Arabidopsis Coexpression Tool:a tool for gene coexpression analysis in Arabidopsis thaliana

Gene coexpression analysis refers to the discovery of sets of genes which exhibit similar expression patterns across multiple transcriptomic data sets, such as microarray experiment data of public repositories. Arabidopsis Coexpression Tool (ACT), a gene coexpression analysis web tool for Arabidopsis thaliana, identifies genes which are correlated to a driver gene. Primary microarray data from ATH1 Affymetrix platform were processed with Single-Channel Array Normalization algorithm and combined to produce a coexpression tree which contains ∼21,000 A. thaliana genes. ACT was developed to present subclades of coexpressed genes, as well as to perform gene set enrichment analysis, being unique in revealing enriched transcription factors targeting coexpressed genes. ACT offers a simple and user-friendly interface producing working hypotheses which can be experimentally verified for the discovery of gene partnership, pathway membership, and transcriptional regulation. ACT analyses have been successful in identifying not only genes with coordinated ubiquitous expressions but also genes with tissue-specific expressions

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties

Study of probiotic microorganisms action in functional foods

Recent studies showed that several Lactic Acid Bacteria (LAB) strains isolated from naturally fermented table olives display probiotic potential (Argyri et al. 2013). Amongst them, two strains, namely Lactobacillus pentosus B281 and Lactobacillus plantarum B282 display significant potential for the production of novel probiotic food products (Argyri et al. 2014). The aim of this thesis was to examine the probiotic potential of strains L. pentosus B281 and L. plantarum B282 in novel functional foods and to investigate the molecular and cellular mechanisms of action. The primary purpose of this study was the development of a rapid and effective method for the molecular detection and identification of the above strains. RAPD fingerprinting was firstly applied for the development of strain specific primers as both strains were recently isolated and no sequence data was available. A multiplex PCR assay was designed for each strain, based on specific primers derived from RAPD analysis. The specificity of the assay was successfully tested against different strains of LAB and dairy products prepared in the laboratory. The stains L. pentosus B281 and L. plantarum B282 were originally isolated from olive microbiota and their performance as starters in the fermentation of green olives was evaluated previously (Argyri et al. 2014). Here, their possible use in yogurt production was examined. Cell enumeration and employment of the novel multiplex PCR methodology showed that both strains were presented in yogurt samples at levels ≥ 6 log CFU/g during fermentation and storage. In addition, the microbiological, physicochemical, and sensory profiles of the novel products were similar to the controls, demonstrating the use of the two strains as starter cultures for the production of novel dairy products with probiotic potential. Then, the adhesion properties and the antiproliferative effects of the strains L. pentosus B281 and L. plantarum B282 were investigated. The results demonstrated that both strains exhibited significantly higher adhesion rates to Caco-2 colon cancer cells compared to the probiotic reference strain L. casei ATCC 393. In addition, the conditioned media of the two strains inhibited growth of human colon cancer cells (Caco-2 and HT-29) by promoting a G1-phase arrest and down-regulation of specific cyclin genes. Finally, the bacterial components appeared to be thermostable, as the anti-proliferative activity of heat-treated conditioned media was similar to the non-heat-treated conditioned media in a time- and dose-dependent manner. This data, enhance our understanding of the probiotic role of the two strains and support their application as starter cultures in the production of novel functional food products. The next aim of the project was to characterize the modes of action of the strains L. pentosus B281 and L. plantarum B282 and establish further their probiotic character. For this reason, we investigated the anti-proliferative activity of viable cells on colon cancer cell lines. The results showed that viable cells of the two strains caused a significant reduction in Caco-2 cell proliferation in a time- and dose-dependent manner. Then, employing the air pouch infection system in mice the immunostimulatory activity of the two strains was evaluated. The results demonstrated that both L. pentosus B281 and L. plantarum B282 induced significant recruitment of leukocytes in the air pouch exudates and the release of a range of pro-inflammatory cytokines including interleukin-1α (IL-1α), IL-1β, and IL-6, as well as the chemokines CCL3 and CCL4.Τα στελέχη Lactobacillus pentosus B281 και Lactobacillus plantarum B282 είναι βακτήρια γαλακτικού οξέος (Lactic Acid Bacteria - LAB), τα οποία απομονώθηκαν από τη μικροχλωρίδα ελιάς και κατέδειξαν προβιοτικές ιδιότητες, όπως είναι η επιβίωση σε προσομοιωμένες συνθήκες του γαστρεντερικού σωλήνα, η αντιμικροβιακή δράση, η ικανότητα προσκόλλησης σε καρκινικά κύτταρα παχέος εντέρου Caco-2, η αντοχή σε αντιβιοτικά και η αιμολυτική δράση (Argyri et al., 2013). Επιπλέον, οι μικροοργανισμοί L. pentosus B281 και L. plantarum B282, χρησιμοποιήθηκαν αποτελεσματικά ως εναρκτήριες καλλιέργειες κατά τη ζύμωση πράσινων ελιών (Argyri et al., 2014; Blana et al., 2014), οι οποίες έγιναν αποδεκτές για κατανάλωση, όπως παρουσιάστηκε σε δοκιμές οργανοληπτικής αξιολόγησης (Argyri et al., 2014). Σκοπός στην παρούσα διατριβή, ήταν να εξεταστεί ο προβιοτικός χαρακτήρας των στελεχών L. pentosus B281 και L. plantarum B282 σε νέα λειτουργικά τρόφιμα και να μελετηθούν οι μοριακοί και κυτταρικοί μηχανισμοί δράσης τους. Αρχικά για τη μοριακή ανίχνευση και ταυτοποίηση των δύο στελεχών σχεδιάστηκαν εξιδεικευμένοι εκκινητές με RAPD ανάλυση και αναπτύχθηκε μια μέθοδος βασιζόμενη στην τεχνική της Multiplex PCR. Η διακριτική ικανότητα της μεθόδου ελέγχθηκε επιτυχώς έναντι διαφορετικών στελεχών LAB καθώς και σε γαλακτοκομικά προϊόντα που παρασκευάστηκαν στο εργαστήριο. Επιπλέον, τα παραπάνω στελέχη, χρησιμοποιήθηκαν αποτελεσματικά ως εναρκτήριες καλλιέργειες για την παρασκευή γιαουρτιού όπως έδειξαν οι μικροβιολογικές, φυσικοχημικές και οργανοληπτικές αναλύσεις που πραγματοποιήθηκαν. Ακόμα, μέσω μικροβιολογικής καταμέτρησης και multiplex PCR ανάλυσης καταδείχτηκε ότι τα στελέχη L. pentosus B281 και L. plantarum B282 επιβίωσαν στα δείγματα γιαουρτιού σε επίπεδα ≥ 6 log CFU/g ακόμη και μετά από 35 ημέρες αποθήκευσης στους 4 °C. Από την διερεύνηση των μηχανισμών δράσης των στελεχών L. pentosus B281 και L. plantarum B282, παρουσιάστηκε αρχικά ότι τα στελέχη παρουσίασαν σημαντική προσκόλληση σε καρκινικά κύτταρα παχέος εντέρου. Η οπτικοποίηση των κυττάρων που προσκολλώνται στα καρκινικά κύτταρα μέσω συνεστιακής μικροσκοπίας φθορισμού επιβεβαίωσε περαιτέρω την παραπάνω παρατήρηση. Ακόμη, αποδείχτηκε η αντί-πολλαπλασιαστική δράση του ρυθμιστικού μέσου των παραπάνω στελεχών έναντι διαφορετικών καρκινικών σειρών παχέος εντέρου, ο ρόλος τους στην κατανομή του κυτταρικού κύκλου και στην έκφραση γονιδίων που σχετίζονται με τον κυτταρικό κύκλο. Επιπροσθέτως, πραγματοποιήθηκε προκαταρτικός χαρακτηρισμός της φύσης των βιοδραστικών μορίων που υπάρχουν στο ρυθμιστικό μέσο των δύο στελεχών και αποδείχτηκε η παρουσία θερμοσταθερών βιοδραστικών ενώσεων. Στη συνέχεια από την διερεύνηση της αντι-πολλαπλασιαστικής και της ανοσοδιεγερτικής δράσης των ζωντανών στελεχών L. pentosus B281 και L. plantarum B282, καταδείχτηκε η αντί-πολλαπλασιαστική δράση τους στην ανθρώπινη καρκινική σειρά παχέος εντέρου Caco-2. Τέλος μέσω του ζωικού μοντέλου του ραχιαίου αεροθύλακα αποδείχτηκε η ανοσοδιεγερτική δράση των παραπάνω στελεχών. Πιο συγκεκριμένα, καταδείχτηκε η στρατολόγηση ουδετερόφιλων και η έκκριση συγκεκριμένων κυτοκινών και χημειοκινών ως ανοσοαπόκριση στη χορήγηση των δύο στελεχών στο ραχιαίο αεροθύλακα ποντικών

Inhibition of Listeria monocytogenes Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Inhibition of <i>Listeria monocytogenes</i> Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties

Fermentation Supernatants of Pleurotus eryngii Mushroom Ameliorate Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide-Induced Caco-2 Cells via Upregulation of Tight Junctions

In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in β-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs’ genes.Funding agency:Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation under the call RESEARCH-CREATE-INNOVATE T1EDK-03404 </p

Approaches in Gene Coexpression Analysis in Eukaryotes

Gene coexpression analysis constitutes a widely used practice for gene partner identification and gene function prediction, consisting of many intricate procedures. The analysis begins with the collection of primary transcriptomic data and their preprocessing, continues with the calculation of the similarity between genes based on their expression values in the selected sample dataset and results in the construction and visualisation of a gene coexpression network (GCN) and its evaluation using biological term enrichment analysis. As gene coexpression analysis has been studied extensively, we present most parts of the methodology in a clear manner and the reasoning behind the selection of some of the techniques. In this review, we offer a comprehensive and comprehensible account of the steps required for performing a complete gene coexpression analysis in eukaryotic organisms. We comment on the use of RNA-Seq vs. microarrays, as well as the best practices for GCN construction. Furthermore, we recount the most popular webtools and standalone applications performing gene coexpression analysis, with details on their methods, features and outputs

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD