Long-Term Dynamic Changes of NMDA Receptors Following an Excitotoxic Challenge.

Excitotoxicity is a form of neuronal death characterized by the sustained activation of N-methyl-D-aspartate receptors (NMDARs) triggered by the excitatory neurotransmitter glutamate. NADPH-diaphorase neurons (also known as nNOS (+) neurons) are a subpopulation of aspiny interneurons, largely spared following excitotoxic challenges. Unlike nNOS (-) cells, nNOS (+) neurons fail to generate reactive oxygen species in response to NMDAR activation, a critical divergent step in the excitotoxic cascade. However, additional mechanisms underlying the reduced vulnerability of nNOS (+) neurons to NMDAR-driven neuronal death have not been explored. Using functional, genetic, and molecular analysis in striatal cultures, we indicate that nNOS (+) neurons possess distinct NMDAR properties. These specific features are primarily driven by the peculiar redox milieu of this subpopulation. In addition, we found that nNOS (+) neurons exposed to a pharmacological maneuver set to mimic chronic excitotoxicity alter their responses to NMDAR-mediated challenges. These findings suggest the presence of mechanisms providing long-term dynamic regulation of NMDARs that can have critical implications in neurotoxic settings.

Resolvin D1 Reduces Lung Infection and Inflammation Activating Resolution in Cystic Fibrosis.

Non-resolving lung inflammation and Pseudomonas aeruginosa infections are the underlying cause of morbidity and mortality in cystic fibrosis (CF). The endogenous lipid mediator resolvin (Rv) D1 is a potent regulator of resolution, and its roles, actions, and therapeutic potential in CF are of interest. Here, we investigated actions and efficacy of RvD1 in preclinical models of cystic fibrosis. Cftr knockout mice with chronic P. aeruginosa lung infection were treated with RvD1 to assess differences in lung bacterial load, inflammation, and tissue damage. Cells from volunteers with CF were treated with RvD1 during ex vivo infection with P. aeruginosa, and effects on phagocytosis and inflammatory signaling were determined. In CF mice, RvD1 reduced bacterial burden, neutrophil infiltration, and histological signs of lung pathology, improving clinical scores of diseases. Mechanistically, RvD1 increased macrophage-mediated bacterial and leukocyte clearance in vivo. The clinical significance of these findings is supported by actions in primary leukocytes and epithelial cells from volunteers with CF where RvD1 enhanced P. aeruginosa phagocytosis and reduced genes and proteins associated to NF-κB activation and leukocyte infiltration. Concentration of RvD1 in sputum from patients with CF was also inversely correlated to those of cytokines and chemokines involved in CF lung pathology. These findings demonstrate efficacy of RvD1 in enhancing resolution of lung inflammation and infections and provide proof of concept for its potential as a prototypic novel pro-resolutive therapeutic approach for CF.

Whole-body exposure to cigarette smoke alters oocyte miRNAs expression in C57BL/6 mice.

Cigarette smoke is toxic for the female reproductive system with particular reference to the ovary. The purpose of the study was to investigate if the microRNAs (miRNAs) pattern could be altered by cigarette smoke exposure in mouse oocytes. For this purpose, C57BL/6 mice were whole-body exposed to three cigarettes daily, 7 days/week, for 2 or 4 months by a specific rodent ventilator. Mice were then superovulated and oocytes collected. MII oocytes pools obtained by single animals were deprived of cumulus cells and used to extract total RNA including miRNAs. TaqMan™ Rodent MicroRNA A Array v2.0 was used to analyze the miRNAs expression profile. The biological functions and the functional networks of the identified up- and downregulated miRNAs were analyzed by ingenuity pathway analysis software. The gene expression of deregulated-miRNAs targets was evaluated. For the first time, the global miRNAs changes in mouse oocyte in response to cigarette smoke exposure were disclosed. Our results revealed significant modulation of miRNAs mainly involved in inflammatory processes, cellular proliferation, and apoptosis. miRNAs expression was altered in a time-dependent manner. Smoke exposure induced an early downregulation of Dicer1. Transcriptional alterations of the modulated miRNAs major targets, estrogen receptor 1, peroxisome proliferator-activated receptor-alpha, and tumor protein 53, as well as that of other key regulatory genes, were evidenced. Cigarette smoke represents a stimulus able to alter miRNAs pattern in mouse oocyte. This study increases our understanding of the ovarian toxicity profile of cigarette smoke, and open new roads toward the identification of biomarkers of oocyte toxicity and dysregulation.

Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. In this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of thede novoceramide biosynthesis, on the aging phenotype of cortical neurons cultured for 22 days, a setting employed as anin vitromodel of senescence. Our findings indicate that, compared to control cultures, 'aged' neurons display dysregulation of [Ca2+]ilevels, mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS positively affected the senescent phenotype, a result associated with recovery of neuronal [Ca2+]isignaling and reduction of mitochondrial dysfunction and ROS generation. The results suggest that thede novoceramide biosynthesis represents a critical intermediate in the molecular and functional cascade leading to neuronal senescence and identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions.

Dynactin pathway-related gene expression is altered by aging, but not by vitrification.

The storage of surplus oocytes by cryopreservation (OC) is a widely used tool in assisted reproductive technology, but there is a great debate about the effects of cryopreservation on oocyte competence. It is known that OC may affect meiotic spindles but remains unclear if OC may increase the risk of aneuploidy. The aim of this study was to test the effects of OC and women aging on the expression of cytokinesis-related genes playing an important role in the segregation of chromosomes (DCTN1, DCTN2, DCTN3, DCTN6 and PLK1). Results highlighted that OC do not modify the expression of the selected genes, whereas women aging modulate the expression of all transcripts, confirming that aging is the crucial factor affecting meiosis and aneuploidy risk. A new role for Dynactin and PLK1 was shed in light, providing information on the ageing process in the oocyte which may be associated to reduced fertility.

Curcumin/Liposome Nanotechnology as Delivery Platform for Anti-inflammatory Activities via NFkB/ERK/pERK Pathway in Human Dental Pulp Treated With 2-HydroxyEthyl MethAcrylate (HEMA).

Curcumin, primary component of the spice turmeric extracted from the rhizomes of Curcuma longa, represents the major anti-oxidant and anti-inflammatory substance found in turmeric, acting thought various mechanisms not completely understood. Curcumin modulates cytokines, growth factors, transcription factors, inflammatory molecules and cell signaling pathways. During restorative dentistry practice, free resin monomers of 2-hydroxyethyl methacrylate (HEMA) propagate through dentin micro-channel and pulp into the bloodstream affecting cellular integrity. The study highlights the significance of application of curcumin bioactive component into liposomal formulations (CurLIP) to restore the homeostasis of dental pulp stem cells (hDPSCs) in response to 3 and 5 mmol L-1 HEMA treatment. Cell proliferation in combination with changes of the morphological features, proinflammatory cytokines secretion as Interleukin (IL) 6, IL8, Monocyte Chemoattractant Protein-1 (MCP1) and Interferon-gamma (IFNγ) were assayed along with the nuclear factor (NF)-kB, an inducible transcription factor involved in the activation of several cell processes associated to extracellular signal-regulated kinases (ERK) and posphorylated (p-) ERK pathway. Our results showed a decreased cell proliferation, morphological changes and upregulation of IL6, IL8, MCP1 and IFNγ in presence of 3 and 5 mmol L-1 HEMA treatment. CurLIP therapy in hDPSCs provokes an increase in cell proliferation and the block of inflammatory cytokines secretion through the inhibitory regulation of NFkB/ERK and pERK signaling cascade. The natural nanocarrier CurLIP influences numerous biochemical and molecular cascades causing anti-inflammatory properties in response to HEMA treatment in human dental pulp stem cells, representing an innovative endodontic formulation able to improve the quality of dental care with a major human community impact.

Pre-conceptional maternal exposure to cyclophosphamide results in modifications of DNA methylation in F1 and F2 mouse oocytes: evidence for transgenerational effects.

Cyclophosphamide (CPM), an agent widely used in breast cancer therapy, has strong gonadotoxic effects. Female reproductive potential after therapy relies on ovulated oocytes deriving from primordial follicles surviving CPM toxic insult. In this study, we investigated in the mouse model whether pre-conceptional maternal exposure to CPM has epigenetic effects on offspring oocytes and if they are inherited. Adult female mice mated following CPM exposure, generated an offspring (F1) with delayed growth, normal fertility and altered methylation of three imprinted genes (H19, Igf2r and Peg3) in their oocytes. These alterations were present in oocytes generated by F2 mice. Pre-conceptional maternal exposure to fertoprotective agents AS101 and crocetin prior to CPM was not able to fully counteract alterations in offspring oocyte imprinting. For the first time, current study evidences that pre-conceptional CPM maternal exposure can affect the competence of offspring's oocytes and warns on possible long-term effects on the health of next generations.

MRAP2 regulates endometrial receptivity and function.

A successful embryo implantation depends on the synchronization between a competent blastocyst and a receptive endometrium. Recently, potential modulators of endometrial receptivity (OVGP1, MRAP2, ZCCHC12, and HAP1) have been reported likely with a functional role during embryo implantation. The aim of this study was to evaluate the gene expression of these genes in the endometrium of infertile women. Eighteen endometrial biopsies, during secretory lutheal phase, were recruited from women with unexplained infertility and women who cannot conceive due to their partners' fertility problems. qRT-PCR was carried out to evaluate MRAP2, OVGP1, ZCCHC12 and HAP1 gene expression. MRAP2 expression was also detected by western blot and it was localized by immunohistochemistry. Morphological analysis was performed by light microscopy. MRAP2 was significantly up-regulated in study vs. control group. Western blot analysis confirmed the observed MRAP2 up-expression. MRAP2 resulted mainly localized in the epithelial cells of uterine glands. Morphological analysis displayed that the epithelium of the uterine glands undergo hypertrophy in women with unexplained infertility in respect to women with male infertility factor. MRAP2 could be considered a mediator of endometrial receptivity likely acting on endometrial stability by binding to MCRs and PKR1.

Aging and the Combined effects of ADRA2B and CB1 deletions on Affective Working Memory.

Many studies have found that memory for affective material is better than memory for neutral information and memory for positive material compared to negative material is better in older adults. Behavioral, neurophysiological as well as single polymorphism differences have been advanced to account for these effects. Here, we aimed to examine whether the combination of two polymorphisms (ADRA2B and CB1) in older adults influences active maintenance and manipulation of emotional information in aging working memory. We examined genotype data from 207 older adults (56 double deletion carriers, 116 single deletion carriers and 35 no deletion carriers) who performed a verbal operation span-like task with positive, negative and neutral words. We found that subjects carrying both ADRA2B and CB1 variants generally remembered a higher number of words. In addition, double carriers showed positivity effects while single carriers showed more general emotional enhancement effects, especially as strings lengthened. These findings are amongst the first to suggest a haplotype account of positivity effects in older adults' memory.

A novel role in skeletal segment regeneration of extracellular vesicles released from periodontal-ligament stem cells.

PURPOSE: The combination of oral derived stem cells and 3-D scaffolds is considered advantageous in bone repair. In particular, collagen membranes possess ideal biological properties and can support infiltration and proliferation of osteoblasts, promoting bone regeneration. Our study aimed to develop a new biocompatible osteogenic construct composed of a commercially available collagen membrane (Evolution [Evo]), human periodontal-ligament stem cells (hPDLSCs) enriched with extracellular vesicles (EVs), or polyethylenimine (PEI)-engineered EVs (PEI-EVs). METHODS: Osteogenic ability and expression of osteogenic genes were evaluated in vitro in hPDLSCs cultured with or without Evo, with Evo and EVs, or PEI-EVs. In addition, the bone-regeneration capacity of Evo, Evo enriched with hPDLSCs, Evo enriched with hPDLSCs and EVs/PEI-EVs was investigated in rats subjected to calvarial defects. RESULTS: Our results showed that Evo enriched with EVs and PEI-EVs showed high biocompatibility and osteogenic properties in vitro and in vivo. In addition, quantitative reverse-transcription polymerase chain reaction demonstrated the upregulation of osteogenic genes, such as TGFB1, MMP8, TUFT1, TFIP11, BMP2, and BMP4, in the presence of PEI-EVs. Upregulation of BMP2/4 was confirmed for Evo enriched with PEI-EVs and hPDLSCs both in vitro by Western blot and in vivo by immunofluorescence. CONCLUSION: Our results indicated that Evo enriched with hPDLSCs and PEI-EVs is able to promote a bone-regeneration process for the treatment of calvarium and ossification defects caused by accidental or surgery trauma. In particular, PEI-EVs had a significant role in activation of the osteogenic process.

The pharmacological perturbation of brain zinc impairs BDNF-related signaling and the cognitive performances of young mice.

Zinc (Zn2+) is a pleiotropic modulator of the neuronal and brain activity. The disruption of intraneuronal Zn2+ levels triggers neurotoxic processes and affects neuronal functioning. In this study, we investigated how the pharmacological modulation of brain Zn2+ affects synaptic plasticity and cognition in wild-type mice. To manipulate brain Zn2+ levels, we employed the Zn2+ (and copper) chelator 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol, CQ). CQ was administered for two weeks to 2.5-month-old (m.o.) mice, and effects studied on BDNF-related signaling, metalloproteinase activity as well as learning and memory performances. CQ treatment was found to negatively affect short- and long-term memory performances. The CQ-driven perturbation of brain Zn2+ was found to reduce levels of BDNF, synaptic plasticity-related proteins and dendritic spine density in vivo. Our study highlights the importance of choosing "when", "where", and "how much" in the modulation of brain Zn2+ levels. Our findings confirm the importance of targeting Zn2+ as a therapeutic approach against neurodegenerative conditions but, at the same time, underscore the potential drawbacks of reducing brain Zn2+ availability upon the early stages of development.

Biofunctionalized Scaffold in Bone Tissue Repair.

Bone tissue engineering is based on bone grafting to repair bone defects. Bone graft substitutes can contribute to the addition of mesenchymal stem cells (MSCs) in order to enhance the rate and the quality of defect regeneration. The stem cell secretome contains many growth factors and chemokines, which could affect cellular characteristics and behavior. Conditioned medium (CM) could be used in tissue regeneration avoiding several problems linked to the direct use of MSCs. In this study, we investigated the effect of human periodontal ligament stem cells (hPDLSCs) and their CM on bone regeneration using a commercially available membrane scaffold Evolution (EVO) implanted in rat calvarias. EVO alone or EVO + hPDLSCs with or without CM were implanted in Wistar male rats subjected to calvarial defects. The in vivo results revealed that EVO membrane enriched with hPDLSCs and CM showed a better osteogenic ability to repair the calvarial defect. These results were confirmed by acquired micro-computed tomography (CT) images and the increased osteopontin levels. Moreover, RT-PCR in vitro revealed the upregulation of three genes (Collagen (COL)5A1, COL16A1 and transforming growth factor (TGF)ß1) and the down regulation of 26 genes involved in bone regeneration. These results suggest a promising potential application of CM from hPDLSCs and scaffolds for bone defect restoration and in particular for calvarial repair in case of trauma.

Butyrylcholinesterase and Acetylcholinesterase polymorphisms in Multiple Sclerosis patients: implication in peripheral inflammation.

Multiple Sclerosis (MS) is an autoimmune disease, having not fully understood aetiology, and both genetic and environmental factors contribute to the pathogenesis of the disease. The cholinergic system has been indicated as a mediator of neuro-immune interactions, as well as an internal regulator of immune responses. The aim of the present research was to assess the associations between BChE and AChE genetic variations and serum cholinergic and inflammatory profiles in 102 Relapsing Remitting-MS patients and 117 healthy controls. An increased frequency of the BChE K-allele in MS patients as compared to controls was found. In addition, data showed that patients had higher BChE enzymatic activity, which is increased by the presence of the polymorphic allele and reduced amounts of circulating ACh. AChE polymorphism was significantly associated to reduced activity in both patients and controls. We propose that serum BChE and AChE activity may be used as a secondary markers to assess the role of non-neuronal cholinergic system in regulating peripheral inflammation via ACh regulation. This pilot study shed light on the role of the non-neuronal cholinergic system in immune cells to better understand MS pathogenesis. The cross-talk between the periphery and the CNS could have a new undescribed crucial role for MS, regarded as a systemic disease.

The ADRA2B gene in the production of false memories for affective information in healthy female volunteers.

False memories are common memory distortions in everyday life and seem to increase with affectively connoted complex information. In line with recent studies showing a significant interaction between the noradrenergic system and emotional memory, we investigated whether healthy volunteer carriers of the deletion variant of the ADRA2B gene that codes for the α2b-adrenergic receptor are more prone to false memories than non-carriers. In this study, we collected genotype data from 212 healthy female volunteers; 91 ADRA2B carriers and 121 non-carriers. To assess gene effects on false memories for affective information, factorial mixed model analysis of variances (ANOVAs) were conducted with genotype as the between-subjects factor and type of memory error as the within-subjects factor. We found that although carriers and non-carriers made comparable numbers of false memory errors, they showed differences in the direction of valence biases, especially for inferential causal errors. Specifically, carriers produced fewer causal false memory errors for scripts with a negative outcome, whereas non-carriers showed a more general emotional effect and made fewer causal errors with both positive and negative outcomes. These findings suggest that putatively higher levels of noradrenaline in deletion carriers may enhance short-term consolidation of negative information and lead to fewer memory distortions when facing negative events.

Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage.

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

Testis Transcriptome Modulation in Klinefelter Patients with Hypospermatogenesis.

