Complete genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198

Within the Streptococcus genus, only Streptococcus thermophilus is considered to be non-pathogenic due to its adaptation to the milk environment. Streptococcus macedonicus is also an intriguing streptococcal species since its most frequent source of isolation to date is fermented foods, mainly of dairy origin. Sequencing of S. macedonicus ACA-DC 198 genome was performed using a combination of 454 GS FLX pyrosequencing and HiSeq 2000 Illumina sequencing. The hybrid assembly between 454 and HiSeq2000 data (>200x coverage) resulted in one continuous genomic scaffold of 2,130,034 bp and a plasmid of 12,728 bp. The genome assembly was validated against a NheI optical map of the S. macedonicus genome. Sequences were annotated with the BaSys and the RAST pipelines and manually curated using Kodon. Final corrections were made based on the quality assessment of the annotation using GenePRIMP. We found 2,192 protein-coding genes on the chromosome, 192 of which were identified as potential pseudogenes, indicating an ongoing genome decay process. This hypothesis is also supported by the approximately 220 kb-smaller genome size of S. macedonicus compared to the S. gallolyticus genomes, despite the high level of gene synteny between the two species. Such a reductive evolutionary process is common for lactic acid bacteria domesticated to the food environment, which in the case of S. thermophilus was also accompanied by the loss of pathogenicity traits. With our in silico analysis we attempt to investigate whether S. macedonicus shows traits that would support its adaptation to the dairy environment at the genomic level

Milk adaptation and pathogenic potential among members of the Streptococcus bovis/Streptococcus equinus complex

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) consists of species commonly found in the gastrointestinal tract (GIT) of herbivores. Certain members of the complex like Streptococcus macedonicus and Streptococcus infantarius are frequently isolated from traditional fermented foods, mostly of dairy origin. However, some species of this complex, like Streptococcus gallolyticus and Streptococcus pasteurianus are known pathogens of humans involved in a range of diseases including endocarditis, menengitis, bacteremia, colon cancer etc. Analysis of the genome of S. macedonicus strain ACA-DC 198 isolated from traditional Greek Kasseri cheese revealed important traits of adaptation to the dairy environment. The strain contains a significant percentage of potential pseudogenes indicating that it may have evolved through genome decay processes. Streptococcus macedonicus has an extra gene cluster for lactose and galactose metabolism unique among the SBSEC members and a typical proteolytic system required for casein hydrolysis. Furthermore, we found evidence in the genome of S. macedonicus suggesting HGT (horizontal gene transfer) events with potential donors Lactococcus lactis and Streptococcus thermophilus. Perhaps the most pronounced among these HGT events is the presence in S. macedonicus of pSMA198 which belongs to the lactococcal pCI305/pWV02 family of plasmids. Our analysis suggests that pSMA198 may have been acquired by S. macedonicus from L. lactis. Analogous traits have been reported for S. infantarius suggesting that at least these two strains present adaptations to the dairy environment among SBSEC. Finally, we were able to identify a number of potential virulence factors (VFs) within the SBSEC members whose presence varied among species of the complex. Streptococcus macedonicus and S. infantarius miss some of the VFs present in S. gallolyticus suggesting a diminished pathogenic potential for the two species

Comparative genomics of the dairy isolate Streptococcus macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus complex

Background: Within the genus Streptococcus, only Streptococcus thermophilus is used as a starter culture in food fermentations. Streptococcus macedonicus though, which belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC), is also frequently isolated from fermented foods mainly of dairy origin. Members of the SBSEC have been implicated in human endocarditis and colon cancer. Here we compare the genome sequence of the dairy isolate S. macedonicus ACA-DC 198 to the other SBSEC genomes in order to assess in silico its potential adaptation to milk and its pathogenicity status. Results: Despite the fact that the SBSEC species were found tightly related based on whole genome phylogeny of streptococci, two distinct patterns of evolution were identified among them. Streptococcus macedonicus, Streptococcus infantarius CJ18 and Streptococcus pasteurianus ATCC 43144 seem to have undergone reductive evolution resulting in significantly diminished genome sizes and increased percentages of potential pseudogenes when compared to Streptococcus gallolyticus subsp. gallolyticus. In addition, the three species seem to have lost genes for catabolizing complex plant carbohydrates and for detoxifying toxic substances previously linked to the ability of S. gallolyticus to survive in the rumen. Analysis of the S. macedonicus genome revealed features that could support adaptation to milk, including an extra gene cluster for lactose and galactose metabolism, a proteolytic system for casein hydrolysis, auxotrophy for several vitamins, an increased ability to resist bacteriophages and horizontal gene transfer events with the dairy Lactococcus lactis and S. thermophilus as potential donors. In addition, S. macedonicus lacks several pathogenicity-related genes found in S. gallolyticus. For example, S. macedonicus has retained only one (i.e. the pil3) of the three pilus gene clusters which may mediate the binding of S. gallolyticus to the extracellular matrix. Unexpectedly, similar findings were obtained not only for the dairy S. infantarius CJ18, but also for the blood isolate S. pasteurianus ATCC 43144. Conclusions: Our whole genome analyses suggest traits of adaptation of S. macedonicus to the nutrient-rich dairy environment. During this process the bacterium gained genes presumably important for this new ecological niche. Finally, S. macedonicus carries a reduced number of putative SBSEC virulence factors, which suggests a diminished pathogenic potential

