The use of customized mouthguards during the training produced protective effects on salivary factors of young athletes.

AIM: Custom-made mouthguards have many advantages compared to the stock and ready-made types, but sport treatments with custom made mouthguards involve changes in ecological factors of the oral cavity. In the present study we investigated the potential protective role of salivary factors, such as pH value, volume, prostaglandin E2 (PGE2) and 8-iso-prostaglandin F2? (8-iso-PGF2?) levels during training with customised mouthguards. MATERIALS AND METHODS: A total of 80 subjects were selected: 40 athletes, of whom 20 practice volleyball and 20 basketball (test group), and 40 subjects who attend a gym at a non-competitive level (control group). The athletes (test group) were analyzed at baseline (T0), pre-training (T1), post-training with custom-made Ethylene-Vinyl-Acetate (EVA) mouthguards (T2), post-training without mouthguards (T3). The control group was analyzed only at baseline (T0). On each player, in the 4 time points, and on the control group at T0, we stimulated saliva for determining PGE2 and 8-iso-PGF2? levels by radioimmunoassay and pH value by a pH meter and volume/ml. Saliva pH was calculated with a pH meter. RESULTS: We observed an inhibition of 8-iso-PGF2? salivary release induced by physical exercise and by use of custom-made mouthguard, while we found an increase in PGE2 salivary level in athletes after training and wearing the mouthguard. Furthermore, in the test of the volume of saliva produced in 5 minutes, a significant inhibition of saliva production emerged in the athletes who did not use the mouthguard during sports activities. CONCLUSION: Sports activity could lead to a reduction in oxidative stress and the use of mouth guards seems even more effective for athletes.

Protective effects induced by the food supplement Fluxonorm® in the lower urinary tract.

OBJECTIVE: Fluxonorm® is a dietary supplement that includes water-soluble extracts of Solidago virga-aurea, Phyllantus niruri, Epilobium angustifolium, Peumus boldus and Ononis spinosa. The aim of the present study was to evaluate the tolerability and efficacy of Fluxonorm® in improving lower urinary tract symptoms in patients with benign prostatic hyperplasia (BPH) in combination with standard of care. PATIENTS AND METHODS: Lower urinary tract symptoms can be improved by a marked anti-inflammatory action on the lower urinary tract (irritative symptoms) and/or by an anti-proliferative action (obstructive symptoms) on the prostate. Thirty patients were enrolled to evaluate the effect of Fluxonorm® on improving lower urinary tract symptoms. All patients complained of lower urinary tract symptoms (LUTS), such as hesitancy, poor flow, intermittent flow, incomplete voiding (obstructive symptoms), as well as increased frequency, nocturia and urgency (storage symptoms). All patients were treated with one tablet of Fluxonorm® (1200 mg) daily for 30 days to corroborate the results of our observation in which the food supplement (800 µg/mL) was also studied on the human prostate cancer PC3 cell line (antiproliferative activity) and on prostaglandin (PG)E2 production (anti-inflammatory activity). In addition, the effect of this compound on cyclooxygenase-2 (COX-2) gene expression was investigated. Finally, a bioinformatic analysis was conducted with the aim of unravelling the mechanism of action underlying the observed bio-pharmacological effects. RESULTS: As hypothesized in our preclinical research, adding Fluxonorm® to the therapy of enrolled patients improved all studied clinical parameters, including maximum flow (Qmax), after one month of treatment. In the preclinical evaluation, this formulation reduced PC3 cell viability and PGE2 production. The effects were also paralleled by reduced COX-2 gene expression and Fluxonorm®'s partly related content of catechin. While docking studies pointed out to the putative inhibition of matrix metalloproteinse-2 by gallic acid, as a further mechanism underlying the observed anti-proliferative effects, in PC3 cells exposed to Fluxonorm®. CONCLUSIONS: Fluxonorm® improved the efficacy of standard therapy, in terms of antioxidant/anti-inflammatory effects, for the management of lower urinary tract symptoms (LUTS). This could be related, albeit partially, to the blunting effect of this compound on PGE2 production.

Coherent anti-Stokes Raman spectroscopy of single and multi-layer graphene.

