Senescence-associated alterations in the extracellular matrix: deciphering their role in the regulation of cellular function.

The extracellular matrix (ECM) is a dynamic structural network that provides a physical scaffolding, as well as biochemical factors that maintain normal tissue homeostasis and thus its disruption is implicated in many pathological conditions. On the other hand, senescent cells express a particular secretory phenotype, affecting the composition and organization of the surrounding ECM and modulating their microenvironment. As accumulation of senescent cells may be linked to the manifestation of several age-related conditions, senescence-associated ECM alterations may serve as targets for novel anti-aging treatment modalities. Here, we will review characteristic changes in the ECM elicited by cellular senescence and we will discuss the complex interplay between ECM and senescent cells, in relation to normal aging and selected age-associated pathologies.

Decreased differentiation capacity and altered expression of extracellular matrix components in irradiation-mediated senescent human breast adipose-derived stem cells.

Radiotherapy is widely used for the treatment of breast cancer. However, we have shown that ionizing radiation can provoke premature senescence in breast stromal cells. In particular, breast stromal fibroblasts can become senescent after irradiation both in vitro and in vivo and they express an inflammatory phenotype and an altered profile of extracellular matrix components, thus facilitating tumor progression. Adipose-derived stem cells (ASCs) represent another major component of the breast tissue stroma. They are multipotent cells and due to their ability to differentiate in multiple cell lineages they play an important role in tissue maintenance and repair in normal and pathologic conditions. Here, we investigated the characteristics of human breast ASCs that became senescent prematurely after their exposure to ionizing radiation. We found decreased expression levels of the specific mesenchymal cell surface markers CD105, CD73, CD44, and CD90. In parallel, we demonstrated a significantly reduced expression of transcription factors regulating osteogenic (i.e., RUNX2), adipogenic (i.e., PPARγ), and chondrogenic (i.e., SOX9) differentiation; this was followed by an analogous reduction in their differentiation capacity. Furthermore, they overexpress inflammatory markers, that is, IL-6, IL-8, and ICAM-1, and a catabolic phenotype, marked by the reduction of collagen type I and the increase of MMP-1 and MMP-13 expression. Finally, we detected changes in proteoglycan expression, for example, the upregulation of syndecan 1 and syndecan 4 and the downregulation of decorin. Notably, all these alterations, when observed in the breast stroma, represent poor prognostic factors for tumor development. In conclusion, we showed that ionizing radiation-mediated prematurely senescent human breast ASCs have a decreased differentiation potential and express specific changes adding to the formation of a permissive environment for tumor growth.

In Vitro Virulence Potential, Surface Attachment, and Transcriptional Response of Sublethally Injured Listeria monocytogenes following Exposure to Peracetic Acid.

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes (motB and flaA) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA. Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

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.

The role of senescence in cancer development.

While research on cancer development is traditionally focusing mainly on the neoplastic cell per se, nowadays the role of tumor stroma in this process is indisputable. The stroma - mainly composed of extracellular matrix (ECM) - is a source of mediators and signals originating from heterotypic cell-cell and cell-matrix interactions that steer the progression of the disease in a context- and a cancer type-dependent manner. With advancing age the stroma exhibits alterations, important being the accumulation of senescent cells. Senescence is often triggered by exogenous stresses, including genotoxic anticancer treatment modalities (such as chemotherapy or radiotherapy) and is manifested as an inhibition of cell proliferation, ascribing to cellular senescence the role of a potent antitumor barrier. On the other hand, senescent cells, through their specific senescence-associated secretory phenotype (SASP) - comprising cytokines, growth factors, ECM components and ECM-degrading enzymes - can establish an immunosuppressive, inflammatory and catabolic microenvironment that may stimulate tumor growth and metastasis. Given that the persistent presence of senescent cells could prove detrimental for tissue homeostasis, inclusion of a senotherapeutic arm in novel anticancer approaches seems compulsory.

Differential Modulation of Listeria monocytogenes Fitness, In Vitro Virulence, and Transcription of Virulence-Associated Genes in Response to the Presence of Different Microorganisms.

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCEListeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.

Long-term exposure to TNF-α leads human skin fibroblasts to a p38 MAPK- and ROS-mediated premature senescence.

