Activity and Selectivity of Novel Chemical Metallic Complexes with Potential Anticancer Effects on Melanoma Cells.

Human malignant melanoma cells from lymph node metastatic site (MeWo) were selected for testing several synthesized and purified silver(I) and gold(I) complexes stabilized by unsymmetrically substituted N-heterocyclic carbene (NHC) ligands, called L20 (N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide) and M1 (4,5-dichloro, N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide), having halogenide (Cl- or I-) or aminoacyl (Gly=N-(tert-Butoxycarbonyl)glycinate or Phe=(S)-N-(tert-Butoxycarbonyl)phenylalaninate) counterion. For AgL20, AuL20, AgM1 and AuM1, the Half-Maximal Inhibitory Concentration (IC50) values were measured, and all complexes seemed to reduce cell viability more effectively than Cisplatin, selected as control. The complex named AuM1 was the most active just after 8 h of treatment at 5 µM, identified as effective growth inhibition concentration. AuM1 also showed a linear dose and time-dependent effect. Moreover, AuM1 and AgM1 modified the phosphorylation levels of proteins associated with DNA lesions (H2AX) and cell cycle progression (ERK). Further screening of complex aminoacyl derivatives indicated that the most powerful were those indicated with the acronyms: GlyAg, PheAg, AgL20Gly, AgM1Gly, AuM1Gly, AgL20Phe, AgM1Phe, AuM1Phe. Indeed, the presence of Boc-Glycine (Gly) and Boc-L-Phenylalanine (Phe) showed an improved efficacy of Ag main complexes, as well as that of AuM1 derivatives. Selectivity was further checked on a non-cancerous cell line, a spontaneously transformed aneuploid immortal keratinocyte from adult human skin (HaCaT). In such a case, AuM1 and PheAg complexes resulted as the most selective allowing HaCaT viability at 70 and 40%, respectively, after 48 h of treatment at 5 µM. The same complexes tested on 3D MeWo static culture induced partial spheroid disaggregation after 24 h of culture, with almost half of the cells dead.

Pleiotropic Outcomes of Glyphosate Exposure: From Organ Damage to Effects on Inflammation, Cancer, Reproduction and Development.

Glyphosate is widely used worldwide as a potent herbicide. Due to its ubiquitous use, it is detectable in air, water and foodstuffs and can accumulate in human biological fluids and tissues representing a severe human health risk. In plants, glyphosate acts as an inhibitor of the shikimate pathway, which is absent in vertebrates. Due to this, international scientific authorities have long-considered glyphosate as a compound that has no or weak toxicity in humans. However, increasing evidence has highlighted the toxicity of glyphosate and its formulations in animals and human cells and tissues. Thus, despite the extension of the authorization of the use of glyphosate in Europe until 2022, several countries have begun to take precautionary measures to reduce its diffusion. Glyphosate has been detected in urine, blood and maternal milk and has been found to induce the generation of reactive oxygen species (ROS) and several cytotoxic and genotoxic effects in vitro and in animal models directly or indirectly through its metabolite, aminomethylphosphonic acid (AMPA). This review aims to summarize the more relevant findings on the biological effects and underlying molecular mechanisms of glyphosate, with a particular focus on glyphosate's potential to induce inflammation, DNA damage and alterations in gene expression profiles as well as adverse effects on reproduction and development.

The Complex Interplay between Endocannabinoid System and the Estrogen System in Central Nervous System and Periphery.

The endocannabinoid system (ECS) is a lipid cell signaling system involved in the physiology and homeostasis of the brain and peripheral tissues. Synaptic plasticity, neuroendocrine functions, reproduction, and immune response among others all require the activity of functional ECS, with the onset of disease in case of ECS impairment. Estrogens, classically considered as female steroid hormones, regulate growth, differentiation, and many other functions in a broad range of target tissues and both sexes through the activation of nuclear and membrane estrogen receptors (ERs), which leads to genomic and non-genomic cell responses. Since ECS function overlaps or integrates with many other cell signaling systems, this review aims at updating the knowledge about the possible crosstalk between ECS and estrogen system (ES) at both central and peripheral level, with focuses on the central nervous system, reproduction, and cancer.

Multi-Systemic Alterations by Chronic Exposure to a Low Dose of Bisphenol A in Drinking Water: Effects on Inflammation and NAD+-Dependent Deacetylase Sirtuin1 in Lactating and Weaned Rats.

Bisphenol A (BPA) is largely used as a monomer in some types of plastics. It accumulates in tissues and fluids and is able to bypass the placental barrier, affecting various organs and systems. Due to huge developmental processes, children, foetuses, and neonates could be more sensitive to BPA-induced toxicity. To investigate the multi-systemic effects of chronic exposure to a low BPA dose (100 µg/L), pregnant Wistar rats were exposed to BPA in drinking water during gestation and lactation. At weaning, newborn rats received the same treatments as dams until sex maturation. Free and conjugated BPA levels were measured in plasma and adipose tissue; the size of cerebral ventricles was analysed in the brain; morpho-functional and molecular analyses were carried out in the liver with a focus on the expression of inflammatory cytokines and Sirtuin 1 (Sirt1). Higher BPA levels were found in plasma and adipose tissue from BPA treated pups (17 PND) but not in weaned animals. Lateral cerebral ventricles were significantly enlarged in lactating and weaned BPA-exposed animals. In addition, apart from microvesicular steatosis, liver morphology did not exhibit any statistically significant difference for morphological signs of inflammation, hypertrophy, or macrovesicular steatosis, but the expression of inflammatory cytokines, Sirt1, its natural antisense long non-coding RNA (Sirt1-AS LncRNA) and histone deacetylase 1 (Hdac1) were affected in exposed animals. In conclusion, chronic exposure to a low BPA dose could increase the risk for disease in adult life as a consequence of higher BPA circulating levels and accumulation in adipose tissue during the neonatal period.

The Epigenetics of the Endocannabinoid System.

The endocannabinoid system (ES) is a cell-signalling system widely distributed in biological tissues that includes endogenous ligands, receptors, and biosynthetic and hydrolysing machineries. The impairment of the ES has been associated to several pathological conditions like behavioural, neurological, or metabolic disorders and infertility, suggesting that the modulation of this system may be critical for the maintenance of health status and disease treatment. Lifestyle and environmental factors can exert long-term effects on gene expression without any change in the nucleotide sequence of DNA, affecting health maintenance and influencing both disease load and resistance. This potentially reversible "epigenetic" modulation of gene expression occurs through the chemical modification of DNA and histone protein tails or the specific production of regulatory non-coding RNA (ncRNA). Recent findings demonstrate the epigenetic modulation of the ES in biological tissues; in the same way, endocannabinoids, phytocannabinoids, and cannabinoid receptor agonists and antagonists induce widespread or gene-specific epigenetic changes with the possibility of trans-generational epigenetic inheritance in the offspring explained by the transmission of deregulated epigenetic marks in the gametes. Therefore, this review provides an update on the epigenetics of the ES, with particular attention on the emerging role in reproduction and fertility.

The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NFκB and STAT3 pathways in cystic fibrosis cells.

OBJECTIVE: N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. MATERIALS AND METHODS: TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid. RESULTS: We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. CONCLUSIONS: Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

BAG3 is involved in neuronal differentiation and migration.

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Pharmacological actions of statins: a critical appraisal in the management of cancer.

Statins, among the most commonly prescribed drugs worldwide, are cholesterol-lowering agents used to manage and prevent cardiovascular and coronary heart diseases. Recently, a multifaceted action in different physiological and pathological conditions has been also proposed for statins, beyond anti-inflammation and neuroprotection. Statins have been shown to act through cholesterol-dependent and -independent mechanisms and are able to affect several tissue functions and modulate specific signal transduction pathways that could account for statin pleiotropic effects. Typically, statins are prescribed in middle-aged or elderly patients in a therapeutic regimen covering a long life span during which metabolic processes, aging, and concomitant novel diseases, including cancer, could occur. In this context, safety, toxicity, interaction with other drugs, and the state of health have to be taken into account in subjects treated with statins. Some evidence has shown a dichotomous effect of statins with either cancer-inhibiting or -promoting effects. To date, clinical trials failed to demonstrate a reduced cancer occurrence in statin users and no sufficient data are available to define the long-term effects of statin use over a period of 10 years. Moreover, results from clinical trials performed to evaluate the therapeutic efficacy of statins in cancer did not suggest statin use as chemotherapeutic or adjuvant agents. Here, we reviewed the pharmacology of the statins, providing a comprehensive update of the current knowledge of their effects on tissues, biological processes, and pathological conditions, and we dissected the disappointing evidence on the possible future use of statin-based drugs in cancer therapy.

