Comparative transcriptional analysis between susceptible and resistant populations of Aedes (Stegomyia) aegypti (Linnaeus, 1762) after malathion exposure.

Aedes aegypti is an important vector of arboviruses, including dengue, chikungunya and Zika. The application of synthetic insecticides is a frequently used strategy to control this insect. Malathion is an organophosphate insecticide that was widely used in Brazil in the 1980s and 1990s to control the adult form of A. aegypti. In situations where resistance to currently used insecticides is detected, the use of malathion may be resumed as a control measure. Many studies have confirmed resistance to malathion, however, comparative studies of differential gene expression of the entire transcriptome of resistant and susceptible insects are scarce. Therefore, understanding the molecular basis of resistance to this insecticide in this species is extremely important. In this paper, we present the first transcriptomic description of susceptible and resistant strains of A. aegypti challenged with malathion. Guided transcriptome assembly resulted in 39,904 transcripts, where 2133 differentially expressed transcripts were detected, and three were validated by RT-qPCR. Enrichment analysis for these identified transcripts resulted in 13 significant pathways (padj < 0.05), 8 associated with down-regulated and 5 with up-regulated transcripts in treated resistant insects. It was possible to divide the transcripts according to the mechanism of action into three main groups: (i) genes involved in detoxification metabolic pathways; (ii) genes of proteins located in the membrane/extracellular region; and (iii) genes related to DNA integration/function. These results are important in advancing knowledge of genes related to resistance mechanisms in this insect, enabling the development of effective technologies and strategies for managing insecticide resistance.

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: a. human pituitary FSH18/21 (hypo-glycosylated); b. human pituitary FSH24 (fully glycosylated); c. Equine FSH (eqFSH) (hypo-glycosylated); and d. Chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10-15 x 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent distinctly different regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways.

Cannabidiol modulates hippocampal genes involved in mitochondrial function, ribosome biogenesis, synapse organization, and chromatin modifications.

BACKGROUND: Cannabidiol (CBD) is one of the main cannabinoids present in Cannabis sativa female flowers. Previous investigation has already provided insights into the CBD molecular mechanism; however, there is no transcriptome data for CBD effects on hippocampal subfields. Here, we investigate transcriptomic changes in dorsal and ventral CA1 of adult mice hippocampus after 100 mg/kg of CBD administration (i.p.) for one or seven consecutive days. METHODS: C57BL/6JUnib mice were treated with either vehicle or CBD for 1 or 7 days. The collected brains were sectioned, and the hippocampal sub-regions were laser microdissected for RNA-Seq analysis. RESULTS: The transcriptome analysis following 7 days of CBD administration indicates the differential expression of 1559 genes in dCA1 and 2924 genes in vCA1. Furthermore, GO/KEGG analysis identified 88 significantly enriched biological process and 26 significantly enriched pathways for dCBD7, whereas vCBD7 revealed 128 enriched BPs and 24 pathways. CONCLUSION: This dataset indicates a widespread decrease of electron transport chain and ribosome biogenesis transcripts in CA1, while chromatin modifications and synapse organization transcripts were increased following CBD administration for 7 days.

Systems Biology Applied to the Study of Papaya Fruit Ripening: The Influence of Ethylene on Pulp Softening.

Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.

Comparative analysis of the transcriptional responses of five Leishmania species to trivalent antimony.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by several species of Leishmania. The resistance phenotype of these parasites depends on the characteristics of each species, which contributes to increased therapeutic failures. Understanding the mechanism used by the parasite to survive under treatment pressure in order to identify potential common and specific therapeutic targets is essential for the control of leishmaniasis. The aim of this study was to investigate the expression profiles and potential shared and specific resistance markers of the main Leishmania species of medical importance [subgenus L. (Leishmania): L. donovani, L. infantum and L. amazonensis; subgenus L. (Viannia): L. panamensis and L. braziliensis)] resistant and sensitive to trivalent stibogluconate (SbIII). METHODS: We conducted comparative analysis of the transcriptomic profiles (only coding sequences) of lines with experimentally induced resistance to SbIII from biological replicates of five Leishmania species available in the databases of four articles based on ortholog attribution. Simultaneously, we carried out functional analysis of ontology and reconstruction of metabolic pathways of the resulting differentially expressed genes (DEGs). RESULTS: Resistant lines for each species had differential responses in metabolic processes, compound binding, and membrane components concerning their sensitive counterpart. One hundred and thirty-nine metabolic pathways were found, with the three main pathways comprising cysteine and methionine metabolism, glycolysis, and the ribosome. Differentially expressed orthologous genes assigned to species-specific responses predominated, with 899 self-genes. No differentially expressed genes were found in common among the five species. Two common upregulated orthologous genes were found among four species (L. donovani, L. braziliensis, L. amazonensis, and L. panamensis) related to an RNA-binding protein and the NAD(P)H cytochrome-B5-oxidoreductase complex, associated with transcriptional control and de novo synthesis of linoleic acid, critical mechanisms in resistance to antimonials. CONCLUSION: Herein, we identified potential species-specific genes related to resistance to SbIII. Therefore, we suggest that future studies consider a treatment scheme that is species-specific. Despite the limitations of our study, this is the first approach toward unraveling the pan-genus genetic mechanisms of resistance in leishmaniasis.