Tumor necrosis factor α (TNF-α) is an inflammatory mediator overexpressed in the skin as a response to ultraviolet radiation, as well as in chronic non-healing wounds. On the other hand, senescent fibroblasts have been shown to accumulate in the skin under these stressful conditions. Accordingly, here we assessed the putative implication of TNF-α in the induction of premature senescence of human adult dermal fibroblasts. We showed that TNF-α led to a rapid transient p38 MAPK activation, while elevation of reactive oxygen species (ROS) only occurred after a chronic exposure to TNF-α. Furthermore, in contrast to the majority of previous reports using various cell models and experimental settings, it was a long-term treatment with TNF-α that resulted in the premature senescence of human dermal fibroblasts, as shown by the reduced proliferative potential and the increased senescence associated ß-galactosidase staining of the cells. TNF-α-senescent cells displayed a permanent phosphorylation of p38 MAPK and an inflammatory and catabolic phenotype. Increased ROS levels were also observed, possibly attributed to the weakened anti-oxidative response evidenced by the underexpression of the Nrf2-regulated genes encoding HO-1 and NQO1. These traits and the overall senescent phenotype were significantly reversed using the known anti-oxidant N-acetyl-L-cysteine or a specific p38 MAPK inhibitor, suggesting the participation of oxidative stress and of the p38 MAPK pathway in TNF-α-triggered premature senescence. Even more, the observed blockade of ROS accumulation in senescent skin fibroblasts by p38 MAPK inhibition indicates a possible link between these two separate events during the manifestation of TNF-α-induced senescence.

Survival, Intestinal Mucosa Adhesion, and Immunomodulatory Potential of Lactobacillus plantarum Strains.

Survival during transit through the gastrointestinal track, intestinal mucosa adhesion, and a potential immunomodulatory effect of Lactobacillus plantarum strains 2035 and ACA-DC 2640 were investigated in a rat model. According to microbiological and multiplex PCR analysis, both strains were detected in feces 24 h after either single-dose or daily administration for 7 days. Intestinal mucosa adhesion of L. plantarum 2035 was noted in the large intestine at 24 h after single-dose administration, while it was not detected at 48 h. Daily dosing, prolonged detection of the strain up to 48 h post-administration, and expanded adhesion to the small intestine. Adhesion of L. plantarum ACA-DC 2640 to the intestinal mucosa after single-dose administration was prolonged and more extended compared to L. plantarum 2035. Daily dosing increased both the levels and the rate of positive cultures of the strains compared to those of the single-dose scheme. In addition, both strains increased total IgG while decreased IgM and IgA serum levels. In conclusion, L. plantarum 2035 and L. plantarum ACA-DC 2640 survived transit through the gastrointestinal track, exhibited transient distinct adhesion to the intestinal mucosa and modulated the systemic immune response.

Human nucleus pulposus intervertebral disc cells becoming senescent using different treatments exhibit a similar transcriptional profile of catabolic and inflammatory genes.

PURPOSE: Chronic low back pain has been associated with intervertebral disc (IVD) degeneration, which is characterized by the accumulation of extracellular matrix (ECM)-degrading proteases and inflammatory molecules in the degenerate tissue. IVD degeneration could be the outcome of natural organismal ageing and/or of the exposure of the disc to cumulative stressful environmental stimuli and is accompanied by an increased population of senescent cells in the tissue. On the other hand, senescent cells are known to secrete proteolytic enzymes and inflammatory molecules, which can contribute to ECM catabolism. The aim of this study was to investigate the transcriptional profile of selected metalloproteinases (MMPs) and inflammatory mediators in human nucleus pulposus IVD cells that became senescent using three different approaches: serial subculturing, exposure to ionizing radiation and p16INK4a overexpression. METHODS: Gene expression was assessed using quantitative RT-PCR and protein levels were determined by western blot analysis. The proliferative potential of the cells, as well as the percentage of senescent cells in the population were estimated by nuclear BrdU incorporation and by senescence-associated ß galactosidase staining, respectively. RESULTS: All senescent cells showed a similar regulation of MMP-1, -2, -3, -9, interleukin (IL) 6, IL8 and interferon γ at the level of transcription, with only some quantitative differentiations observed in p16INK4a-overexpressing cells. CONCLUSIONS: Data described here suggest that senescent cells may have similar functions in IVD homeostasis, irrespective of the origin of senescence induction.

