Optimization of Thermoultrasound Process of Soursop (Annona muricata) Nectar and Comparison of Its Physicochemical Properties and In Vitro Bioaccessibility of Antioxidants with Pasteurized Sample.

Research background: Soursop nectar contains antioxidants and is preserved by pasteurization. However, this technology impairs its physicochemical properties and bioactive compounds. An alternative is therefore thermoultrasound, which could counteract these effects. The thermosonicated nectar was compared with a pasteurized one and the in vitro bioaccessibility of antioxidants was estimated. Experimental approach: The soursop nectar (25 %) was processed and the response surface methodology was used to determine the optimal conditions for thermoultrasound treatment (TUS). The TUS (75-90 % amplitude, 3.15-15 min) was applied, and 2 % stevia and 6 % agave inulin were added as sweeteners. The microbiological, physicochemical, enzymatic and antioxidant properties were analyzed. The properties of thermosonicated nectar obtained under optimal conditions were compared with pasteurized nectar. In addition to the above determinations, microstructure, total dietary fiber (TDF) and in vitro bioaccessibility of antioxidants were determined. Results and conclusions: The response variables that fit the mathematical model were L*, b*, chroma (C*), total phenolic content (TPC) and antioxidant activity determined by ABTS•+, DPPH˙ and Fe(III) reducing antioxidant power (FRAP). The L* and DPPH˙ were affected by quadratic time and TPC by time (p<0.0001). The optimum TUS condition was 82 % amplitude for 9.15 min and the responses variables were L*, b* and C* (45.48, 3.55 and 3.62, respectively), TPC expressed as gallic acid equivalents (38.40 mg/100 mL), ABTS•+ expressed as Trolox equivalents (TE) (31.28 µmol/100 mL), DPPH˙ expressed as TE (124.22 µmol/100 mL) and FRAP expressed as Fe(II) (3.06 µmol/100 mL). Compared to the pasteurized sample, thermosonicated sample had high values of L* (45.56), h° (-56.49), TPC (26.63 mg/100 mL), ABTS•+ and DPPH˙ (22.03 and 129.22 µmol/100 mL, respectively), FRAP (3.10 µmol/100 mL) and low pectin methylesterase (PME) activity (0.28 U/mL). For in vitro bioaccessibility, thermosonicated nectar showed high absorption of TPC (15.26/100 mL) and high antioxidant activity determined by ABTS (34.92 µmol/100 mL) and FRAP (7.88 µmol/100 mL). Novelty and scientific contribution: The thermoultrasound improves the physicochemical properties and in vitro bioaccessibility of antioxidants in soursop nectar. On the other hand, as an alternative, this beverage offers low-calorie alternative with prebiotic properties that benefits consumer health.

Antiretroviral Therapy-Induced Dysregulation of Gene Expression and Lipid Metabolism in HIV+ Patients: Beneficial Role of Antioxidant Phytochemicals.

Human immunodeficiency virus (HIV) infection has continued to be the subject of study since its discovery nearly 40 years ago. Significant advances in research and intake of antiretroviral therapy (ART) have slowed the progression and appearance of the disease symptoms and the incidence of concomitant diseases, which are the leading cause of death in HIV+ persons. However, the prolongation of ART is closely related to chronic degenerative diseases and pathologies caused by oxidative stress (OS) and alterations in lipid metabolism (increased cholesterol levels), both of which are conditions of ART. Therefore, recent research focuses on using natural therapies to diminish the effects of ART and HIV infection: regulating lipid metabolism and reducing OS status. The present review summarizes current information on OS and cholesterol metabolism in HIV+ persons and how the consumption of certain phytochemicals can modulate these. For this purpose, MEDLINE and SCOPUS databases were consulted to identify publications investigating HIV disease and natural therapies and their associated effects.

Ligand-Based Virtual Screening and Molecular Docking of Benzimidazoles as Potential Inhibitors of Triosephosphate Isomerase Identified New Trypanocidal Agents.