PLASTAMINATION: Outcomes on the Central Nervous System and Reproduction.

BACKGROUND: Environmental exposures to non-biodegradable and biodegradable plastics are unavoidable. Microplastics (MPs) and nanoplastics (NPs) from the manufacturing of plastics (primary sources) and the degradation of plastic waste (secondary sources) can enter the food chain directly or indirectly and, passing biological barriers, could target both the brain and the gonads. Hence, the worldwide diffusion of environmental plastic contamination (PLASTAMINATION) in daily life may represent a possible and potentially serious risk to human health. OBJECTIVE: This review provides an overview of the effects of non-biodegradable and the more recently introduced biodegradable MPs and NPs on the brain and brain-dependent reproductive functions, summarizing the molecular mechanisms and outcomes on nervous and reproductive organs. Data from in vitro, ex vivo, non-mammalian and mammalian animal models and epidemiological studies have been reviewed and discussed. RESULTS: MPs and NPs from non-biodegradable plastics affect organs, tissues and cells from sensitive systems such as the brain and reproductive organs. Both MPs and NPs induce oxidative stress, chronic inflammation, energy metabolism disorders, mitochondrial dysfunction and cytotoxicity, which in turn are responsible for neuroinflammation, dysregulation of synaptic functions, metabolic dysbiosis, poor gamete quality, and neuronal and reproductive toxicity. In spite of this mechanistic knowledge gained from studies of non-biodegradable plastics, relatively little is known about the adverse effects or molecular mechanisms of MPs and NPs from biodegradable plastics. CONCLUSION: The neurological and reproductive health risks of MPs/NPs exposure warrant serious consideration, and further studies on biodegradable plastics are recommended.

COVID-19 Pandemic: 1-Year Follow-Up in Children and Adolescents with Neuropsychiatric Disorders.

INTRODUCTION: Few studies have focused on the long-term effects of the COVID-19 pandemic on mental health. The objective of our work was to evaluate the changes in emotional and behavioral symptoms in patients with neuropsychiatric disorders and the impact on parenting stress 1 year after the first national lockdown. METHODS: We enrolled 369 patients aged 1.5-18 years of age referred to the Child and Adolescent Neuropsychiatry Unit of the University Hospital of Salerno (Italy) by their parents. We asked their parents to complete two standardized questionnaires for the assessment of emotional/behavioral symptoms (Child Behavior CheckList, CBCL) and parental stress (Parenting Stress Index, PSI) prior to the pandemic (Time 0), during the first national lockdown (Time 1) and after 1 year (Time 2), and we monitored the changes in symptoms over time. RESULTS: After 1 year from the start of the first national lockdown, we found a significant increase of internalizing problems, anxiety, depression, somatization, and social and oppositional-defiant problems in older children (6-18 years), and a significant increase of somatization, anxiety problems, and sleep problems in younger children (1.5-5 years). We also observed a significant relationship between emotional/behavioral symptoms and parental stress. CONCLUSION: Our study showed that parental stress levels increased compared to the pre-pandemic months and continues to persist over time, while internalizing symptoms of children and adolescents showed a significant worsening during 1 year follow-up from the first COVID-19 lockdown.

The Other Side of Plastics: Bioplastic-Based Nanoparticles for Drug Delivery Systems in the Brain.

Plastics have changed human lives, finding a broad range of applications from packaging to medical devices. However, plastics can degrade into microscopic forms known as micro- and nanoplastics, which have raised concerns about their accumulation in the environment but mainly about the potential risk to human health. Recently, biodegradable plastic materials have been introduced on the market. These polymers are biodegradable but also bioresorbable and, indeed, are fundamental tools for drug formulations, thanks to their transient ability to pass through biological barriers and concentrate in specific tissues. However, this "other side" of bioplastics raises concerns about their toxic potential, in the form of micro- and nanoparticles, due to easier and faster tissue accumulation, with unknown long-term biological effects. This review aims to provide an update on bioplastic-based particles by analyzing the advantages and drawbacks of their potential use as components of innovative formulations for brain diseases. However, a critical analysis of the literature indicates the need for further studies to assess the safety of bioplastic micro- and nanoparticles despite they appear as promising tools for several nanomedicine applications.

The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.

Interaction of endocannabinoid system and steroid hormones in the control of colon cancer cell growth.

Increasing evidence suggest the role of the cannabinoid receptors (CBs) in the control of cell survival or death and signaling pathways involved in tumor progression. Cancer cell lines are characterized by a subtle modulation of CB levels which produces a modified responsiveness to specific ligands, but the molecular mechanisms underlying these events are poorly and partially understood. We previously provided evidence that the endocannabinoid (EC) anandamide (AEA) exerts anti-proliferative effect likely by modulation of the expression of genes involved in the cellular fate. In this study we focused on the role of the CB1 receptor, ECs, and steroids in the mechanisms involved in colorectal cancer (CRC) cell growth inhibition in vitro. We demonstrated that, in DLD1 and SW620 cells, 17ß-estradiol induced a specific and strong up-regulation of the CB1 receptor by triggering activation of the CB1 promoting region, localized at the exon 1 of the CNR1 gene. Moreover, treatment of DLD1 and SW620 cells with Met-F-AEA, a stable AEA-analogous, or URB597, a selective inhibitor of FAAH, induced up-regulation of CB1 expression by co-localization of PPARγ and RXRα at the promoting region. Finally, increased availability of AEA, of both exogenous and endogenous sources, induced the expression of estrogen receptor-beta in both cell lines. Our results partially elucidated the role of EC system in the molecular mechanisms enrolled by steroids in the inhibition of colon cancer cell growth and strongly suggested that targeting the EC system could represent a promising tool to improve the efficacy of CRC treatments.

Stem Cells from Healthy and Tendinopathic Human Tendons: Morphology, Collagen and Cytokines Expression and Their Response to T3 Thyroid Hormone.

The aim of this study was to investigate the effect of triiodothyronine (T3) on tendon specific markers and cytokines expression of stem cells extracted from human tendons. Indeed, thyroid hormones have been reported to be protective factors, maintaining tendons' homeostasis, whereas tendinopathy is believed to be related to a failed healing response. Healthy and tendinopathic human tendons were harvested to isolate tendon stem/progenitor cells (TSPCs). TSPCs obtained from pathological samples showed gene expression and morphological modifications at baseline in comparison with cells harvested from healthy tissues. When cells were maintained in a medium supplemented with T3 (10-6 M), only pathological populations showed a significant upregulation of tenogenic markers (DCN, TNC, COL1A1, COL3A1). Immunostaining revealed that healthy cells constantly released type I collagen, typical of tendon matrix, whereas pathological ones overexpressed and secreted type III collagen, typical of scarred and impaired tissue. Pathological cells also overexpressed pro- and anti-inflammatory cytokines, suggesting an impaired balance in the presence of T3, without STAT3 activation. Moreover, DKK-1 was significantly high in the culture medium of pathological cell cultures and was reversed by T3. This study opens perspectives on the complex biochemical alteration of cells from pathological tendons, which may lead to the chronic disease context with an impaired extracellular matrix.

Neuroinflammation: Molecular Mechanisms And Therapeutic Perspectives.