The main genetic cause of male infertility is represented by the Klinefelter Syndrome (KS), a condition accounting for 3% of all cases of infertility and up to15% of cases of azoospermia. KS is generally characterized by azoospermia; approximately 10% of cases have severe oligozoospermia. Among these, the 30-40% of patients show hypospermatogenesis. The mechanisms leading to adult testis dysfunctions are not completely understood. A microarray transcriptome analysis was performed on testis biopsies obtained from three KS patients with hypospermatogenesis and three control subjects. KS testis showed a differential up- and down-regulation of 303 and 747 transcripts, respectively, as compared to controls. The majority of down-regulated transcripts were involved in spermiogenesis failure and testis morphological defects, whereas up-regulated genes were responsible for testis apoptotic processes. Functional analysis of the transcriptionally altered genes indicated a deregulation in cell death, germ cell function and morphology as well as blood-testis-barrier maintenance and Leydig cells activity. These data support a complex scenario in which spermatogenic impairment is the result of functional and morphological alterations in both germinal and somatic components of KS testis. These findings could represent the basis for evaluating new markers of KS spermatogenesis and potential targets of therapeutic intervention to preserve residual spermatogenesis.

A Neural "Tuning Curve" for Multisensory Experience and Cognitive-Perceptual Schizotypy.

Our coherent perception of external events is enabled by the integration of inputs from different senses occurring within a range of temporal offsets known as the temporal binding window (TBW), which varies from person to person. A relatively wide TBW may increase the likelihood that stimuli originating from different environmental events are erroneously integrated and abnormally large TBW has been found in psychiatric disorders characterized by unusual perceptual experiences. Despite strong evidence of inter-individual differences in TBW, both within clinical and nonclinical populations, the neurobiological underpinnings of this variability remain unclear. We adopted an integrated strategy linking TBW to temporal dynamics in functional magnetic resonance imaging (fMRI)-resting-state activity and cortical excitation/inhibition (E/I) balance. E/I balance was indexed by glutamate/Gamma-AminoButyric Acid (GABA) concentrations and common variation in glutamate and GABA genes in a healthy sample. Stronger resting-state long-range temporal correlations, indicated by larger power law exponent (PLE), in the auditory cortex, robustly predicted narrower audio-tactile TBW, which was in turn associated with lower cognitive-perceptual schizotypy. Furthermore, PLE was highest and TBW narrowest for individuals with intermediate levels of E/I balance, with shifts towards either extreme resulting in reduced multisensory temporal precision and increased schizotypy, effectively forming a neural "tuning curve" for multisensory experience and schizophrenia risk. Our findings shed light on the neurobiological underpinnings of multisensory integration and its potentially clinically relevant inter-individual variability.

Mitochondrial heteroplasmy profiling in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. Mutations of the mitochondrial genome lead to an altered energetic metabolism with negative effects on oocyte developmental competence. In this study, mtDNA heteroplasmy at an intra-oocyte level and between the different analyzed human oocytes (n = 12) was identified by a next-generation sequencing (NGS) protocol previously developed by this research group and submitted to GenBank. This method highlighted, in particular, variants in the genes involved in the respiratory chain providing a direct indication of the cell-specific damage within the mitochondrial genome as predictor of the oocyte quality.

Stemness Characteristics of Periodontal Ligament Stem Cells from Donors and Multiple Sclerosis Patients: A Comparative Study.

Multiple sclerosis (MS) is the most prevalent and progressive autoimmune disease that affects the central nervous system, and currently, no drug is available for the treatment. Stem cell therapy has received substantial attention in MS treatment. Recently, we demonstrated the immunosuppressive effects of mesenchymal stem cells derived from neural crest-originated human periodontal ligament tissue (hPDLSCs) in an in vivo model of MS. In the present study, we comparatively investigated the stemness properties of hPDLSCs derived from healthy donors and relapsing-remitting MS patients. Stem cell marker expression, cell proliferation, and differentiation capacity were studied. We found that both donor- and MS patient-derived hPDLSCs at early passage 2 showed similar expression of surface antigen markers and cell proliferation rate. Significant level of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation capacities was observed in both donor- and MS patient-derived hPDLSCs. Interestingly, these cells maintained the stemness properties even at late passage 15. Senescence markers p16 and p21 expression was considerably enhanced in passage 15. Our results propose that hPDLSCs may serve as simple and potential autologous stem cell niche, which may help in personalized stem cell therapy for MS patients.

Complete sequence of human mitochondrial DNA obtained by combining multiple displacement amplification and next-generation sequencing on a single oocyte.

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. In this study, the complete mtDNA sequence obtained for the first time by multiple displacement amplification approach in combination with next-generation sequencing from a single human oocyte is reported (GenBank accession no. KT364276). The analysis of oocyte mitochondrial mutations could provide a better understanding of the genetic variants correlated with the oocyte quality.