Nrf2-mediated fibroblast reprogramming drives cellular senescence by targeting the matrisome

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor

Plant-Derived Senotherapeutics for the Prevention and Treatment of Intervertebral Disc Degeneration and Aging

Chronic low back pain, a major cause of disability with a great global socioeconomic impact, has been inextricably associated with intervertebral disc degeneration. On the other hand, an enhanced number of senescent cells has been identified in aged and degenerated intervertebral discs and their senescence-associated secretory phenotype (SASP) has been connected with qualitative/quantitative alterations in the extracellular matrix and ultimately with the disturbance of tissue homeostasis. Given that selective elimination of senescent cells (by the so-called senolytics) or amendment of their secretome towards a less catabolic/inflammatory phenotype (by molecules known as senomorphics) has been reported to alleviate symptoms of several age-associated diseases and to improve tissue quality during aging, here we will review the emerging role of senolytic and senomorphic agents derived from plants and natural products against intervertebral disc degeneration. The mode of action of these senotherapeutics, as well as the challenges in their practical application, will also be explicitly discussed in an attempt to direct their more targeted and effective use in exclusive or combinatorial therapeutic schemes for the prevention and/or treatment of disc degenerative disorders

Study of the effect of osmotic stress on intervertebral disc cells' proliferation and senescence

The proper function of intervertebral disc cells is necessary for the maintenance of a healthy tissue. Given the unusual physicochemical conditions of this particular tissue in comparison to other tissues (acidic pH, low oxygen concentration, nutirients’ deficiency and high osmolality), the deeper understanding of disc cells’ physiology and responses in the concept of their peculiar extracellular microenvironment is of great importance. The aim of this dissertation was to investigate the effect of different stresses confronted by the intervertebral disc in its native environment on cellular proliferation and senescence. Cells’ proliferation rate was estimated directly with the development of growth curves and indirectly by measuring cell’s ability for novel DNA synthesis, as assessed by tritiated thymidine incorporation. Cell cycle analysis was performed by flow cytometry after labeling of the cells with propidium iodide or simultaneous labeling with propidium iodide and an antibody raised against the phopshorylated form of histone H3, which is a known mitotic marker. Protein expression or activation was examined by Western analysis, while for intracellular localization immunofluorescence experiments were performed. The participation of protein-targets in particular pathways was studied using specific pharmacological inhibitors, cell lines not expressing p53 or ATM kinase and a siRNA molecule for p53 gene silencing. Single cell gel electrophoresis (comet assay) was used in order to explore the possibility of DNA damage and cells’ repairing capacity was measured after transfection of the cells with a vector carrying a reporter gene which was previously damaged exogenously. Finally, senescent cells were characterized microscopically, with immunofluorescence experiments for bromodeoxyuridine incorporation and after SA β-gal staining for senescent cells. In the present thesis we showed that the physicochemical conditions of intervertebral disc’s extracellular environment affect the proliferation and the biochemical behavior of its cells. We found that hyperosmotic stress provokes a rapid p38-MAPK-dependent cell cycle arrest at the G2 phase and a delayed G0/G1 arrest regulated by p53. We revealed for the first time that high salinity causes DNA damage to disc cells which respond to this insult by activating a typical response to genotoxic stress that is enhanced under hypertonic conditions. We finally demonstrated that the continuing presence of high osmolality in the extracellular environment leads the cells to premature senescence in vitro.Η καλή λειτουργία των κυττάρων του μεσοσπονδύλιου δίσκου είναι απαραίτητη για τη διατήρηση της υγείας του ιστού. Δεδομένης της ιδιαιτερότητας των φυσικοχημικών συνθηκών που επικρατούν στο συγκεκριμένο ιστό σε σχέση με άλλους ιστούς (όξινο pH, χαμηλή συγκέντρωση οξυγόνου, έλλειψη θρεπτικών συστατικών και υψηλή ωσμωμοριακότητα) είναι σημαντική η βαθύτερη κατανόηση της φυσιολογίας και των αποκρίσεων των κυττάρων του δίσκου μέσα στο πλαίσιο του ιδιαίτερου εξωκυτταρικού τους μικροπεριβάλλοντος. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η διερεύνηση της επίδρασης των διαφόρων μορφών στρες που φυσιολογικά υφίσταται ο μεσοσπονδύλιος δίσκος στον πολλαπλασιασμό και τη γήρανση των κυττάρων του. Η εκτίμηση του ρυθμού πολλαπλασιασμού των κυττάρων έγινε άμεσα με καμπύλες ανάπτυξης και έμμεσα μέσω της ικανότητας των κυττάρων για σύνθεση νέου DNA, όπως αυτή υπολογίζεται με πειράματα ενσωμάτωσης τριτιωμένης θυμιδίνης. Η ανάλυση του κυτταρικού κύκλου έγινε με κυτταρομετρία ροής μετά από την επισήμανση των κυττάρων με ιωδιούχο προπίδιο ή την ταυτόχρονη επισήμανσή τους με ιωδιούχο προπίδιο και με αντίσωμα έναντι της φωσφορυλιωμένης μορφής της ιστόνης H3, το οποίο επισημαίνει ειδικά τα μιτωτικά κύτταρα. Η μελέτη της έκφρασης ή ενεργοποίησης των πρωτεϊνών έγινε με ανάλυση Western και ο ενδοκυτταρικός τους εντοπισμός με ανοσοφθορισμό. Η συμμετοχή των πρωτεϊνών-στόχων σε συγκεκριμένες κυτταρικές αποκρίσεις διερευνήθηκε με τη χρήση ειδικών φαρμακολογικών αναστολέων, κυτταρικών σειρών που δεν εκφράζουν την p53 ή την κινάση ATM και μικρών παρεμβαλλόμενων μορίων siRNA για την αποσιώπηση του γονιδίου της p53. Η πιθανότητα πρόκλησης βλαβών στο DNA των κυττάρων μελετήθηκε με ηλεκτροφόρηση ολόκληρων κυττάρων σε πήκτωμα αγαρόζης (comet assay), ενώ η ικανότητά τους για επιδιόρθωση εκτιμήθηκε μετά από επιμόλυνση των κυττάρων με πλασμίδιο που φέρει γονίδιο αναφοράς και στο οποίο είχαν προκληθεί προηγουμένως βλάβες εξωγενώς. Τέλος, ο χαρακτηρισμός των γηρασμένων κυττάρων έγινε με μικροσκοπική παρατήρηση, με πειράματα ενσωμάτωσης βρωμοδεοξυουριδίνης και με τη χρήση της ειδικής χρώσης γηρασμένων κυττάρων SA β-gal. Στην παρούσα διδακτορική διατριβή δείχθηκε ότι οι φυσικοχημικές συνθήκες που επικρατούν στο εξωκυτταρικό περιβάλλον του μεσοσπονδύλιου δίσκου επηρεάζουν τον πολλαπλασιασμό και τη βιοχημική συμπεριφορά των κυττάρων του. Ειδικότερα έγινε σαφές ότι το υπερωσμωτικό στρες προκαλεί μια ταχεία αναστολή του κυτταρικού κύκλου στη φάση G2 που εξαρτάται από την p38-MAPK και μια πιο καθυστερημένη αναστολή του κυτταρικού κύκλου στη φάση G0/G1, η οποία ρυθμίζεται από την p53. Για πρώτη φορά αποκαλύφθηκε η πρόκληση βλαβών στο DNA των κυττάρων αυτού του ιστού από την υψηλή αλατότητα αλλά και η ενισχυμένη ικανότητά τους για επιδιόρθωση σε υπερτονικό περιβάλλον μέσω ενός κλασικού μονοπατιού απόκρισης σε γενοτοξικό στρες. Τέλος, βρέθηκε ότι η συνεχής παρουσία υψηλής ωσμωμοριακότητας στο εξωκυτταρικό περιβάλλον οδηγεί σε πρόωρη κυτταρική γήρανση in vitro. Μελλοντικό στόχο αποτελεί η διερεύνηση των παραπάνω φαινομένων in vivo

Whole-Genome Sequence of the Cheese IsolateStreptococcus macedonicus679

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Bone turnover markers in gingival crevicular fluid and blood serum of patients with fixed orthodontic appliances.

AIM Bone remodelling can be followed through the bone turnover markers (BTMs). Aim of the present study was to record the fluctuation of an osteoclastic and an osteoblastic BTM [C-terminal telopeptide of type I collagen (CTX) and N-terminal pro-peptide of type I pro-collagen (PINP), respectively] in both the gingival crevicular fluid (GCF) and the serum of orthodontic patients before and after the initial application of orthodontic forces. MATERIALS AND METHODS Twenty-one Caucasian patients were prospectively evaluated. GCF and blood samples were collected in order to measure the selected biomarkers by ELISA at three time-points: exactly before, 5 days, and 14 days after bonding of the appliances. Standardized sample handling and patient preparation procedures were adopted in order to reduce pre-analytical variability. RESULTS GCF and serum CTX levels were found to be independent of age, although higher in the serum of female subjects. PINP levels were found higher in the serum of patients ≥25 years old, as well as in the GCF of males. A positive correlation between serum and GCF baseline PINP levels was observed. LIMITATIONS The effect of orthodontic treatment on bone remodelling might not be absolutely representative of the local bone microenvironment as the levels of the specific BTMs where measured within the GCF of the lower front teeth. CONCLUSIONS This is the first time PINP and CTX have been evaluated in the GCF and serum of orthodontic patients with fixed appliances. No statistically significant alterations of CTX and PINP levels in the GCF and the serum of patients were recorded over time during the initial stages of orthodontic treatment