BACKGROUND: Neuroinflammation is a key component in the etiopathogenesis of neurological diseases and brain aging. This process involves the brain immune system that modulates synaptic functions and protects neurons from infection or damage. Hence, the knowledge of neuroinflammation related pathways and modulation by drugs or natural compounds is functional to developing therapeutic strategies aimed at preserving, maintaining and restoring brain health. OBJECTIVE: This review article summarizes the basics of neuroinflammation and related signaling pathways, the success of the dietary intervention in clinical practice and the possible development of RNA-based strategies for treating neurological diseases. METHODS: Pubmed search from 2012 to 2022 with the keywords neuroinflammation and molecular mechanisms in combination with diet, miRNA and non-coding RNA. RESULTS: Glial cells-play a crucial role in neuroinflammation, but several pathways can be activated in response to different inflammatory stimuli, inducing cell death by apoptosis, pyroptosis or necroptosis. The dietary intervention has immunomodulatory effects and could limit the inflammatory process induced by microglia and astrocytes. Thus by inhibiting neuroinflammation and improving the symptoms of a variety of neurological diseases, diet exerts pleiotropic neuroprotective effects independently from the spectrum of pathophysiological mechanisms underlying the specific disorder. Furthermore, data from animal models revealed that altered expression of specific noncoding RNAs, in particular microRNAs, contributes to neuroinflammatory diseases; consequently, RNA-based strategies may be promising to alleviate the consequences of neuroinflammation. CONCLUSION: Further studies are needed to identify the molecular pathways and the new pharmacological targets in neuroinflammation to lay the basis for more effective and selective therapies to be applied, in parallel to dietary intervention, in the treatment of neuroinflammation-based diseases.

Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis.

The hypothalamus-pituitary-testis axis controls the production of spermatozoa, and the kisspeptin system, comprising Kiss1 and Kiss1 receptor (Kiss1R), is the main central gatekeeper. The activity of the kisspeptin system also occurs in testis and spermatozoa, but currently the need of peripheral kisspeptin to produce gametes is not fully understood. Hence, we characterized kisspeptin system in rat spermatozoa and epididymis caput and cauda and analyzed the possible presence of Kiss1 in the epididymal fluid. The presence of Kiss1 and Kiss1R in spermatozoa collected from epididymis caput and cauda was evaluated by Western blot; significant high Kiss1 levels in the caput (p < 0.001 vs. cauda) and constant levels of Kiss1R proteins were observed. Immunofluorescence analysis revealed that the localization of Kiss1R in sperm head shifts from the posterior region in the epididymis caput to perforatorium in the epididymis cauda. In spermatozoa-free epididymis, Western blot revealed higher expression of Kiss1 and Kiss1R in caput (p < 0.05 vs. cauda). Moreover, immunohistochemistry revealed that Kiss1 and Kiss1R proteins were mainly localized in the secretory epithelial cell types and in contractile myoid cells, respectively. Finally, both dot blot and Elisa revealed the presence of Kiss1 in the epididymal fluid collected from epididymis cauda and caput, indicating that rat epididymis and spermatozoa possess a complete kisspeptin system. In conclusion, we reported for the first time in rodents Kiss1R trafficking in spermatozoa during the epididymis transit and Kiss1 measure in the epididymal fluid, thus suggesting a possible role for the system in spermatozoa maturation and storage within the epididymis.

Supercritical emulsion extraction fabricated PLA/PLGA micro/nano carriers for growth factor delivery: Release profiles and cytotoxicity.

Controlled delivery of human growth factors is still a challenge in tissue engineering protocols, and poly-lactic acid and poly-lactic-co-glycolic acid carriers have been recently proposed for this purpose. Here, the microencapsulation of two human growth factors, namely Growth Differentiation Factor -5 (hGDF-5) and Transforming Growth Factor ß1 (hTGF-ß1) was tested, by processing different emulsions with Supercritical Emulsion Extraction (SEE) technology. Polymer molecular weight, co-polymer ratio and surfactant amount in aqueous phases as well as phases mixing rate were varied to fabricate carriers with suitable size and loadings. Carriers with different mean sizes from 0.4 ± 0.09 µm up to 3 ± 0.9 µm were obtained by SEE technology when processing emulsions with different formulations; carriers were loaded with 3 µg/g and 7 µg/g for hGDF-5 and hTGF-ß1 with controlled growth factor release over 25 days. Carriers displayed extremely low cytotoxicity when evaluated in Chinese Hamster Ovary cells (CHO-K1). Further, they also exhibited reduced cytotoxicity with respect to carriers obtained by conventional evaporation techniques, and low reactivity on human peripheral blood mononuclear cells (hPBMCs), suggesting their safety and potential use in tissue engineering protocols.

Bisphenol A induces DNA damage in cells exerting immune surveillance functions at peripheral and central level.

Bisphenol A (BPA) is a synthetic xenoestrogen diffused worldwide. Humans are chronically exposed to low doses of BPA from food and drinks, thus BPA accumulates in tissues posing human health risk. In this study, we investigated the effects of BPA on peripheral blood mononuclear cells (PBMC) from human healthy donors, and in glia and microglia of rat offspring at postnatal day 17 (17PND) from pregnant females who received BPA soon after coupling and during lactation and weaning. Results indicated that BPA affected Phytoemagglutinin (PHA) stimulated PBMC proliferation causing an S-phase cell cycle accumulation at nanomolar concentrations while BPA was almost ineffective in resting PBMC. Furthermore, BPA induced chromosome aberrations and the appearance of shattered cells characterized by high number of fragmented and pulverized chromosomes, suggesting that the compound could cause a massive genomic rearrangement by inducing catastrophic events. The BPA-induced DNA damage was observed mainly in TCD4+ and TCD8+ subsets of T lymphocytes and was mediated by the increase of ERK1/2 phosphorylation, p21/Waf1 and PARP1 protein expression. Intriguingly, we observed for the first time that BPA-induced effects were associated to a sex specific modulation of ERα and ERß in human PBMC. Immunofluorescence analysis of rat hippocampus corroborated in vitro findings showing that BPA induced É£H2AX phosphorylation in microglia and astrocytosis by decreasing ERα expression within the dentate gyrus. Overall these results suggest that BPA can alter immune surveillance functions at both peripheral and central level with a potential risk for cancer, neuroinflammation and neurodegeneration.

Demineralized bone matrix paste formulated with biomimetic PLGA microcarriers for the vancomycin hydrochloride controlled delivery: Release profile, citotoxicity and efficacy against S. aureus.

Infection and resulting bone defects caused by Staphylococcus aureus is one of the major issues in orthopaedic surgeries. Vancomycin hydrochloride (VaH) is largely used to manage these events. Here, a human derived bone paste supplemented with biopolymer microcarriers for VaH sustained delivery to merge osteoinductive and antimicrobial actions is described. In detail, different emulsion formulations were tested to fabricate micro-carriers of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) by a proprietary technology (named Supercritical Emulsion Extraction). These carriers (mean size 827 ± 68 µm; loading 47 mgVaH/gPLGA) were assembled with human demineralized bone matrix (DBM) to obtain an antimicrobial bone paste system (250 mg/0.5 cm3 w/v, carrier/DBM). Release profiles in PBS indicated a daily drug average release of about 4 µg/mL over two weeks. This concentration was close to the minimum inhibitory concentration and able to effectively inhibit the S. aureus growth in our experimental sets. Carriers cytotoxicity tests showed absence of adverse effects on cell viability at the concentrations used for paste assembly. This approach points toward the potential of the DBM-carrier-antibiotic system in hampering the bacterial growth with accurately controlled antibiotic release and opens perspectives on functional bone paste with PLGA carriers for the controlled release of bioactive molecules.

A new role of anandamide in human sperm: focus on metabolism.

The endocannabinoid system and the presence of CB1 receptor (CB1-R) target of the anandamide were identified in human sperm, however the anandamide action in this context needs to be further elucidated. At this purpose we analyzed the effects of anandamide on human sperm capacitation and motility. Afterwards, we focused on lipid and glucose sperm metabolism and also investigated the interrelationship between anandamide and insulin secretion by sperm. By intracellular free Ca(2+) content assay and proteins tyrosine phosphorylation, we evidenced that anandamide did not induce capacitation process and a negative effect was obtained on sperm motility. The blockage of CB1-R by the specific antagonist SR141716 increased both capacitation and sperm motility suggesting an involvement of the CB1-R in the acquisition of sperm fertilizing activity. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities, suggest that anandamide exerts a lipogenetic effect on human sperm lipid metabolism. Concerning the glucose metabolism, anandamide increases GSK3 phosphorylation indicating that it is involved in the accumulation of energy substrates. G6PDH activity was not affected by anandamide. Interestingly, AEA is involved in insulin secretion by sperm. As insulin had been demonstrated to be an autocrine factor that triggers capacitation, the endocannabinoid might be inserted in the signaling cascade that induces this process. Altogether these findings highlight a pivotal involvement of the CB1-R in the control of sperm energy homeostasis and propose a new site of action for endocannabinoids in the control of energy metabolism.