Human type 2 diabetes mellitus-associated transcriptional disturbances in a high-sugar diet long-term exposed Drosophila melanogaster.

Type 2 Diabetes mellitus (T2DM) is a multifactorial and polygenic disorder with the molecular bases still idiopathic. Experimental analyses and tests are quite limited upon human samples due to the access, variability of patient's conditions, and the size and complexity of the genome. Therefore, high-sugar diet exposure is commonly used for modeling T2DM in non-human animals, which includes invertebrate organisms like the fruit fly Drosophila melanogaster. Interestingly, high-sugar diet (HSD) induces delayed time for pupation and reduced viability in fruit fly larvae hatched from a 30% sucrose-containing medium (HSD-30%). Here we carried out an mRNA-deep sequencing study to identify differentially transcribed genes in adult fruit fly hatched and reared from an HSD-30%. Seven days after hatching, flies reared on control and HSD-30% were used to glucose and triglyceride level measurements and RNA extraction for sequencing. Remarkably, glucose levels were about 2-fold higher than the control group in fruit flies exposed to HSD-30%, whereas triglycerides levels increased 1.7-fold. After RNA-sequencing, we found that 13.5% of the genes were differentially transcribed in the dyslipidemic and hyperglycaemic insects. HSD-30% up-regulated genes involved in ribosomal biogenesis (e.g. dTOR, ERK and dS6K) and down-regulated genes involved in energetic process (e.g. Pfk, Gapdh1, and Pyk from pyruvate metabolism; kdn, Idh and Mdh2 from the citric acid cycle; ATPsynC and ATPsynẞ from ATP synthesis) and insect development. We found a remarkable down-regulation for Actin (Act88F) that likely impairs muscle development. Moreover, HSD-30% up-regulated both the insulin-like peptides 7 and 8 and down-regulated the insulin receptor substrate p53, isoform A and insulin-like peptide 6 genes, whose functional products are insulin signaling markers. All these features pointed together to a tightly correlation of the T2DM-like phenotype modeled by the D. melanogaster and an intricate array of phenomena, which includes energetic processes, muscle development, and ribosomal synthesis as that observed for the human pathology.

Expression analysis of progesterone­regulated miRNAs in cells derived from human glioblastoma.

Glioblastomas (GBMs) are the most frequent and malignant type of brain tumor. It has been reported that progesterone (P4) regulates the progression of GBMs by modifying the expression of genes that promote cell proliferation, migration and invasion; however, it is not fully understood how these processes are regulated. It is possible that P4 mediates some of these effects through changes in the microRNA (miRNA) expression profile in GBM cells. The present study investigated the effects of P4 on miRNAs expression profile in U­251MG cells derived from a human GBM. U­251MG cells were treated for 6 h with P4, RU486 (an antagonist of the intracellular progesterone receptor), the combined treatment (P4+RU486) and cyclodextrin (vehicle) and then a miRNA microarray analysis conducted. The expression analysis revealed a set of 190 miRNAs with differential expression in the treatments of P4, RU486 and P4+RU486 in respect to the vehicle and P4 in respect to P4+RU486, of which only 16 were exclusively regulated by P4. The possible mRNA targets of the miRNAs regulated by P4 could participate in the regulation of proliferation, cell cycle progression and cell migration of GBMs. The present study provided insight for understanding epigenetic modifications regulated by sex hormones involved in GBM progression, and for identifying potential therapeutic strategies for these brain tumors.

Transcriptomic studies on Purpureocillium lilacinum reveal molecular mechanisms of response to fluconazole and itraconazole.