Monitoring Autophagy Immunohistochemically and Ultrastructurally during Human Head and Neck Carcinogenesis. Relationship with the DNA Damage Response Pathway.

Autophagy is a catabolic process that preserves cellular homeostasis. Its exact role during carcinogenesis is not completely defined. Specifically in head and neck cancer, such information from clinical settings that comprise the whole spectrum of human carcinogenesis is very limited. Towards this direction, we examined the in situ status of the autophagy-related factors, Beclin-1, microtubule-associated protein 1 light chain 3, member B (LC3B) and sequestosome 1/p62 (p62) in clinical material covering all histopathological stages of human head and neck carcinogenesis. This material is unique as each panel of lesions is derived from the same patient and moreover we have previously assessed it for the DNA damage response (DDR) activation status. Since Beclin-1, LC3B and p62 reflect the nucleation, elongation and degradation stages of autophagy, respectively, their combined immunohistochemical (IHC) expression profiles could grossly mirror the autophagic flux. This experimental approach was further corroborated by ultrastructural analysis, applying transmission electron microscopy (TEM). The observed Beclin-1/LC3B/p62 IHC patterns, obtained from serial sections analysis, along with TEM findings are suggestive of a declined authophagic activity in preneoplastic lesions that was restored in full blown cancers. Correlating these findings with DDR status in the same pathological stages are indicative of: (i) an antitumor function of autophagy in support to that of DDR, possibly through energy deprivation in preneoplastic stages, thus preventing incipient cancer cells from evolving; and (ii) a tumor-supporting role in the cancerous stage.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells.

Various Listeria monocytogenes strains may contaminate a single food product, potentially resulting in simultaneous exposure of consumers to multiple strains. However, due to bias in strain recovery, L. monocytogenes strains isolated from foods by selective enrichment (SE) might not always represent those that can better survive the immune system of a patient. We investigated the effect of cocultivation in tryptic soy broth with 0.6% yeast extract (TSB-Y) at 10°C for 8 days on (i) the detection of L. monocytogenes strains during SE with the ISO 11290-1:1996/Amd 1:2004 protocol and (ii) the in vitro virulence of strains toward the Caco-2 human colon epithelial cancer cell line following exposure to simulated gastric fluid (SGF; pH 2.0)-HCl (37°C). We determined whether the strains which were favored by SE would be effective competitors under the conditions of challenges related to gastrointestinal passage of the pathogen. Interstrain competition of L. monocytogenes in TSB-Y determined the relative population of each strain at the beginning of SE. This in turn impacted the outcome of SE (i.e., favoring survival of competitors with better fitness) and the levels exposed subsequently to SGF. However, strong growth competitors could be outcompeted after SGF exposure and infection of Caco-2 cells by strains outgrown in TSB-Y and underdetected (or even missed) during enrichment. Our data demonstrate a preferential selection of certain L. monocytogenes strains during enrichments, often not reflecting a selective advantage of strains during infection. These findings highlight a noteworthy scenario associated with the difficulty of matching the source of infection (food) with the L. monocytogenes isolate appearing to be the causative agent during listeriosis outbreak investigations.IMPORTANCE This report is relevant to understanding the processes involved in selection and prevalence of certain L. monocytogenes strains in different environments (i.e., foods or sites of humans exposed to the pathogen). It highlights the occurrence of multiple strains in the same food as an important aspect contributing to mismatches between clinical isolates and infection sources during listeriosis outbreak investigations.

The differential proliferative response of fetal and adult human skin fibroblasts to TGF-ß is retained when cultured in the presence of fibronectin or collagen.

BACKGROUND: Transforming growth factor-ß is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-ß inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-ß on fetal and adult cells. METHODS: Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-ß was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. RESULTS: Fetal skin fibroblast-proliferation was inhibited by TGF-ß, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-ß on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. CONCLUSIONS: Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-ß when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. GENERAL SIGNIFICANCE: The interplay between TGF-ß and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Deficiency in the α1 subunit of Na+/K+-ATPase enhances the anti-proliferative effect of high osmolality in nucleus pulposus intervertebral disc cells.

