Loss of the tumour suppressor LKB1/STK11 uncovers a leptin-mediated sensitivity mechanism to mitochondrial uncouplers for targeted cancer therapy.

Non-small cell lung cancer (NSCLC) constitutes one of the deadliest and most common malignancies. The LKB1/STK11 tumour suppressor is mutated in ∼ 30% of NSCLCs, typically lung adenocarcinomas (LUAD). We implemented zebrafish and human lung organoids as synergistic platforms to pre-clinically screen for metabolic compounds selectively targeting LKB1-deficient tumours. Interestingly, two kinase inhibitors, Piceatannol and Tyrphostin 23, appeared to exert synthetic lethality with LKB1 mutations. Although LKB1 loss alone accelerates energy expenditure, unexpectedly we find that it additionally alters regulation of the key energy homeostasis maintenance player leptin (LEP), further increasing the energetic burden and exposing a vulnerable point; acquired sensitivity to the identified compounds. We show that compound treatment stabilises Hypoxia-inducible factor 1-alpha (HIF1A) by antagonising Von Hippel-Lindau (VHL)-mediated HIF1A ubiquitination, driving LEP hyperactivation. Importantly, we demonstrate that sensitivity to piceatannol/tyrphostin 23 epistatically relies on a HIF1A-LEP-Uncoupling Protein 2 (UCP2) signaling axis lowering cellular energy beyond survival, in already challenged LKB1-deficient cells. Thus, we uncover a pivotal metabolic vulnerability of LKB1-deficient tumours, which may be therapeutically exploited using our identified compounds as mitochondrial uncouplers.

Design and Synthesis of Novel Antioxidant 2-Substituted-5,7,8-Trimethyl-1,4-Benzoxazine Hybrids: Effects on Young and Senescent Fibroblasts.

The exponential growth of the aged population worldwide is followed by an increase in the prevalence of age-related disorders. Oxidative stress plays central role in damage accumulation during ageing and cell senescence. Thus, a major target of today's anti-ageing research has been focused on antioxidants counteracting senescence. In the current work, six novel 5,7,8-trimethyl-1,4-benzoxazine/catechol or resorcinol hybrids were synthesized connected through a methoxymethyl-1,2,3-triazolyl or a 1,2,3-triazoly linker. The compounds were evaluated for their antioxidant capacity in a cell-free system and for their ability to reduce intracellular ROS levels in human skin fibroblasts, both young (early-passage) and senescent. The most efficient compounds were further tested in these cells for their ability to induce the expression of the gene heme oxygenase-1 (ho-1), known to regulate redox homeostasis, and cellular glutathione (GSH) levels. Overall, the two catechol derivatives were found to be more potent than the resorcinol analogues. Furthermore, these two derivatives were shown to act coordinately as radical scavengers, ROS inhibitors, ho-1 gene expression inducers, and GSH enhancers. Interestingly, one of the two catechol derivatives was also found to enhance human skin fibroblast viability. The properties of the synthesized compounds support their potential use in cosmetic applications, especially in products targeting skin ageing.

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.

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.

New long-standing metabolites of 17α-methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine.

Novel metabolites of the anabolic androgenic steroid 17α-methyltestosterone have been detected in HepG2 cell in vitro metabolic model and in human urine. Their detection was accomplished through targeted gas chromatography-(tandem) mass spectrometry analysis that has been based on microscale synthesized standards. The related synthesis and the gas chromatography-(tandem) mass spectrometry characterization of the analytical standards are described. All newly presented metabolites have a fully reduced steroid A-ring with either an 17,17-dimethyl-18-nor-Δ13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17-dimethyl-18-nor-Δ13 structure may be considered as side products of phase II metabolic sulfation of the 17ß-hydroxy group of methyltestosterone or its reduced tetrahydro-methyltestosterone metabolites 17α-methyl-5ß-androstane-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol that produce the known epimeric 17ß-methyl-5ß-androstane-3α,17α-diol and 17ß-methyl-5α-androstane-3α,17α-diol metabolites. The prospective of these new metabolites to increase detection time windows and improve identification was investigated by applying the World Anti-doping Agency TD2021IDCR criteria. The new metabolites, presented herein, complement the current knowledge on the 17α-methyltestosterone metabolism and in some cases can be used as additional long-term markers in the frame of sport doping drug testing.

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.

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.

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.

A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer.

BACKGROUND: Prostate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed. METHODS: Androgen-dependent (LNCaP) and -independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy. RESULTS: We demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression. CONCLUSIONS: We identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.

A Novel Experimental Rat Model for the In Vivo Assessment of Myocardial Ischemia Based on Single Photon Emission Computed Tomography.