Trypanosoma cruzi (T. cruzi) is a parasite that affects humans and other mammals. T. cruzi depends on glycolysis as a source of adenosine triphosphate (ATP) supply, and triosephosphate isomerase (TIM) plays a key role in this metabolic pathway. This enzyme is an attractive target for the design of new trypanocidal drugs. In this study, a ligand-based virtual screening (LBVS) from the ZINC15 database using benzimidazole as a scaffold was accomplished. Later, a molecular docking on the interface of T. cruzi TIM (TcTIM) was performed and the compounds were grouped by interaction profiles. Subsequently, a selection of compounds was made based on cost and availability for in vitro evaluation against blood trypomastigotes. Finally, the compounds were analyzed by molecular dynamics simulation, and physicochemical and pharmacokinetic properties were determined using SwissADME software. A total of 1604 molecules were obtained as potential TcTIM inhibitors. BP2 and BP5 showed trypanocidal activity with half-maximal lytic concentration (LC50) values of 155.86 and 226.30 µM, respectively. Molecular docking and molecular dynamics simulation analyzes showed a favorable docking score of BP5 compound on TcTIM. Additionally, BP5 showed a low docking score (-5.9 Kcal/mol) on human TIM compared to the control ligand (-7.2 Kcal/mol). Both compounds BP2 and BP5 showed good physicochemical and pharmacokinetic properties as new anti-T. cruzi agents.

Consumption Patterns of Energy Drinks in Adolescents and Their Effects on Behavior and Mental Health: A Systematic Review.

The aim of the current review was to analyze primary studies about energy drink consumption patterns in adolescents and their relationship with mental health. PubMed, PLOS ONE, PsycINFO, and ScienceDirect databases were searched to identify articles related to adverse effects of energy drinks in adolescents and young adults aged 11 to 18 years. Psychological and behavioral measures were based on validated screening tools used in various contexts, and bias was detected in energy drink consumption patterns. In regard to sex, boys consumed more energy drinks than girls, and a strong, positive association was reported between consumption and probability of risky behaviors; tendency for anxiety, depression, and impulsivity; poor academic performance; and sleep disturbances. A progressive increase in consumption was also noted of 25% to 75% within 5 years. Findings suggest that standardized consumption pattern assessment be included in evaluations of mental health to determine potential causal relationships. [Journal of Psychosocial Nursing and Mental Health Services, 60(2), 41-47.].

(-)-Epicatechin protects from amebic liver abscess development in hamster.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x106E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites.

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.

Biological activity of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide against E. histolytica and their analysis as potential thioredoxin reductase inhibitors.

Amoebiasis is caused by the protozoan Entamoeba histolytica that affects millions of people throughout the world. The standard treatment is metronidazole, however, this drug causes several side effects, and is also mutagenic and carcinogenic. Therefore, the search for therapeutic alternatives is necessary. Quinoxaline 1,4-di-N-oxides (QdNOs) derivatives have been shown to exhibit activity against different protozoan. In the present study, the effects of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide (7-carboxylate QdNOs) derivatives on E. histolytica proliferation, morphology, ultrastructure, and oxidative stress were evaluated, also their potential as E. histolytica thioredoxin reductase (EhTrxR) inhibitors was analyzed. In vitro tests showed that 12 compounds from n-propyl and isopropyl series, were more active (IC50 = 0.331 to 3.56 µM) than metronidazole (IC50 = 4.5 µM). The compounds with better biological activity have a bulky, trifluoromethyl and isopropyl group at R1-, R2-, and R3-position, respectively. The main alterations found in trophozoites treated with some of these compounds included changes in chromatin, cell granularity, redistribution of vacuoles with cellular debris, and an increase in reactive oxygen species. Interestingly, docking studies suggested that 7-carboxylate QdNOs derivatives could interact with amino acid residues of the NADPH-binding domain and/or the redox-active site of EhTrxR. Enzymatic assays demonstrated that selected 7-carboxylate QdNOs inhibits EhTrxR disulfide reductase activity, and diaphorase activity shows that these compounds could act as electron acceptor substrates for the enzyme. Taken together, these data indicate that among the mechanisms involved in the antiamoebic effect of the 7-carboxylate QdNOs derivatives studied, is the induction of oxidative stress and the inhibition of EhTrxR activity.

Effect of Ultrasound on In Vitro Bioaccessibility of Phenolic Compounds and Antioxidant Capacity of Blackberry (Rubus fruticosus) Residues cv. Tupy.

Agro-industries residues of fruit are a source of antioxidant compounds with a possible health impact. The aim of this study was to evaluate the total phenolic content (TPC), total monomeric anthocyanins (TMA), individual phenolic compounds (IPC), antioxidant capacity and in vitro intestinal bioaccessibility from blackberry residues (BR), and ultrasonicated blackberry residues (US-BR). The results showed that BR had higher TPC (4,016.43 mg GAE/100 g DM), TMA (364.53 mg Cy-3-Gl/100 g) antioxidant capacity by ABTS (5,422.38 mg AAE/100 g DM) and FRAP (12511.44 µmol Fe(II)/100 g DM) than US-BR. TPC and TMA obtained by US-BR were more bioaccessible (70 and 51%, respectively) compared to BR (37 and 34%, respectively). The use of ultrasound can aid the extraction of total phenolic compounds and improve their bioaccessibility. After acid hydrolysis, a high amount of individual phenolic compounds (IPC) in US-BR (chlorogenic acid, caffeic acid, apigenin, luteolin and kaempferol) was obtained compared with BR. Before in vitro digestion, total individual compounds (TIC) content was lower in the BR (29.49 mg/100 g DM) than US-BR (92.36 mg/100 g DM) and there was 5 and < 1%, respectively. Therefore, the food industry would use residues of blackberry fruits as a source of antioxidant compounds with possible health benefits.

Fenton Discoloration of Ultrasonicated Purple Cactus Pear Juice.

The aim of this study was to evaluate the stability of color, betaxanthin, and betacyanin pigments in the presence of Cu(II)-dependent hydroxyl radicals (HO•) from ultrasonicated purple cactus pear juice at amplitudes of 40%, 60%, and 80%, in comparison to untreated sample. L* parameter of juice treated at 40% and 80% amplitude for 25 and 15 min, respectively (11.3 and 9.3, respectively), were significantly higher compared to the control; b* and hue parameters of juice treated at 80%, 25 min showed values of 1.7 and 0.1, respectively. Color differences (ΔE) were lower (<3) for juices treated at high amplitude (80%) and short times (3-5 min). Juice treated at 40% 15 min, 60% 25 min, 80% 15 and 25 min presented high values of betacyanins (281.7 mg·L-1, 255.9 mg·L-1, 294.4 mg·L-1, and 276.7 mg·L-1, respectively). Betaxanthin values were higher in the juices treated at 40% 5 min and 80% 15 and 25 min (154.2 mg·L-1, 135.2 mg·L-1, and 128.5 mg·L-1, respectively). Purple cactus pear juice exhibited significant chelating activity of copper ions and great stability when exposed to HO•.

Effect of electric field on the characteristics of crude avocado oil and virgin olive.

The objective of this study was to analyze the effect of an electric field treatment (voltage 9 kV cm-1, frequency 720 Hz and time of 5 and 25 min) as method of preservation on two edible oils. Unsaturated fatty acid oxidation in the crude avocado oil and virgin olive oil was analyzed by Fourier transform infrared spectroscopy in the mid infrared region and by quality parameters (acidity, peroxide and iodine). The electric field is a suitable method to preserve the crude oils composition with minimal modifications without the synthetic antioxidant addition.

Effect of Thermoultrasound on the Antioxidant Compounds and Fatty Acid Profile of Blackberry (Rubus fruticosus spp.) Juice.

Blackberry (Rubus fruticosus spp.) fruit has high antioxidant activity due to its significant content of anthocyanins and antioxidant compounds. Among emerging technologies for food preservation, thermoultrasound is a technique that reduces microbial loads and releases compounds with antioxidant properties. The objective of this study was to determine the antioxidant content and fatty acid profile of blackberry juice subjected to thermoultrasound treatment in comparison to pasteurized juice. Blackberry juice and n-hexane extracts from a control (untreated juice), pasteurized, and thermoultrasonicated samples were evaluated for antioxidant activity, fatty acid profile, and antioxidant content. The juice treated with thermoultrasound exhibited significantly (p < 0.05) higher levels of total phenols (1011 mg GAE/L), anthocyanins (118 mg Cy-3-GlE/L); antioxidant activity by ABTS (44 mg VCEAC/L) and DPPH (2665 µmol TE/L) in comparison to the control and pasteurized samples. Oil extract from thermoultrasound juice also had the highest antioxidant activity (177.5 mg VCEAC/L and 1802.6 µmol TE/L). The fatty acid profile of the n-hexane extracts showed the presence of myristic, linolenic, stearic, oleic, and linoleic acids and was not affected by the treatments except for stearic acid, whose amount was particularly higher in the control. Our results demonstrated that thermoultrasound can be an alternative technology to pasteurization that maintains and releases antioxidant compounds and preserves the fatty acids of fruit juice.

Application of Ultrasound in a Closed System: Optimum Condition for Antioxidants Extraction of Blackberry (Rubus fructicosus) Residues.

Blackberry processing generates up to 20% of residues composed mainly of peel, seeds and pulp that are abundant in flavonoids. The objective of this study was to optimize the ultrasound conditions, in a closed system, for antioxidants extraction, using the response surface methodology. Blackberry (Rubus fructicosus) residues were analyzed for total phenolics, total anthocyanins, and antioxidant activity by ABTS and DPPH. The selected independent variables were ultrasound amplitude (X1: 80%-90%) and extraction time (X2: 10-15 min), and results were compared with conventional extraction methods. The optimal conditions for antioxidants extraction were 91% amplitude for 15 min. The results for total phenolic content and anthocyanins and antioxidant activity by ABTS and DPPH were of 1201.23 mg gallic acid equivalent (GAE)/100 g dry weight basis (dw); 379.12 mg/100 g·dw; 6318.98 µmol Trolox equivalent (TE)/100 g·dw and 9617.22 µmol TE/100 g·dw, respectively. Compared to solvent extraction methods (water and ethanol), ultrasound achieved higher extraction of all compounds except for anthocyanins. The results obtained demonstrated that ultrasound is an alternative to improve extraction yield of antioxidants from fruit residues such as blackberry.

Effect of electric field treatment on unsaturated fatty acid in crude avocado oil.

The objective of this study was to evaluate the stability of the fatty acids in avocado oil when the product is subjected to different conditions of electric field treatment (voltage: 5 kV cm(-1); frequency: 720 Hz; treatment time: 5, 10, 15, 20, and 25 min). Fatty acids were analyzed by Fourier transform infrared spectroscopy in the mid-infrared region. Electric field is a suitable method to preserve the oil quality and composition with minimal modifications in unsaturated fatty acids.

S-Dihydrodaidzein and 3-(1,3-benzoxazol-2-yl)-benzamide, Two New Potential ß-estrogen Receptor Ligands with Anti-adipogenic Activity.

BACKGROUND: The elucidation of molecular pathways associated with adipogenesis has evidenced the relevance of estrogen and estrogen receptor beta (ERß). The positive effects of ERß ligands on adipogenesis, energy expenditure, lipolysis, food intake, and weight loss, make ERß an attractive target for obesity control. From ligand-based virtual screening, molecular docking, and molecular dynamic simulations, six new likely ERß ligands (C1 to C6) have been reported with potential for pharmacological obesity treatment. OBJECTIVE: In this study, the effect of molecules C1-C6 on adipogenesis using the murine 3T3-L1 cell line was evaluated. METHODS: Cell viability was assessed by MTT assays. Lipid accumulation and gene expression were investigated by ORO staining and real-time quantitative RT-PCR experiments, respectively. RESULTS: Cell viability was not significantly affected by C1-C6 at concentrations up to 10 µM. Interestingly, treatment with 10 µM of C1 (S-Dihydrodaidzein) and C2 (3-(1,3-benzoxazol-2-yl)- benzamide) for 72 h inhibited adipocyte differentiation; moreover, ORO staining evidenced a reduced intracellular lipid accumulation (40% at day 7). Consistently, mRNA expression of the adipogenic markers, PPARγ and C/EBPα, was reduced by 50% and 82%, respectively, in the case of C1, and by 83% and 59%, in the case of C2. CONCLUSION: Altogether, these results show the two new potential ß-estrogen receptor ligands, C1 and C2, to exhibit anti-adipogenic activity. They could further be used as lead structures for the development of more efficient drugs for obesity control.

Primary Peritoneal Serous Cancer: A Case Report of a Tumor in the Rectovaginal Septum.

Peritoneal cancer is the invasion by malignant cells of serous membrane that lines the abdominal cavity, the viscera, and the coelom of the amniotes. Histologically, it is indistinguishable from ovarian counterpart, although in the former, it commonly involves the ovary only superficially, or it may totally lack an ovarian component, but with extensive involvement of the peritoneum, calcified perihepatic peritoneal nodules, or involvement of the omentum, in most cases. The current study describes the case of a 54-year-old female patient referring a history of colitis and dairy intolerance. A transvaginal ultrasound and a computed tomography (CT) scan revealed a tumor measuring 70 × 61 × 63 mm. CA-125 serum levels were 880 U/ml. Laparotomy surgery was indicated, and tumor was found at the level of the rectovaginal septum without evidence of metastasis. Tumor dissection and protective colostomy with loop sigmoid colon were performed. A pathological study gave a diagnosis of a high-grade peritoneal serous carcinoma with a micropapillary pattern. The present study describes the case of papillary serous peritoneal cancer presented as a single tumor mass without extensive involvement of the peritoneum. Additionally, the need for routine tests for its diagnosis and documenting hormonal alterations as the cause of its origin are suggested.

Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica.

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.

Total Antioxidant Capacity in Obese and Non-Obese Subjects and Its Association with Anthropo-Metabolic Markers: Systematic Review and Meta-Analysis.

The total antioxidant capacity (TAC) has been related to the development of and complications associated with chronic diseases, but its importance during obesity is not entirely clear. We conducted a systematic review and meta-analysis to clarify whether there are differences or similarities in the TAC between subjects with obesity (SO) and subjects with normal weight (NW). Following the recommendations of PRISMA and Cochrane, we performed a systematic search in the PubMed, Scopus, Web of Science, Cochrane, and PROSPERO databases, identifying 1607 studies. Among these, 22 studies were included in the final analysis, comprising 3937 subjects (1665 SO and 2272 NW) in whom serum TAC was measured, and from these 19,201 subjects, the correlation of serum TAC with anthropo-metabolic parameters was also estimated. The Newcastle-Ottawa method was used for the evaluation of the risk of bias. Using a random-effect model (REM), TAC was reduced in SO independently of age (SMD, -0.86; 95% CI -1.38 to -0.34; p = 0.0012), whereas malondialdehyde (SMD, 1.50; 95% CI 0.60 to 2.41), oxidative stress index (SMD, 1.0; 95% CI 0.16 to 1.84), and total oxidant status (SMD, 0.80; 0.22 to 1.37) were increased. There were seven significant pooled correlations of TAC with anthropometric and metabolic parameters: weight (r = -0.17), hip circumference (r= -0.11), visceral adipose index (r = 0.29), triglycerides (r = 0.25), aspartate aminotransferase (r = 0.41), alanine aminotransferase (r = 0.38), and uric acid (r = 0.53). Our results confirm a decrease in TAC and an increase in markers of oxidative stress in SO and underpin the importance of these serum biomarkers in obesity.

Virtual Screening of Benzimidazole Derivatives as Potential Triose Phosphate Isomerase Inhibitors with Biological Activity against Leishmania mexicana.

Leishmania mexicana (L. mexicana) is a causal agent of cutaneous leishmaniasis (CL), a "Neglected disease", for which the search for new drugs is a priority. Benzimidazole is a scaffold used to develop antiparasitic drugs; therefore, it is interesting molecule against L. mexicana. In this work, a ligand-based virtual screening (LBVS) of the ZINC15 database was performed. Subsequently, molecular docking was used to predict the compounds with potential binding at the dimer interface of triosephosphate isomerase (TIM) of L. mexicana (LmTIM). Compounds were selected on binding patterns, cost, and commercial availability for in vitro assays against L. mexicana blood promastigotes. The compounds were analyzed by molecular dynamics simulation on LmTIM and its homologous human TIM. Finally, the physicochemical and pharmacokinetic properties were determined in silico. A total of 175 molecules with docking scores between -10.8 and -9.0 Kcal/mol were obtained. Compound E2 showed the best leishmanicidal activity (IC50 = 4.04 µM) with a value similar to the reference drug pentamidine (IC50 = 2.23 µM). Molecular dynamics analysis predicted low affinity for human TIM. Furthermore, the pharmacokinetic and toxicological properties of the compounds were suitable for developing new leishmanicidal agents.

Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model.

Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.

Proteomic and Functional Analysis of the Effects of Quinoxaline Derivatives on Entamoeba histolytica.

Quinoxalines are heterocyclic compounds that contain a benzene ring and a pyrazine ring. The oxidation of both nitrogen of the pyrazine ring results in quinoxaline derivatives (QdNO), which exhibit a variety of biological properties, including antiparasitic activity. However, its activity against Entamoeba histolytica, the protozoan that causes human amebiasis, is poorly understood. Recently, our group reported that various QdNOs produce morphological changes in E. histolytica trophozoites, increase reactive oxygen species, and inhibit thioredoxin reductase activity. Notably, T-001 and T-017 derivatives were among the QdNOs with the best activity. In order to contribute to the characterization of the antiamebic effect of QdNOs, in this work we analyzed the proteomic profile of E. histolytica trophozoites treated with the QdNOs T-001 and T-017, and the results were correlated with functional assays. A total number of 163 deregulated proteins were found in trophozoites treated with T-001, and 131 in those treated with T-017. A set of 21 overexpressed and 24 under-expressed proteins was identified, which were mainly related to cytoskeleton and intracellular traffic, nucleic acid transcription, translation and binding, and redox homeostasis. Furthermore, T-001 and T-017 modified the virulence of trophozoites, since they altered their erythrophagocytosis, migration, adhesion and cytolytic capacity. Our results show that in addition to alter reactive oxygen species, and thioredoxin reductase activity, T-001 and T-017 affect essential functions related to the actin cytoskeleton, which eventually affects E. histolytica virulence and survival.