Filamentous fungus Purpureocillium lilacinum is an emerging pathogen that infects immunocompromised and immunocompetent individuals and is resistant to several azole molecules. Although azole resistance mechanisms are well studied in Aspergillus sp. and Candida sp., there are no studies to date reporting P. lilacinum molecular response to these molecules. The aim of this study was to describe P. lilacinum molecular mechanisms involved in antifungal response against fluconazole and itraconazole. Transcriptomic analyses showed that gene expression modulation takes place when P. lilacinum is challenged for 12 h with fluconazole (64 µg/mL) or itraconazole (16 µg/mL). The antifungals acted on the ergosterol biosynthesis pathway, and two homologous genes coding for cytochrome P450 51 enzymes were upregulated. Genes coding for efflux pumps, such as the major facilitator superfamily transporter, also displayed increased expression in the treated samples. We propose that P. lilacinum develops antifungal responses by raising the expression levels of cytochrome P450 enzymes and efflux pumps. Such modulation could confer P. lilacinum high levels of target enzymes and could lead to the constant withdrawal of antifungals, which would force an increase in the administration of antifungal medications to achieve fungal morbidity or mortality. The findings in this work could aid in the decision-making for treatment strategies in cases of P. lilacinum infection.

Toxicogenomic Analysis.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

Physiological and Transcriptomic Changes in the Hypothalamic-Neurohypophysial System after 24 h of Furosemide-Induced Sodium Depletion.

BACKGROUND/AIMS: Furosemide is a loop diuretic widely used in clinical practice for the treatment of oedema and hypertension. The aim of this study was to determine physiological and molecular changes in the hypothalamic-neurohypophysial system as a consequence of furosemide-induced sodium depletion. METHODS: Male rats were sodium depleted by acute furosemide injection (10 and 30 mg/kg) followed by access to low sodium diet and distilled water for 24 h. The renal and behavioural consequences were evaluated, while blood and brains were collected to evaluate the neuroendocrine and gene expression responses. RESULTS: Furosemide treatment acutely increases urinary sodium and water excretion. After 24 h, water and food intake were reduced, while plasma angiotensin II and corticosterone were increased. After hypertonic saline presentation, sodium-depleted rats showed higher preference for salt. Interrogation using RNA sequencing revealed the expression of 94 genes significantly altered in the hypothalamic paraventricular nucleus (PVN) of sodium-depleted rats (31 upregulated and 63 downregulated). Out of 9 genes chosen, 5 were validated by quantitative PCR in the PVN (upregulated: Ephx2, Ndnf and Vwf; downregulated: Caprin2 and Opn3). The same genes were also assessed in the supraoptic nucleus (SON, upregulated: Tnnt1, Mis18a, Nr1d1 and Dbp; downregulated: Caprin2 and Opn3). As a result of these plastic transcriptome changes, vasopressin expression was decreased in PVN and SON, whilst vasopressin and oxytocin levels were reduced in plasma. CONCLUSIONS: We thus have identified novel genes that might regulate vasopressin gene expression in the hypothalamus controlling the magnocellular neurons secretory response to body sodium depletion and consequently hypotonic stress.

Bioinformatic analysis of RHO family of GTPases identifies RAC1 pharmacological inhibition as a new therapeutic strategy for hepatocellular carcinoma.

OBJECTIVE: The RHO family of GTPases, particularly RAC1, has been linked with hepatocarcinogenesis, suggesting that their inhibition might be a rational therapeutic approach. We aimed to identify and target deregulated RHO family members in human hepatocellular carcinoma (HCC). DESIGN: We studied expression deregulation, clinical prognosis and transcription programmes relevant to HCC using public datasets. The therapeutic potential of RAC1 inhibitors in HCC was study in vitro and in vivo. RNA-Seq analysis and their correlation with the three different HCC datasets were used to characterise the underlying mechanism on RAC1 inhibition. The therapeutic effect of RAC1 inhibition on liver fibrosis was evaluated. RESULTS: Among the RHO family of GTPases we observed that RAC1 is upregulated, correlates with poor patient survival, and is strongly linked with a prooncogenic transcriptional programme. From a panel of novel RAC1 inhibitors studied, 1D-142 was able to induce apoptosis and cell cycle arrest in HCC cells, displaying a stronger effect in highly proliferative cells. Partial rescue of the RAC1-related oncogenic transcriptional programme was obtained on RAC1 inhibition by 1D-142 in HCC. Most importantly, the RAC1 inhibitor 1D-142 strongly reduce tumour growth and intrahepatic metastasis in HCC mice models. Additionally, 1D-142 decreases hepatic stellate cell activation and exerts an anti-fibrotic effect in vivo. CONCLUSIONS: The bioinformatics analysis of the HCC datasets, allows identifying RAC1 as a new therapeutic target for HCC. The targeted inhibition of RAC1 by 1D-142 resulted in a potent antitumoural effect in highly proliferative HCC established in fibrotic livers.

