Biological Activities of Citrus-Derived Extracellular Vesicles on Human Cells: The Role of Preservation.

Extracellular vesicles (EVs) have been identified as important mediators for cell-to-cell communication. Citrus-based EVs in particular offer an excellent platform for nutraceutical delivery systems, as their endemic cargo includes micronutrients (e.g., ascorbic acid), which contribute to their antioxidant capacity. Despite being extensively investigated as to their therapeutic and diagnostic potential, their cargo is inherently unstable and thus directly affected by their storage and preservation. In this study, EVs were isolated from citrus fruit using tangential flow filtration and evaluated for their physicochemical characteristics, antioxidant activity and effects on human cells. To assess how their isolation and preservation methods affect these properties, the EVs were tested immediately after isolation (from fresh and freeze-thawed juices) or following freeze-drying. A measurable biological effect of cryoprotection on citrus-derived EVs was evident, whether during or after isolation. This was more pronounced in the cell-based assays, ranging from -4% to +32% in human skin fibroblast proliferation. Nevertheless, the effects on human cancer cells varied depending on the cell line. Although these results should be considered preliminary observations, subject to further investigation, it is safe to state that any type of preservation is expected to impact the EVs' biological activity.

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.

Galectin-3 interferes with tissue repair and promotes cardiac dysfunction and comorbidities in a genetic heart failure model.

Galectin-3, a biomarker for heart failure (HF), has been associated with myocardial fibrosis. However, its causal involvement in HF pathogenesis has been questioned in certain models of cardiac injury-induced HF. To address this, we used desmin-deficient mice (des-/-), a model of progressive HF characterized by cardiomyocyte death, spontaneous inflammatory responses sustaining fibrosis, and galectin-3 overexpression. Genetic ablation or pharmacological inhibition of galectin-3 led to improvement of cardiac function and adverse remodeling features including fibrosis. Over the course of development of des-/- cardiomyopathy, monitored for a period of 12 months, galectin-3 deficiency specifically ameliorated the decline in systolic function accompanying the acute inflammatory phase (4-week-old mice), whereas a more pronounced protective effect was observed in older mice, including the preservation of diastolic function. Interestingly, the cardiac repair activities during the early inflammatory phase were restored under galectin-3 deficiency by increasing the proliferation potential and decreasing apoptosis of fibroblasts, while galectin-3 absence modulated macrophage-fibroblast coupled functions and suppressed both pro-fibrotic activation of cardiac fibroblasts and pro-fibrotic gene expression in the des-/- heart. In addition, galectin-3 also affected the emphysema-like comorbid pathology observed in the des-/- mice, as its absence partially normalized lung compliance. Collectively galectin-3 was found to be causally involved in cardiac adverse remodeling, inflammation, and failure by affecting functions of cardiac fibroblasts and macrophages. In concordance with this role, the effectiveness of pharmacological inhibition in ameliorating cardiac pathology features establishes galectin-3 as a valid intervention target for HF, with additive benefits for treatment of associated comorbidities, such as pulmonary defects. Schematic illustrating top to bottom, the detrimental role of galectin-3 (Gal3) in heart failure progression: desmin deficiency-associated spontaneous myocardial inflammation accompanying cardiac cell death (reddish dashed border) is characterized by infiltration of macrophages (round cells) and up-regulation of Lgals3 (encoding secretable galectin-3, green) and detrimental macrophage-related genes (Ccr2 and Arg1). In this galectin-3-enriched milieu, the early up-regulation of profibrotic gene expression (Tgfb1, Acta2, Col1a1), in parallel to the suppression of proliferative activities and a potential of senescence induction by cardiac fibroblasts (spindle-like cells), collectively promote des-/- cardiac fibrosis and dysfunction establishing heart failure (left panel). Additionally, galectin-3+ macrophage-enrichment accompanies the development of emphysema-like lung comorbidities. In the absence of galectin-3 (right panel), the effect of macrophage-fibroblast dipole and associated events are modulated (grey color depicts reduced expression or activities) leading to attenuated cardiac pathology in the des-/-Lgals3-/- mice. Pulmonary comorbidities are also limited.

Biological Activities of Ceratonia siliqua Pod and Seed Extracts: A Comparative Analysis of Two Cretan Cultivars.

Ceratonia siliqua L., commonly known as the carob tree, appears in most Mediterranean countries, often cultivated for the collection of its fruits to be used as food for humans and animals. This study was aimed at the phytochemical characterization of two common Cretan C. siliqua cultivars and the biological evaluation of deseeded pod and seed extracts regarding their putative use in cosmetics. Gas and liquid chromatographic techniques were used to assess their essential oil, fatty acid, and carbohydrate profiles. Cell-free assays, including free-radical scavenging; the inhibition of tyrosinase and collagenase; the blocking of advanced glycation end product (AGE) formation; along with assays in human skin fibroblast cultures, i.e., reactive oxygen species suppression, glutathione stimulation, and protection from oxidative stress and from ultraviolet (UVB) radiation, were also used. Extracts from both cultivars were found to possess antioxidant capacity, tyrosinase- and collagenase-inhibitory activities, an ability to block glucose-induced AGEs, and in certain cases, UVB absorbance and photoprotective activities. Seed extracts were in general more active, while the use of 30% aqueous methanol seemed to be more efficient than n-hexane for extraction. Serial partition of the most active extracts resulted in fractions with enriched biological activities. These properties make Cretan carob extracts and their fractions suitable candidates for use in cosmetics.

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.

Cytotoxicity and estrogenicity of a novel 3-dimensional printed orthodontic aligner.

INTRODUCTION: Orthodontic aligners printed with in-office 3-dimensional (3D) procedures have been described, but no data on their biocompatibility exist. This study investigates the cytotoxicity and estrogenicity of a 3D-printed orthodontic aligner by assessing its biological and behavioral effects. METHODS: Ten sets of 1 type of aligner were immersed in sterile deionized water for 14 days, and the cytotoxicity and estrogenicity of released factors were assessed via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays on human gingival fibroblasts and the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231 breast cancer cell lines. 17ß-Estradiol and bisphenol-A were used as positive controls. The statistical analysis of data was performed with generalized linear models at a 0.05 level of significance. RESULTS: No signs of cytotoxicity were seen for the aligner samples for concentrations (v/v) of 20% (P = 0.32), 10% (P = 0.79), or 5% (P = 0.76). The antioxidant activity expressed as the capacity to reduce intracellular levels of reactive oxygen species was not affected in the aligner samples (P = 0.08). No significant estrogenicity was induced by the aligner samples compared with eluents from the negative control for both MCF-7 (P = 0.65) and MDA-MB-231 (P = 0.78). As expected, 17ß-Estradiol and bisphenol-A stimulated MCF-7 cell proliferation, whereas no effect was observed on MDA-MB-231 cells. CONCLUSIONS: In conclusion, if any factors were released during the 14-day aging of 3D-printed aligners in water, these were not found to be cytotoxic for human gingival fibroblasts and did not affect their intracellular reactive oxygen species levels. Moreover, no estrogenic effects of these putative eluates were observed based on an E-screen assay.

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.

Reacquisition of a spindle cell shape does not lead to the restoration of a youthful state in senescent human skin fibroblasts.

Senescent fibroblasts are characterized by their inability to proliferate and by a pro-inflammatory and catabolic secretory phenotype, which contributes to age-related pathologies. Furthermore, senescent fibroblasts when cultured under classical conditions in vitro are also characterized by striking morphological changes, i.e. they lose the youthful spindle-like appearance and become enlarged and flattened, while their nuclei from elliptical become oversized and highly lobulated. Knowing the strong relation between cell shape and function, we cultured human senescent fibroblasts on photolithographed Si/poly(vinyl alcohol) (PVA) micro-patterned surfaces in order to restore the classical spindle-like geometry and subsequently to investigate whether the changes in senescent cells' morphology are the cause of their functional alterations. Interestingly, under these conditions senescent cells' nuclei do not revert to the classical elliptical phenotype. Furthermore, enforced spindle-shaped senescent cells retained their deteriorated proliferative ability, and maintained the increased gene expression of the cell cycle inhibitors p16Ink4a and p21Waf1. In addition, Si/PVA-patterned-grown senescent fibroblasts preserved their senescence-associated phenotype, as evidenced by the overexpression of inflammatory and catabolic genes such as IL6, IL8, ICAM1 and MMP1 and MMP9 respectively, which was further manifested by an intense downregulation of fibroblasts' most abundant extracellular matrix component Col1A, compared to their young counterparts. These data indicate that the restoration of the spindle-like shape in senescent human fibroblasts is not able to directly alter major functional traits and restore the youthful phenotype.

Short- and long-term treatment with TNF-α inhibits the induction of osteoblastic differentiation in cyclic tensile-stretched periodontal ligament fibroblasts.

BACKGROUND: Cyclic tensile stretching (CTS) induces osteoblastic differentiation of periodontal ligament fibroblasts (PDLF). On the other hand, increased concentrations of tumour necrosis factor-α (TNF-α) are found in inflammatory conditions, leading to periodontal disease and tooth loss. Accordingly, our aim was to investigate the short- and long-term effect of TNF-α on the response of human PDLF to CTS and its implication on osteoblastic differentiation. METHODS: PDLF were either pre-incubated for 4 hours or were repeatedly exposed to TNF-α for up to 50 days and then subjected to CTS. Gene expression was determined by quantitative real-time polymerase chain reaction. Activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and cell proliferation by bromodeoxyuridine incorporation. Intracellular reactive oxygen species were determined by the 2´, 7´-dichlorofluorescein-diacetate assay and osteoblastic differentiation by Alizarin Red-S staining after an osteo-inductive period of 21 days. RESULTS: CTS of PDLF induced an immediate upregulation of the c-fos transcription factor and, further downstream the overexpression of alkaline phosphatase and osteopontin, two major osteoblast marker genes. A 4-hour pre-incubation with TNF-α repressed these effects. Similarly, long-term propagation of PDLF along with TNF-α diminished their osteoblastic differentiation capacity and suppressed cells' CTS-elicited responses. The observed phenomena were not linked with TNF-α-induced premature senescence or oxidative stress. While CTS induced the activation of MAPKs, involved in mechanotransduction, TNF-α treatment provoked a small delay in the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. CONCLUSION: Increased concentrations of TNF-α, such as those recorded in many inflammatory diseases, suppress PDLF's immediate responses to mechanical forces compromising their osteoblastic differentiation potential, possibly leading to tissue's impaired homeostasis.

Effect of hyperglycaemic conditions on the response of human periodontal ligament fibroblasts to mechanical stretching.

OBJECTIVES: The aim of the present study was to investigate the impact of high glucose concentration on the response of human periodontal ligament fibroblasts (PDLFs) to cyclic tensile strain. MATERIALS AND METHODS: Human PDLFs were incubated under normal or high glucose conditions, and then were subjected to cyclic tensile stretching (8 per cent extension, 1 Hz). Gene expression was determined by quantitative real-time polymerase chain reaction. Intracellular reactive oxygen species (ROS) were determined by the 2',7'-dichlorofluorescein-diacetate assay, activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and osteoblastic differentiation was estimated with Alizarin Red-S staining. RESULTS: Cyclic tensile stretching of PDLF leads to an immediate activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as to the increased expression of the transcription factor c-fos, known to regulate many osteogenesis-related genes. At later time points, the alkaline phosphatase and osteopontin genes were also upregulated. Hyperglycaemic conditions inhibited these effects. High glucose conditions were unable to increase ROS levels, but they increased the medium's osmolality. Finally, increase of osmolality mimics the inhibitory effect of hyperglycaemia on MAPK activation, c-fos and osteoblast-specific gene markers' upregulation, as well as osteogenic differentiation capacity. CONCLUSION: Our findings indicate that under high glucose conditions, human PDLFs fail to adequately respond to mechanical deformation, while their strain-elicited osteoblast differentiation ability is deteriorated. The aforementioned effects are most probably mediated by the increased osmolality under hyperglycaemic conditions.

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.

Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should be 103 min-1 g-1 FAE for kcat and mM-1 min-1 g-1 FAE for kcat/Km. This correction does not affect any conclusions drawn within the article.

In Vitro Assessment of Cytotoxicity and Estrogenicity of Vivera® Retainers.

AIM: To investigate the cytotoxicity and estrogenicity of Vivera® retainers by assessing their biological behavioral effects as-received from the manufacturer and after retrieved from patients. MATERIALS AND METHODS: In this, in vitro investigation six sets (maxillary and mandibular) of Vivera® retainers, three as received and three retrieved after four weeks of use by patients of an orthodontic postgraduate clinic, were immersed in the normal saline solution for 14 days following different modes of sterilization. The estrogenicity assays involved two cell lines, namely the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231. Following a 6 day incubation with the solutions to be tested, at concentrations varying from 5% to 20% v/v in medium supplemented with 2% fetal calf serum devoid of endogenous estrogens, estrogenicity was assessed by cell counting; p-Estradiol was used as positive control. The statistical analysis of data was performed with two-way analysis of variance (ANOVA) with appliance and concentration as predictors. Differences were further investigated with the Tukey multiple comparison tests at the 0.05 level of significance. RESULTS: No significant MCF-7 proliferation was induced by the three samples compared either to the eluents from as-received retainers or to the negative control. As expected, p-estradiol induced a potent stimulation of MCF-7 cell proliferation, while no effect was observed on MDA-MB-231 cells. CONCLUSION: Under the conditions of this experiment eluents of as-received and retrieved Vivera® retainers did not seem to exhibit xenoestrogenic activity. CLINICAL SIGNIFICANCE: Vivera® retainers can be used as part-time removable oral appliances following the manufacturer's instructions.

Antiproliferative Effects of Red and White Wine Extracts in PC-3 Prostate Cancer Cells.

Experimental and epidemiological studies have shown that antioxidant polyphenols can act as chemopreventive agents against prostate cancer. Cabernet Sauvignon and Rombola wine were extracted in order to obtain fractions containing different classes of compounds. All extracts inhibited the androgen-insensitive human prostate cancer cells (PC-3) proliferation in a dose-dependent manner. The most potent compounds were selected to be further tested.Treatment of PC-3 cells with the selected wine extracts marginally increased the cell distribution in S phase, while producing a remarkable induction of autophagy. Finally, the levels of glutathione along with the concentration of hydrogen peroxide and nitrogen oxide were modulated in the treated cells. Herein, we show that red and wine extracts have direct effects on the proliferation, survival, oxidative status, and the induction of autophagy of PC-3 cells. Our data may have important implications for the design of a more effective adjuvant treatment for prostate cancer patients.

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 µM for PFA, 329.9 µM for FA). PFA was not cytotoxic at 0.8-100 µM (IC50 220.23 µM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 µM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

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.