Spontaneous Raman spectroscopy is a powerful characterization tool for graphene research. Its extension to the coherent regime, despite the large nonlinear third-order susceptibility of graphene, has so far proven challenging. Due to its gapless nature, several interfering electronic and phononic transitions concur to generate its optical response, preventing to retrieve spectral profiles analogous to those of spontaneous Raman. Here we report stimulated Raman spectroscopy of the G-phonon in single and multi-layer graphene, through coherent anti-Stokes Raman Scattering. The nonlinear signal is dominated by a vibrationally non-resonant background, obscuring the Raman lineshape. We demonstrate that the vibrationally resonant coherent anti-Stokes Raman Scattering peak can be measured by reducing the temporal overlap of the laser excitation pulses, suppressing the vibrationally non-resonant background. We model the spectra, taking into account the electronically resonant nature of both. We show how coherent anti-Stokes Raman Scattering can be used for graphene imaging with vibrational sensitivity.

Tracking the Connection between Disorder and Energy Landscape in Glasses Using Geologically Hyperaged Amber.

Fossil amber offers the unique opportunity to investigate an amorphous material that has been exploring its energy landscape for more than 110 million years of natural aging. By applying different X-ray scattering methods to amber before and after annealing the sample to erase its thermal history, we identify a link between the potential energy landscape and the structural and vibrational properties of glasses. We find that hyperaging induces a depletion of the vibrational density of states in the terahertz region, also ruling the sound dispersion and attenuation properties of the corresponding acoustic waves. Critically, this is accompanied by a densification with structural implications different in nature from that caused by hydrostatic compression. Our results, rationalized within the framework of fluctuating elasticity theory, reveal how upon approaching the bottom of the potential energy landscape (9% decrease in the fictive temperature) the elastic matrix becomes increasingly less disordered (6%) and longer-range correlated (22%).

Raman spectroscopy of graphene under ultrafast laser excitation.

The equilibrium optical phonons of graphene are well characterized in terms of anharmonicity and electron-phonon interactions; however, their non-equilibrium properties in the presence of hot charge carriers are still not fully explored. Here we study the Raman spectrum of graphene under ultrafast laser excitation with 3 ps pulses, which trade off between impulsive stimulation and spectral resolution. We localize energy into hot carriers, generating non-equilibrium temperatures in the ~1700-3100 K range, far exceeding that of the phonon bath, while simultaneously detecting the Raman response. The linewidths of both G and 2D peaks show an increase as function of the electronic temperature. We explain this as a result of the Dirac cones' broadening and electron-phonon scattering in the highly excited transient regime, important for the emerging field of graphene-based photonics and optoelectronics.

Effects of central fibroblast growth factor 21 and irisin in anxiety-like behavior.

Adipose tissue and skeletal muscle are organs capable of secreting many bioactive molecules, such as adipomiokines that could be possibly involved in mood disorders. In the present work, we investigated the possible behavioral effects of a single intracerebroventricular (i.c.v.) injection of two adipomiokines, fibrobroblast growth factor (FGF)-21 (0.5-5.0 µg) and irisin (0.4-0.6 µg), in male rats tested in the open field and elevated plus maze tests. Prefrontal cortex levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and the gene expression of catechol-O-methyltransferase (COMT), dopamine transport (DAT) and tyrosine hydroxylase (TH), were measured by high performance liquid chromatography (HPLC) analysis and real-time reverse transcription polymerase chain reaction (RT-PCR). Both FGF-21 and irisin administration induced anxiogenic behavior, increased DA levels in prefrontal cortex, decreased COMT, DAT and increased TH gene expression. In conclusion, in the present study we demonstrated behavioral effects induced by central FGF-21 and irisin injections that could involve increased DA signaling in the prefrontal cortex.

Effects of central fibroblast growth factor 21 (FGF21) in energy balance.

Fibroblast growth factor 21 (FGF21) is known as a major metabolic regulator of glucose and lipid homeostasis. Continuous intracerebroventricular (i.c.v.) administration of FGF21 was found to modulate feeding and energy expenditure in rats with diet-induced obesity, suggesting a central effect by the peptide. In this context, in the present work, we studied the effects of a single central FGF21 administration (0.5-5 µg) on feeding and energy expenditure by evaluating locomotor activity, interscapular brown adipose tissue (BAT) weight, gene expression of uncoupling protein-1 (UCP-1) in BAT and plasma norepinephrine (NE) levels in Sprague-Dawley fed rats. In addition, we evaluated the effects of FGF21 on orexigenic [agouti-related peptide (AgRP) and neuropeptide Y (NPY)] and anorexigenic [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] peptides, in the hypothalamus, and dopamine (DA) and serotonin (5-hydroxytriptamine, 5-HT) levels in nucleus accumbens (NAc). We confirmed that central FGF21 administration induced a significant increase in food intake, possibly mediated by increased NPY and AgRP, and decreased POMC and CART gene expression. Moreover, FGF21 could modulate the motivational aspects of feeding, possibly through stimulated NAc DA levels. On the other hand, our findings of decreased locomotor activity, BAT weight, UCP-1 gene expression and plasma NE levels support a role for FGF21 in decreasing energy expenditure.

Crocus sativus, Serenoa repens and Pinus massoniana extracts modulate inflammatory response in isolated rat prostate challenged with LPS.

Prostatitis is a common prostate disease that could be promoted by bacterial or non-bacterial infectious agents. In addition, inflammatory pathways involved in prostatitis have been increasingly studied, and herbal extracts endowed with anti-inflammatory effects are under investigation, individually or in combination, for their efficacy in alleviating the burden of inflammation, with possible improvements in symptoms. Serenoa repens (Serenoa), in combination with Crocus sativus (Crocus) and Pinus massoniana (Pinus), has previously shown to improve sexual function and limit urinary symptoms in patients suffering from concomitant erectile dysfunction and lower urinary tract symptoms. In this context, the aim of the present study is to evaluate the efficacy of Serenoa, Crocus and Pinus extracts, either alone or in combination, on immortalized prostate cells (PC3) and in an experimental model of bacterial prostatitis constituted by ex vivo prostate specimens challenged with lipopolysaccharide (LPS). We found that the tested extracts were able to reduce ROS production by PC3 cells and NFkB and PGE2 activity in prostate specimens challenged with LPS. In addition, the pharmacological association of the extracts displayed synergistic effects indicating a rational use of the mixture of the tested extracts as a novel anti-oxidant and anti-inflammatory formulation in bacterial prostatitis. Finally, we performed analytical and in vitro evaluation to better characterize the phytochemical profile and the mechanism of action of selected secondary metabolites.

In vitro protective effects of resveratrol and stilbene alkanoic derivatives on induced oxidative stress on C2C12 and MCF7 cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which has been extensively studied for a wide range of biological effects. A large number of stilbene-containing derivatives have displayed antioxidant and antiproliferative activities on various cancer cell lines. In this study, a series of stilbene hybrids 1-9, previously reported as peroxisome proliferator-activated receptor (PPAR) agonists, were assessed at micromolar concentrations using MTT cell viability assay in C2C12 and MCF7 cell lines. The modulation of oxidative stress was also evaluated by measuring the protective effects on reactive oxygen species (ROS) production induced or not by oxidative stimulus. Among these, compounds 2 and 8 showed significant radical scavenging activity.

Direct observation of subpicosecond vibrational dynamics in photoexcited myoglobin.

Determining the initial pathway for ultrafast energy redistribution within biomolecules is a challenge, and haem proteins, for which energy can be deposited locally in the haem moiety using short light pulses, are suitable model systems to address this issue. However, data acquired using existing experimental techniques that fail to combine sufficient structural sensitivity with adequate time resolution have resulted in alternative hypotheses concerning the interplay between energy flow among highly excited vibrational levels and potential concomitant electronic processes. By developing a femtosecond-stimulated Raman set-up, endowed with the necessary tunability to take advantage of different resonance conditions, here we visualize the temporal evolution of energy redistribution over different vibrational modes in myoglobin. We establish that the vibrational energy initially stored in the highly excited Franck-Condon manifold is transferred with different timescales into low- and high-frequency modes, prior to slow dissipation through the protein. These findings demonstrate that a newly proposed mechanism involving the population dynamics of specific vibrational modes settles the controversy on the existence of transient electronic intermediates.

Central apelin-13 administration modulates hypothalamic control of feeding.

The 77 amino prepropeptide apelin has been isolated from bovine stomach tissue and several smaller fragments, including apelin-13, showed high affinity for the orphan APJ receptor. The distribution of apelinergic fibers and receptors in the hypothalamus may suggest a role of apelin-13 on energy balance regulation, albeit the studies reporting the acute effects of apelin on feeding control are inconsistent. Considering the possible involvement of apelinergic system on hypothalamic appetite controlling network, in the present study we evaluated in the rat the effects of intrahypothalamic apelin-13 injection on food intake and the involvement of orexigenic and anorexigenic hypothalamic peptides and neurotransmitters. Eighteen rats (6 for each group of treatment) were injected into the ARC with either vehicle or apelin-13 (1-2 µg/rat). Food intake and hypothalamic peptide and neurotransmitter levels were evaluated 2 and 24 h after injection. Compared to vehicle, apelin-13 administration increased food intake both 2 and 24 h following treatment. This effect could be related to inhibited cocaine- and amphetamine-regulated transcript (CART) gene expression and serotonin (5-hydroxytryptamine, 5-HT) synthesis and release, and increased orexin A gene expression in the hypothalamus.

Electronic resonances in broadband stimulated Raman spectroscopy.

Spontaneous Raman spectroscopy is a formidable tool to probe molecular vibrations. Under electronic resonance conditions, the cross section can be selectively enhanced enabling structural sensitivity to specific chromophores and reaction centers. The addition of an ultrashort, broadband femtosecond pulse to the excitation field allows for coherent stimulation of diverse molecular vibrations. Within such a scheme, vibrational spectra are engraved onto a highly directional field, and can be heterodyne detected overwhelming fluorescence and other incoherent signals. At variance with spontaneous resonance Raman, however, interpreting the spectral information is not straightforward, due to the manifold of field interactions concurring to the third order nonlinear response. Taking as an example vibrational spectra of heme proteins excited in the Soret band, we introduce a general approach to extract the stimulated Raman excitation profiles from complex spectral lineshapes. Specifically, by a quantum treatment of the matter through density matrix description of the third order nonlinear polarization, we identify the contributions which generate the Raman bands, by taking into account for the cross section of each process.

A natural formula containing lactoferrin, Equisetum arvensis, soy isoflavones and vitamin D3 modulates bone remodeling and inflammatory markers in young and aged rats.

A pivotal role in osteoporosis development is played by radical oxygen species (ROS), the increased production of which is related to inhibited osteoblastic activity and bone formation. A new field of research could involve medicinal plants with antioxidant and protective effects in osteoporosis. Furthermore, considering the multifactorial metabolic aspects of osteoporosis, the pharmacological association of multiple medicinal plants could improve patient response. The aim of the present study is to evaluate in vitro and in vivo the protective effects of a natural formula containing lactoferrin 12%, Equisetum arvensis ES 54%, soy isoflavones 34% and vitamin D3 0.002%, in PBMC and C2C12 cells and in the bone matrix of young (3-month-old) and aged (12-month-old) female Sprague-Dawley rats, following chronic (21 days) administration. In this context, we assayed the activities of several inflammation and bone homeostasis mediators, such as IL-6, TNFα, PGE2, osteoprotegerin, RANK, RANKL and NFkB. In vitro studies showed that natural formula (5-1000µg/ml) was able to significantly inhibit ROS and PGE2 production. In the same concentration range, the natural formula inhibited both TNFα and IL-6 gene expression. In the in vivo studies, we administered to young and aged female rats the natural formula at 5mg/rat for 21 days, finding a significant reduction in inflammatory PGE2 and NFkB activity. Nevertheless, we observed a significant increase in osteoprotegerin/RANKL ratio only in aged rats, compared to the respective control group. In conclusion, our findings corroborate the rational use of natural formula in the prevention and management of osteoporotic disease.

An easy-to-handle microfluidic device suitable for immunohistochemical procedures in mammalian cells grown under flow conditions.

Microfluidic, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

A natural formulation (imoviral™) increases macrophage resistance to LPS-induced oxidative and inflammatory stress in vitro.

Imoviral™ is a natural product formulation containing a mixture of uncaria, shiitake and ribes extracts. All ingredients are recognized as antioxidant, anti-inflammatory agent and immunomodulant. In order to evaluate the rational basis of extract mixture as immunomodulatory agent, we tested the effect of Imoviral™ formulation on macrophage response to lipopolysaccharide (LPS)-induced stress. The effect was evaluated as variation of reactive oxygen species (ROS) and prostaglandin E2 (PGE2) production and as cytokine gene expression. The extract did not affect cell viability up to 250 µg/ml. Treatment with extract (10-150 µg/ml) reduced ROS and PGE2 production as well as IL-8 and TNF-α gene expression. A pre-treatment with extract blunted LPS-induced production of ROS and PGE2, markers of oxidative and inflammatory stress, as well as the gene expression of all cytokines tested, indicators, in vitro, of immune response activation. In conclusion, we demonstrated that Imoviral™ formulation could be a useful tool to modulate the immune function, reducing the oxidative and inflammatory markers related to bacterial attack. Experimental data suggest that Imoviral™ extract mixture could also represent a preventive pharmacological strategy to enhance cell resistance to bacterial infections.

Acoustic dynamics of network-forming glasses at mesoscopic wavelengths.

The lack of long-range structural order in amorphous solids induces well known thermodynamic anomalies, which are the manifestation of distinct peculiarities in the vibrational spectrum. Although the impact of such anomalies vanishes in the long wavelength, elastic continuum limit, it dominates at length scales comparable to interatomic distances, implying an intermediate transition regime still poorly understood. Here we report a study of such mesoscopic domains by means of a broadband version of picosecond photo-acoustics, developed to coherently generate and detect hypersonic sound waves in the sub-THz region with unprecedented sampling efficiency. We identify a temperature-dependent fractal v(3/2) frequency behaviour of the sound attenuation, pointing to the presence of marginally stable regions and a transition between the two above mentioned limits. The essential features of this behaviour are captured by a theoretical approach based on random spatial variation of the shear modulus, including anharmonic interactions.

Spectrally tailored narrowband pulses for femtosecond stimulated Raman spectroscopy in the range 330-750 nm.

Spectral compression of femtosecond pulses by second harmonic generation in the presence of substantial group velocity dispersion provides a convenient source of narrowband Raman pump pulses for femtosecond stimulated Raman spectroscopy (FSRS). We discuss here a simple and efficient modification that dramatically increases the versatility of the second harmonic spectral compression technique. Adding a spectral filter following second harmonic generation produces narrowband pulses with a superior temporal profile. This simple modification i) increases the Raman gain for a given pulse energy, ii) improves the spectral resolution, iii) suppresses coherent oscillations associated with slowly dephasing vibrations, and iv) extends the useful tunable range to at least 330-750 nm.

Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter.

Non-activated macrophages express low levels of A(2A)Rs and lipopolysaccharides (LPS) upregulates A(2A)R expression in an NF-κB-dependent manner. The murine A(2A)R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5' untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A(2A)Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402-417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPß sites did not. Site-directed mutagenesis of CREB (309-320), STAT1 (526-531) and AP2 (566-569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582-588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A(2A)R expression in macrophages in response to inflammatory stimuli.

Effects of visfatin/PBEF/NAMPT on feeding behaviour and hypothalamic neuromodulators in the rat.

Visfatin, also known as pre-B cell colony enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (NAMPT), is a cytokine that is produced by adipose tissue, skeletal muscle, liver and immune cells. We studied the effects of visfatin/PBEF/NAMPT on feeding behavior, hypothalamic steady state concentrations of aminergic neurotransmitters and hypothalamic mRNA levels of anorexigenic peptides, such as cocaine- and amphetamine-regulated transcript (CART) peptide, corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and orexigenic peptides, such as agouti-related peptide (AgRP) and neuropeptide Y (NPY). Forty-eight rats were injected in the arcuate nucleus (ARC) of the hypothalamus with either saline or visfatin/PBEF/NAMPT (3 microg). Food intake was recorded 1, 2 and 24 h following injection, and either dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytryptamine, 5-HT) or peptide gene expression were evaluated 2 and 24 h after visfatin/PBEF/NAMPT administration. Compared to vehicle, visfatin/PBEF/NAMPT significantly increased food intake, as evaluated 1, 2 and 24 h post-injection. Visfatin/PBEF/NAMPT treatment led to a significant decrease of DA steady state concentration, CART and CRH mRNA levels. Consequently, visfatin/PBEF/NAMPT could play an orexigenic role in the ARC, and the effect could be mediated by modulation of DA, CART and CRH activity in the hypothalamus.

A multiherbal formulation influencing immune response in vitro.

AIM: Aim of this study was to evaluate the effects of phytocomplexes of Uncaria, Shiitake and Ribes in terms of viability and inflammatory response on immune cell-derived cultures. METHODS: Standardized extracts of Uncaria, Shitake and Ribes and their commercial formulation were tested on cell lines PBMC, U937 and macrophage. The activity was evaluated in terms of cell viability (MTT test), variations of oxidative marker release (ROS and PGE2) and modulatory effects on immune response (gene expression of IL-6, IL-8 and TNFα, RT-PCR). RESULTS: Cell viability was not affected by extracts, except subtle variations observed only at higher doses (>250 µg/mL). The extract mixture was well tolerated, with no effects on cell viability up to doses of 500 µg/mL. Pre-treatment of macrophages with subtoxic doses of the extracts reduced the basal release of oxidative markers and enhanced the cell response to exogenous oxidant stimulation, as revealed by ROS and PGE2 release reduction. The same treatment on macrophage resulted in a selective modulation of the immune response, as shown by an increase of IL-6 mRNA and, partially, IL-8 mRNA, while a reduction was observed for TNFα mRNA. CONCLUSION: Data confirm that extracts and their formulations can act as regulator of the immune system with mechanisms involving the oxidative stress and the release of selected proinflammatory cytokines.