Intervertebral disc cells are constantly exposed to a hyperosmotic environment. Among cellular responses towards this stress is the inhibition of proliferation through the activation of p38 MAPK and p53. In an effort to further elucidate the biochemical pathways triggered by hyperosmotic stress, we assessed the high osmolality-induced transcriptional changes of bovine nucleus pulposus cells using whole-genome arrays. A 5- and a 24-h hyperosmotic treatment led to the differential expression of >100 and >200 genes, respectively, including nine genes encoding transporters (SLC4A11, SLC5A3, ATP1A1, SLC38A2, KCNK17, KCTD20, KCTD11, SLC7A5, and CLCA2). Differences in the transcriptional profile of these selected genes, as indicated by the microarrays experiments, were validated by qRT-PCR in 2D and 3D cell cultures, under hyperosmolar salt and sorbitol conditions, revealing the presence of a common triggering signal for osmotic adaptation. The key signaling molecules p38 MAPK and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters. Finally, siRNA-mediated knocking-down of each one of the three transporters with the highest and sustained over-expression (i.e., SLC4A11, SLC5A3, and ATP1A1) had a distinct outcome on the transcriptional profile of the other transporters, on p38 MAPK and p53 phosphorylation and consequently on cell cycle progression. The inhibition of ATP1A1 had the most prominent effect on the transcription of the rest of the transporters and was found to enhance the anti-proliferative effect of hyperosmotic conditions through an increased G2/M cell cycle block, ascribing to this pump a central role in the osmoregulatory response of nucleus pulposus cells.

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.

Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level.

PURPOSE: Mechanical loading represents an integral part of intervertebral disc (IVD) homeostasis. This review aims to summarise recent knowledge on the effects of mechanical loads on the IVD and the disc cells, taking into consideration the changes that IVDs undergo during ageing and degeneration, from the macroscopic to the cellular and subcellular level. METHODS: Non-systematic literature review. RESULTS: Several scientific papers investigated the external loads that act on the spine and the resulting stresses inside the IVD, which contribute to estimate the mechanical stimuli that influence the cells that are embedded within the disc matrix. As disc cell responses are also influenced by their biochemical environment, recent papers addressed the role that degradation pathways play in the regulation of (1) cell viability, proliferation and differentiation and (2) matrix production and turnover. Special emphasis was put on the intracellular-signalling pathways, as mechanotransduction pathways play an important role in the maintenance of normal disc metabolism and in disc degenerative pathways. CONCLUSIONS: Disc cells are exposed to a wide range of mechanical loads, and the biochemical environment influences their responses. Degeneration-associated alterations of the disc matrix change the biochemical environment of disc cells and also the mechanical properties of the disc matrix. Recent studies indicate that these factors interact and regulate disc matrix turnover.

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.

Synthesis, cytotoxic activity evaluation and mechanistic investigation of novel 3,7-diarylsubstituted 6-azaindoles.

A number of new disubstituted 6-azaindoles have been designed and synthesized bearing a crucial structural modification in respect to an analogous antiproliferative hit compound. The synthesis was performed using 2-amino-3-nitro-4-picoline, that was suitably modified and converted to 7-chloro-3-iodo-6-azaindole and this central scaffold was used for successive Suzuki-type couplings, to result in the target compounds. The evaluation of the cytotoxic activity was performed against four human cancer cell lines, as well as a normal human fibroblast strain. Certain compounds possessed strong anticancer activity without affecting normal cells. At subcytotoxic concentrations for cancer cells, these compounds displayed an anti-proliferative effect by arresting the cells at the G2/M phase of the cell cycle, which could be associated with the observed decrease in the phosphorylation levels of the MEK1- ERK1/2 pathway and/or the activation of the p53-p21WAF1 axis.

Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

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

The species Streptococcus macedonicus is associated with the food environment, especially with fermented dairy products. Here we present the complete 2.1-Mb genome sequence of strain ACA-DC 198, which was isolated from naturally fermented Greek kasseri cheese.

Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea.

Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration.