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SummaryBackgroundCyclic 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.MethodsPDLF 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.ResultsCTS 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.ConclusionIncreased 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.

Simple Summary Ionizing radiation (a typical remedy for breast cancer) results in the premature senescence of the adjacent to the neoplastic cells stromal fibroblasts. Here, we showed that these senescent fibroblasts are characterized by the down-regulation of the small leucine-rich proteoglycan decorin, a poor prognostic factor for the progression of the disease. Decorin down-regulation is mediated by secreted growth factors in an autocrine and paracrine (due to the interaction with breast cancer cells) manner, with bFGF and VEGF being the key players of this regulation in young and senescent breast stromal fibroblasts. Autophagy activation increases decorin mRNA levels, indicating that impaired autophagy is implicated in the reduction in decorin in this cell model. Decorin down-regulation acts additively to the already tumor-promoting phenotype of ionizing radiation-induced prematurely senescent human stromal fibroblasts, confirming that stromal senescence is a side-effect of radiotherapy that should be taken into account in the design of anticancer treatments. Abstract Down-regulation of the small leucine-rich proteoglycan decorin in the stroma is considered a poor prognostic factor for breast cancer progression. Ionizing radiation, an established treatment for breast cancer, provokes the premature senescence of the adjacent to the tumor stromal fibroblasts. Here, we showed that senescent human breast stromal fibroblasts are characterized by the down-regulation of decorin at the mRNA and protein level, as well as by its decreased deposition in the pericellular extracellular matrix in vitro. Senescence-associated decorin down-regulation is a long-lasting process rather than an immediate response to γ-irradiation. Growth factors were demonstrated to participate in an autocrine manner in decorin down-regulation, with bFGF and VEGF being the critical mediators of the phenomenon. Autophagy inhibition by chloroquine reduced decorin mRNA levels, while autophagy activation using the mTOR inhibitor rapamycin enhanced decorin transcription. Interestingly, the secretome from a series of both untreated and irradiated human breast cancer cell lines with different molecular profiles inhibited decorin expression in young and senescent stromal fibroblasts, which was annulled by SU5402, a bFGF and VEGF inhibitor. The novel phenotypic trait of senescent human breast stromal fibroblasts revealed here is added to their already described cancer-promoting role via the formation of a tumor-permissive environment.

The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferulate (VFA) with Larabinose in detergentless microemulsions. The effect of parameters such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation was investigated. FaeA1 offered the highest transesterification yield (52.2 ± 4.3%) after 8 h of incubation at 50 °C using 80 mM VFA, 55 mM L-arabinose and 0.02 mg FAE mL−1 in a mixture comprising of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. The ability of L-arabinose ferulate (AFA) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was significant (IC50 386.5 μM). AFA was not cytotoxic even at high concentrations (1 mM) however was found to be pro-oxidant at concentrations higher than 20 μM when the antioxidant activity was determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in human skin fibroblasts.

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.

Summary 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.

The genus Strychnos (Loganiaceae) is well-known as a rich source of various bioactive metabolites. In continuation of our phytochemical studies on plants from Amazonia, we examined Strychnosaff. darienensis, collected in Peru. This species has been traditionally used in South America and is still presently used as a drug by the Yanesha tribe in Peru. Phytochemical investigation of this plant led to the isolation and structure elucidation by &Nu, uclear&Mu, agnetic Resonance and High Resolution Mass Spectroscopy of 14 compounds that belong to the categories of phenolic acids [p-hydroxybenzoic acid (1) and vanillic acid (2)], flavonoids [luteolin, (3),3-O-methyl quercetin (4), strychnobiflavone (5), minaxin (6) and 3&rsquo, 4&rsquo, 7-trihydroxy-flavone (7)], lignans [syringaresinol-&beta, D-glucoside (8), balanophonin (9) and ficusal (10)] and alkaloids [venoterpine (11), 11-methoxyhenningsamine (12), diaboline (13) and 11-methoxy diaboline (14)]. The isolated flavonoids&mdash, a class known for its anti-aging activities&mdash, were further evaluated for their biological activities on normal human skin fibroblasts. Among them, only (6), and to a lesser extent (7), exhibited cytotoxicity at 100 &micro, g/ml. All five flavonoids suppressed intracellularreactive oxygen species (ROS) levels, either basal or following stimulation with hydrogen peroxide or both. Moreover, luteolin and strychnobiflavone protected skin fibroblasts against ultraviolet (UV)-irradiation-induced cell death. The isolated flavonoids could prove useful bioactive ingredients in the cosmetic industry.

Objective: To compare the mechanotransduction caused by cyclic and static mechanical strains in human periodontal ligament fibroblasts (hPDLFs) cultured under identical conditions. Materials and methods: hPDLFs, originating from the same donors, were exposed either to cyclic or to static tensile strain using specially designed devices and under identical culture conditions. Activation of all members of mitogen-activated protein kinases (MAPKs) was monitored by western immunoblot analysis. Expression levels of immediate/early genes c-fos and c-jun were assessed with quantitative real-time polymerase chain reaction. Results: Time course experiments revealed that both types of stresses activate the three members of MAPK, that is ERK, p38, and JNK, with cyclic stress exhibiting a slightly more extended activation. Further downstream, both stresses upregulate the immediate/early genes c-fos and c-jun , encoding components of the activator protein-1 (AP-1), a key transcription factor in osteoblastic differentiation; again cyclic strain provokes a more intense upregulation. Six hours after the application of both strains, MAPK activation and gene expression return to basal levels. Finally, cells exposed to cyclic stress for longer periods are distributed approximately perpendicular to the axis of the applied strain, whereas cells exposed to static loading remain in a random orientation in culture. Conclusion: The findings of the present study indicate similar, although not identical, immediate/early responses of hPDLs to cyclic and static stretching, with cyclic strain provoking a more intense adaptive response of these cells to mechanical deformation.

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.

Cellular senescence, being the result of serial subculturing or of exogenous stresses, is considered to be a potent anticancer mechanism. However, it has been proposed that senescent cells may enhance the growth of adjacent malignant epithelial cells. On the other hand, exposure of tumors to repeated low doses of γ-irradiation is a common treatment regime. Nevertheless, γ-irradiation also affects the neighboring stromal cells and the interaction of the latter with cancer cells. Accordingly, in this study, we have exposed confluent cultures of human lung fibroblasts to repeated subcytotoxic doses of 4 Gy of γ-irradiation. We found that a single dose immediately activates a DNA damage response, leading to an intense, but reversible, cell cycle arrest. After a series of doses (total dose approximately 50 Gy) cellular senescence was accelerated, as shown by permanent growth arrest and the upregulation of specific biochemical and morphological senescence-associated markers. This process was found to be p53-dependent. Next, we studied the effect of these prematurely senescent cells on the growth of human malignant lung cell lines (A549 and H1299) and found that the presence of irradiation-mediated senescent cells strongly enhances the growth of these cancer cells in vitro and in immunocompromised (SCID) mice in vivo. This effect seems not to be related to an induction of epithelial-to-mesenchymal transdifferentiation but, to a significant extent, to the increased expression of matrix metalloproteases (MMPs), as a specific MMP inhibitor significantly restrains the growth of cancer in the presence of senescent fibroblasts. These findings indicate that lung fibroblasts that become senescent after ionizing radiation may contribute to lung cancer progression.