Mexican Ganoderma Lucidum Extracts Decrease Lipogenesis Modulating Transcriptional Metabolic Networks and Gut Microbiota in C57BL/6 Mice Fed with a High-Cholesterol Diet.

Prevention of hyperlipidemia and associated diseases is a health priority. Natural products, such as the medicinal mushroom Ganoderma lucidum (Gl), have demonstrated hypocholesterolemic, prebiotic and antidiabetic properties. However, the underlying transcriptomic mechanisms by which Gl exerts bioactivities are not completely understood. We report a comprehensive hepatic and renal transcriptome profiling of C57BL/6 mice under the consumption of a high-cholesterol diet and two standardized Gl extracts obtained from basidiocarps cultivated on conventional substrate (Gl-1) or substrate containing acetylsalicylic acid (ASA; Gl-2). We showed that Gl extracts modulate relevant metabolic pathways involving the restriction of lipid biosynthesis and the enrichment of lipid degradation and secretion. The Gl-2 extract exerts a major modulation over gene expression programs showing the highest similarity with simvastatin druggable-target-genes and these are enriched more in processes related to human obesity alterations in the liver. We further show a subset of Gl-modulated genes correlated with Lactobacillus enrichment and the reduction of circulating cholesterol-derived fats. Moreover, Gl extracts induce a significant decrease of macrophage lipid storage, which occurs concomitantly with the down-modulation of Fasn and Elovl6. Collectively, this evidence suggests a new link between Gl hypocholesterolemic and prebiotic activity, revealing thereby that standardized Mexican Gl extracts are a novel transcriptome modulator to prevent metabolic disorders associated with hypercholesterolemia.

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines.

BACKGROUND: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing. METHODS: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search. RESULTS: The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation. CONCLUSIONS: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Analysis of the endometrial transcriptome at the time of implantation in women receiving a single post-ovulatory dose of levonorgestrel or mifepristone

Abstract Background: Levonorgestrel (LNG) is a progesterone receptor agonist used in both regular and emergency hormonal contraception; however, its effects on the endometrium as a contraceptive remain widely unknown and under public debate. Objective: To analyze the effects of LNG or mifepristone (MFP), a progesterone receptor antagonist and also known as RU-486, administered at the time of follicle rupture (FR) on endometrial transcriptome during the receptive period of the menstrual cycle. Methods: Ten volunteers ovulatory women were studied during two menstrual cycles, a control cycle and a consecutively treated cycle; in this last case, women were randomly allocated to two groups of 5 women each, receiving one dose of LNG (1.5 mg) or MFP (50 mg) the day of the FR by ultrasound. Endometrial biopsies were taken 6 days after drug administration and prepared for microarray analysis. Results: Genomic functional analysis in the LNG-treated group showed as activated the bio-functions embryo implantation and decidualization, while these bio-functions in the T-MFP group were predicted as inhibited. Conclusions: The administration of LNG as a hormonal emergency contraceptive resulted in an endometrial gene expression profile associated with receptivity. These results agree on the concept that LNG does not affect endometrial receptivity and/or embryo implantation when used as an emergency contraceptive.

Analysis of the Endometrial Transcriptome at the Time of Implantation in Women Receiving a Single Post-Ovulatory Dose of Levonorgestrel or Mifepristone.

BACKGROUND: Levonorgestrel (LNG) is a progesterone receptor agonist used in both regular and emergency hormonal contraception; however, its effects on the endometrium as a contraceptive remain widely unknown and under public debate. OBJECTIVE: To analyze the effects of LNG or mifepristone (MFP), a progesterone receptor antagonist and also known as RU-486, administered at the time of follicle rupture (FR) on endometrial transcriptome during the receptive period of the menstrual cycle. METHODS: Ten volunteers ovulatory women were studied during two menstrual cycles, a control cycle and a consecutively treated cycle; in this last case, women were randomly allocated to two groups of 5 women each, receiving one dose of LNG (1.5 mg) or MFP (50 mg) the day of the FR by ultrasound. Endometrial biopsies were taken 6 days after drug administration and prepared for microarray analysis. RESULTS: Genomic functional analysis in the LNG-treated group showed as activated the bio-functions embryo implantation and decidualization, while these bio-functions in the T-MFP group were predicted as inhibited. CONCLUSIONS: The administration of LNG as a hormonal emergency contraceptive resulted in an endometrial gene expression profile associated with receptivity. These results agree on the concept that LNG does not affect endometrial receptivity and/or embryo implantation when used as an emergency contraceptive.

Transcriptomic determinants of the response of ST-111 Pseudomonas aeruginosa AG1 to ciprofloxacin identified by a top-down systems biology approach.

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in diverse environments and causes a variety of human infections. Pseudomonas aeruginosa AG1 (PaeAG1) is a high-risk sequence type 111 (ST-111) strain isolated from a Costa Rican hospital in 2010. PaeAG1 has both blaVIM-2 and blaIMP-18 genes encoding for metallo-ß-lactamases, and it is resistant to ß-lactams (including carbapenems), aminoglycosides, and fluoroquinolones. Ciprofloxacin (CIP) is an antibiotic commonly used to treat P. aeruginosa infections, and it is known to produce DNA damage, triggering a complex molecular response. In order to evaluate the effects of a sub-inhibitory CIP concentration on PaeAG1, growth curves using increasing CIP concentrations were compared. We then measured gene expression using RNA-Seq at three time points (0, 2.5 and 5 h) after CIP exposure to identify the transcriptomic determinants of the response (i.e. hub genes, gene clusters and enriched pathways). Changes in expression were determined using differential expression analysis and network analysis using a top-down systems biology approach. A hybrid model using database-based and co-expression analysis approaches was implemented to predict gene-gene interactions. We observed a reduction of the growth curve rate as the sub-inhibitory CIP concentrations were increased. In the transcriptomic analysis, we detected that over time CIP treatment resulted in the differential expression of 518 genes, showing a complex impact at the molecular level. The transcriptomic determinants were 14 hub genes, multiple gene clusters at different levels (associated to hub genes or as co-expression modules) and 15 enriched pathways. Down-regulation of genes implicated in several metabolism pathways, virulence elements and ribosomal activity was observed. In contrast, amino acid catabolism, RpoS factor, proteases, and phenazines genes were up-regulated. Remarkably, > 80 resident-phage genes were up-regulated after CIP treatment, which was validated at phenomic level using a phage plaque assay. Thus, reduction of the growth curve rate and increasing phage induction was evidenced as the CIP concentrations were increased. In summary, transcriptomic and network analyses, as well as the growth curves and phage plaque assays provide evidence that PaeAG1 presents a complex, concentration-dependent response to sub-inhibitory CIP exposure, showing pleiotropic effects at the systems level. Manipulation of these determinants, such as phage genes, could be used to gain more insights about the regulation of responses in PaeAG1 as well as the identification of possible therapeutic targets. To our knowledge, this is the first report of the transcriptomic analysis of CIP response in a ST-111 high-risk P. aeruginosa strain, in particular using a top-down systems biology approach.

The combination of coffee compounds attenuates early fibrosis-associated hepatocarcinogenesis in mice: involvement of miRNA profile modulation.

Aberrant microRNA expression implicates on hepatocellular carcinoma (HCC) development. Conversely, coffee consumption reduces by ~40% the risk for fibrosis/cirrhosis and HCC, while decaffeinated coffee does not. It is currently unknown whether these protective effects are related to caffeine (CAF), or to its combination with other common and/or highly bioavailable coffee compounds, such as trigonelline (TRI) and chlorogenic acid (CGA). We evaluated whether CAF individually or combined with TRI and/or CGA alleviates fibrosis-associated hepatocarcinogenesis, examining the involvement of miRNA profile modulation. Then, male C3H/HeJ mice were submitted to a diethylnitrosamine/carbon tetrachloride-induced model. Animals received CAF (50 mg/kg), CAF+TRI (50 and 25 mg/kg), CAF+CGA (50 and 25 mg/kg) or CAF+TRI+CGA (50, 25 and 25 mg/kg), intragastrically, 5×/week, for 10 weeks. Only CAF+TRI+CGA combination reduced the incidence, number and proliferation (Ki-67) of hepatocellular preneoplastic foci while enhanced apoptosis (cleaved caspase-3) in adjacent parenchyma. CAF+TRI+CGA treatment also decreased hepatic oxidative stress and enhanced the antioxidant Nrf2 axis. CAF+TRI+CGA had the most pronounced effects on decreasing hepatic pro-inflammatory IL-17 and NFκB, contributing to reduce CD68-positive macrophage number, stellate cell activation, and collagen deposition. In agreement, CAF+TRI+CGA upregulated tumor suppressors miR-144-3p, miR-376a-3p and antifibrotic miR-15b-5p, frequently deregulated in human HCC. CAF+TRI+CGA reduced the hepatic protein levels of pro-proliferative EGFR (miR-144-3p target), antiapoptotic Bcl-2 family members (miR-15b-5p targets), and the number of PCNA (miR-376a-3p target) positive hepatocytes in preneoplastic foci. Our results suggest that the combination of most common and highly bioavailable coffee compounds, rather than CAF individually, attenuates fibrosis-associated hepatocarcinogenesis by modulating miRNA expression profile.

Transcriptional response of Fusarium oxysporum and Neocosmospora solani challenged with amphotericin B or posaconazole.

Some species of fusaria are well-known pathogens of humans, animals and plants. Fusarium oxysporum and Neocosmospora solani (formerly Fusarium solani) cause human infections that range from onychomycosis or keratitis to severe disseminated infections. In general, these infections are difficult to treat due to poor therapeutic responses in immunocompromised patients. Despite that, little is known about the molecular mechanisms and transcriptional changes responsible for the antifungal resistance in fusaria. To shed light on the transcriptional response to antifungals, we carried out the first reported high-throughput RNA-seq analysis for F. oxysporum and N. solani that had been exposed to amphotericin B (AMB) and posaconazole (PSC). We detected significant differences between the transcriptional profiles of the two species and we found that some oxidation-reduction, metabolic, cellular and transport processes were regulated differentially by both fungi. The same was found with several genes from the ergosterol synthesis, efflux pumps, oxidative stress response and membrane biosynthesis pathways. A significant up-regulation of the C-22 sterol desaturase (ERG5), the sterol 24-C-methyltransferase (ERG6) gene, the glutathione S-transferase (GST) gene and of several members of the major facilitator superfamily (MSF) was demonstrated in this study after treating F. oxysporum with AMB. These results offer a good overview of transcriptional changes after exposure to commonly used antifungals, highlights the genes that are related to resistance mechanisms of these fungi, which will be a valuable tool for identifying causes of failure of treatments.

Comparative high-throughput analysis of the Trypanosoma cruzi response to organometallic compounds.

There is an urgent need to develop new drugs against Chagas' disease. In addition, the mechanisms of action of existing drugs have not been completely worked out at the molecular level. High throughput approaches have been demonstrated to be powerful tools not only for understanding the basic biology of Trypanosoma cruzi, but also for the identification of drug targets such as proteins or pathways that are essential for parasite infection and survival within the mammalian host. Here, we have applied these tools towards the discovery of the effects of two organometallic compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, on the transcriptome and proteome of T. cruzi epimastigotes. These approaches have not yet been reported for any other prospective metal-based anti T. cruzi drug. We found differentially expressed transcripts and proteins in treated parasites. Pd-dppf-mpo treatment resulted in more modulated transcripts (2327 of 10 785 identified transcripts) than Pt-dppf-mpo treatment (201 of 10 773 identified transcripts) suggesting a mechanism of action for Pd-dppf-mpo at the transcriptome level. Similar numbers of differentially expressed proteins (342 and 411 for Pd-dppf-mpo and Pt-dppf-mpo respectively) were also observed. We further functionally categorized differentially expressed transcripts and identified cellular processes and pathways significantly impacted by treatment with the compounds. Transcripts involved in DNA binding, protein metabolism, transmembrane transport, oxidative defense, and the ergosterol pathways were found to be modulated by the presence of the compounds. Our transcriptomic dataset also contained previously validated essential genes. These data allowed us to hypothesize a multimodal mechanism of action for the trypanocidal activity of Pd-dppf-mpo and Pt-dppf-mpo, and a differential contribution of the metal moiety of each compound.

In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX).

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 µg/mL and 200 µg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 µg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 µg/mL or 200 µg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.