein expression in the follicular fluid, which can influences the outcome of pregnancy. These proteins may be potential targets for better diagnostics and new therapeutic intervention in affected women, as well as assisting in comprehending the physiopathologic mechanisms underlying endometriosis.

Ion mobility spectrometry focusing on speciation analysis of metals/metalloids bound to carbonic anhydrase.

In the present work, traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) was applied to speciation analysis of metalloproteins. The influence of pH on complexation conditions between some metals and bovine carbonic anhydrase was evaluated from pH 6 to 9, as well as the time involved in their complexation (0-24 h). Employing TWIMS-MS, two conformational states of bovine carbonic anhydrase were observed with charge states of +12 and +11; these configurations being evaluated in terms of the folded state of the apo form and this protein (at charge state +11) being linked to barium, lead, copper, and zinc in their divalent forms. Metalloprotein speciation analysis was carried out for copper (Cu(+) and Cu(2+)), lead (Pb(2+) and Pb(4+)), and selenium (Se(4+) and Se(6+)) species complexed with bovine carbonic anhydrase. Mobilities of all complexed species were compared, also considering the apo form of this protein.

Proteomic Investigation over the Antimicrobial Photodynamic Therapy Mediated by Rose Bengal Against Staphylococcus aureus.

In order, understanding the antimicrobial action of photodynamic therapy and how this technique can contribute to its application in the control of pathogens. The objective of the study was to employ a proteomic approach to investigate the protein profile of Staphylococcus aureus after antimicrobial photodynamic therapy mediated by rose bengal (RB-aPDT). S. aureus was treated with RB (10 nmoL L-1 ) and illuminated with green LED (0.17 J cm-2 ) for cell viability evaluation. Afterward, proteomic analysis was employed for protein identification and bioinformatic tools to classify the differentially expressed proteins. The reduction in S. aureus after photoinactivation was ~2.5 log CFU mL-1 . A total of 12 proteins (four up-regulated and eight down-regulated) correspond exclusively to alteration by RB-aPDT. Functionally, these proteins are distributed in protein binding, structural constituent of ribosome, proton transmembrane transporter activity and ATPase activity. The effects of photodamage include alterations of levels of several proteins resulting in an activated stress response, altered membrane potential and effects on energy metabolism. These 12 proteins required the presence of both light and RB suggesting a unique response to photodynamic effects. The information about this technique contributes valuable insights into bacterial mechanisms and the mode of action of photodynamic therapy.

Hit-to-lead optimization of a pyrazinylpiperazine series against Leishmania infantum and Leishmania braziliensis.

An early hit-to-lead optimization of a novel pyrazinylpiperazine series against L. infantum and L. braziliensis has been performed after an extensive SAR focusing on the benzoyl fragment of hit (4). Deletion of the meta-Cl of (4) led to the obtention of the para-hydroxyl derivative (12), on which the design of most monosubstituted derivatives of the SAR was based. Further optimization of the series, involving disubstituted benzoyl fragments and the hydroxyl substituent of (12), allowed the obtention of a total of 15 compounds with increased antileishmanial potency (IC50 < 10 µM), nine of which displayed activity in the low micromolar range (IC50 < 5 µM). This optimization ultimately identified the ortho, meta-dihydroxyl derivative (46) as an early lead for this series (IC50 (L. infantum) = 2.8 µM, IC50 (L. braziliensis) = 0.2 µM). Additional assessment of some selected compounds against other trypanosomatid parasites revealed that this series is selective towards Leishmania parasites, and in silico ADMET predictions revealed satisfactory profiles for these compounds, allowing further lead optimization of the pyrazinylpiperazine class against Leishmania.

Synthesis of 1,2,3-Triazole-Containing Methoxylated Cinnamides and Their Antileishmanial Activity against the Leishmania braziliensis Species.

Leishmaniasis is a group of infectious diseases caused by protozoan parasites that belong to the genus Leishmania. Currently, there is no human vaccine, and the available treatments are associated with toxicity, high cost, and the emergence of resistant strains. These factors highlight the need to identify new antileishmanial candidates. In this study, we synthesized twenty-four methoxylated cinnamides containing 1,2,3-triazole fragments and evaluated their antileishmanial activity against the Leishmania braziliensis species, which is the main etiological agent responsible for American Tegumentary Leishmaniasis (ATL). The cinnamides were synthetically prepared using nucleophilic acyl substitution and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The compounds were characterized using infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. We performed preliminary studies to evaluate the biological activity of these compounds against L. braziliensis promastigotes and axenic amastigotes. Compound 28, N-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazole-4-yl) methyl)-3,4-dimethoxy cinnamide, demonstrated relevant antileishmanial activity with low toxicity in murine cells. The selectivity index values for this compound were superior compared with data obtained using amphotericin B. Furthermore, this cinnamide derivative reduced the infection percentage and number of recovered amastigotes in L. braziliensis-infected macrophages. It also induced an increase in reactive oxygen species production, depolarization of the mitochondrial potential, and disruption of the parasite membrane. Taken together, these findings suggest that this synthetic compound holds potential as an antileishmanial candidate and should be considered for future studies in the treatment of ATL.

Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele.

STUDY QUESTION: What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? SUMMARY ANSWER: In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. WHAT IS KNOWN ALREADY: Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. STUDY DESIGN, SIZE AND DURATION: A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006-2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. LIMITATIONS AND REASONS FOR CAUTION: A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. WIDER IMPLICATIONS OF THE FINDINGS: Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the São Paulo Federal University.

The follicular microenviroment as a predictor of pregnancy: MALDI-TOF MS lipid profile in cumulus cells.

PURPOSE: This research proposed to study the changes in lipid composition in cumulus cells (CCs) from women who achieved pregnancy compared with women who did not, after in vitro fertilization treatment. This approach has the potential to provide novel information on the lipid metabolism of the CCs and as an additional method to predict pregnancy. METHOD: Fifty-four samples from couples with tubal and male factor infertility and where the female partner was age 35 or younger were divided in two groups according to their level of hCG 14 days after embryo transfer as follows: (1) 23 samples in pregnant group and (2) 31 samples in non-pregnant group. Lipid extraction was performed by the Bligh-Dyer protocol, and lipid profiles were obtained by MALDI-TOF MS. Mass spectra data were processed with MassLynx, and statistical analysis was performed using MarkerLynx extended statistic. OPLS-DA model was built. RESULTS: S-plot Analysis revealed three ions as potential markers in the pregnant group, and five ions in the non-pregnant group. These ions were identified in the human metabolome database (HMDB) as phosphatidylcholine in the pregnant group and as phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol species in the non-pregnant group. These lipids might be involved in cell proliferation and differentiation, apoptosis and GAP junction regulation. CONCLUSION: We conclude that MALDI-TOF MS can be used as an informative and fast analytical strategy to obtain and study the lipid profile of cumulus cells and can potentially be used as a supporting tool to predict pregnancy based on the metabolic state of the CCs.