BACKGROUND/AIM: Myocardial infarction, an acute medical situation with a high mortality rate worldwide, has been extensively studied in modern cardiovascular research, using different experimental models. However, a deep understanding of myocardial activity loss has not been fully investigated. We have developed a novel experimental rat model for noninvasive assessment of myocardial ischemia based on single photon emission computed tomography (SPECT/CT), in order to further understand and evaluate myocardial activity before and after surgical induction of myocardial ischemia. MATERIALS AND METHODS: Thirty adult female Wistar rats underwent open thoracotomy with (n=20) or without (n=10) surgical ligation of the left anterior descending coronary artery (LAD). The myocardial ischemia was confirmed with ECG and myocardial viability was evaluated via SPECT/CT at 7 days before as well as at 7 and 14 days post-surgery, after which animals were sacrificed and myocardial ischemic injury was further assessed histologically. RESULTS: All animals were evaluated with anatomical and functional criteria based on the SPECT/CT imaging results. A successful surgical technique causing ischemia and loss of myocardial function in all animals undergoing a LAD ligation was established. Furthermore, evaluation of the viable myocardium with SPECT/CT confirmed the reduction of functional myocardial cells of the left ventricle post-infarction, which was also documented histologically. CONCLUSION: Using our technique, the validity of this animal model to induce and evaluate myocardial ischemia was demonstrated. Our choice to apply SPECT-CT qualitative and quantitative evaluation of myocardial function leads to a new approach in experimentation with an anticipated significant impact in the ongoing cardiovascular laboratory research.

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.

Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation.

Although UVB radiation is mainly absorbed by the epidermis, ~5-10% of its photons reach and affect the upper part of the dermis. Physiologically relevant UVB doses, able to provoke erythema, induce apoptosis in human dermal fibroblasts in vitro, as well as in the dermis of SKH-1 mice. Given the sparse and even contradictory existing information on the effect of UVB radiation on dermal fibroblasts' viability, aim of this work was to unravel the crucial signaling pathways regulating the survival of UVB-treated human dermal fibroblasts. We found that UVB radiation immediately stimulates the phosphorylation of MAPK family members, as well as Akt, and is genotoxic leading to the delayed ATM-p53 axis activation. Akt phosphorylation after UVB radiation is EGFR-mediated and EGFR inhibition leads to a further decrease of viability, while the Akt activator SC79 rescues fibroblasts to an extent by a mechanism involving Nrf2 activation. The known Nrf2 activator sulforaphane also exerts a partial protective effect, although by acting in a distinct mechanism from SC79. On the other hand, inhibition of JNKs or of the ATM-p53 axis leads to a complete loss of viability after UVB irradiation. Interestingly, JNKs activation is necessary for p53 phosphorylation, while the ATM-p53 pathway is required for the long-term activation of JNKs and Akt, reassuring the protection from UVB. Although UVB radiation results in intense and prolonged increase of intracellular ROS levels, classical anti-oxidants, such as Trolox, are unable to affect Akt, JNKs, or p53 phosphorylation and to reverse the loss of fibroblasts' viability. Collectively, here we provide evidence that the main viability-regulating UVB-triggered biochemical pathways act synergistically towards the protection of human dermal fibroblasts, with EGFR/Akt and Nrf2 serving as auxiliary anti-apoptotic machineries, while JNKs/ATM-p53 activation and interplay being overriding and indispensable for the perpetuation of cellular defense and the maintenance of cell viability.

Identification of Novel Circular RNAs of the Human Protein Arginine Methyltransferase 1 (PRMT1) Gene, Expressed in Breast Cancer Cells.

Circular RNAs (circRNAs) constitute a type of RNA formed through back-splicing. In breast cancer, circRNAs are implicated in tumor onset and progression. Although histone methylation by PRMT1 is largely involved in breast cancer development and metastasis, the effect of circular transcripts deriving from this gene has not been examined. In this study, total RNA was extracted from four breast cancer cell lines and reversely transcribed using random hexamer primers. Next, first- and second-round PCRs were performed using gene-specific divergent primers. Sanger sequencing followed for the determination of the sequence of each novel PRMT1 circRNA. Lastly, bioinformatics analysis was conducted to predict the functions of the novel circRNAs. In total, nine novel circRNAs were identified, comprising both complete and truncated exons of the PRMT1 gene. Interestingly, we demonstrated that the back-splice junctions consist of novel splice sites of the PRMT1 exons. Moreover, the circRNA expression pattern differed among these four breast cancer cell lines. All the novel circRNAs are predicted to act as miRNA and/or protein sponges, while five circRNAs also possess an open reading frame. In summary, we described the complete sequence of nine novel circRNAs of the PRMT1 gene, comprising distinct back-splice junctions and probably having different molecular properties.

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.

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.

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.

Accumulation of zoledronic acid in rabbit intervertebral discs.

Low back pain is a major chronic musculoskeletal disorder, caused mainly due the degeneration of the intervertebral discs (IVDs). Bisphosphonates (BPs), like zoledronic acid (ZOL), are used in osteoporosis management; however, their accumulation in the IVDs and their physiological role has not been addressed so far. To this end, an SPE-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) analytical method, using on-cartridge derivatization, has been developed and validated. The analytical method presented excellent linearity (R2 > 0.992), high recoveries (67.8-82.6%), increased repeatability (0.5-9.9%) and low LOD values (21 ng g-1 in the case of IVDs) in all matrices studied. The injection of ZOL in a rabbit animal model resulted in rapid accumulation in blood plasma and the skin, followed by quick clearance. On the other hand, no ZOL was detected in nucleus pulposus, the core of the IVD, while in the peripheral annulus fibrosus a lower and delayed accumulation, as well as dispersal was found. These variations are most probably due to the avascular nature of IVD, allowing only the diffusion of small molecules in and out of the tissue, and/or to the unique physicochemical environment of IVDs. Finally, ZOL, at the concentrations found, did not affect cells' viability or the increase of reactive oxygen species (ROS).

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.