A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.

BACKGROUND: DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. METHOD: An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. RESULTS: For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. COMPARISON WITH EXISTING METHODS: This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. CONCLUSIONS: The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.

Western blot expression of 5-lipoxygenase in the brain from striped dolphins (stenella coeruleoalba) and bottlenose dolphins (tursiops truncatus) with or without encephalitis/meningo-encephalitis of infectious nature.

Dolphin Morbillivirus (DMV), Toxoplasma gondii and Brucella ceti are pathogens of major concern for wild cetaceans. Although a more or less severe encephalitis/meningo-encephalitis may occur in striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) infected by the aforementioned agents, almost no information is available on the neuropathogenesis of brain lesions, including the neuronal and non-neuronal cells targeted during infection, along with the mechanisms underlying neurodegeneration. We analyzed 5-lipoxygenase (5-LOX) expression in the brain of 11 striped dolphins and 5 bottlenose dolphins, affected or not by encephalitic lesions of various degrees associated with DMV, T. gondii and B. ceti. All the 8 striped dolphins with encephalitis showed a more consistent 5-LOX expression than that observed in the 3 striped dolphins showing no morphologic evidence of brain lesions, with the most prominent band intensity being detected in a B. ceti-infected animal. Similar results were not obtained in T. gondii-infected vs T. gondii-uninfected bottlenose dolphins. Overall, the higher 5-LOX expression found in the brain of the 8 striped dolphins with infectious neuroinflammation is of interest, given that 5-LOX is a putative marker for neurodegeneration in human patients and in experimental animal models. Therefore, further investigation on this challenging issue is also needed in stranded cetaceans affected by central neuropathies.

Breathing pattern and head posture: changes in craniocervical angles.

AIM: The aim of this study was to observe the influence of oral breathing on head posture and to establish possible postural changes observing the variation of craniocervical angles NSL/OPT and NSL/CVT between oral breathing subjects and physiological breathing subjects. METHODS: A cross-sectional study was conducted. The sample included 115 subject, 56 boys and 59 girls, 5-22-year-old. Among these, 80 were classified as oral breathers and 35 as physiological breathers. The diagnosis of oral breathing was carried out thanks to characteristic signs and symptoms evaluated on clinical examination, the analysis of characteristic X-ray images, ENT examination with active anterior rhinomanometric (AAR) test. The structural and postural analysis was carried out, calculating the craniofacial angles NSL/OPT and NSL/CVT. RESULTS: Both NSL/OPT and NSL/CVT appear to be significantly greater to those observed in physiological breathing patients. This means that patients who tend to breathe through the mouth rather than exclusively through the nose show a reduction of cervical lordosis and a proinclination of the head. CONCLUSION: Our study confirms that the oral breathing modifies head position. The significant increase of the craniocervical angles NSL/OPT and NSL/CVT in patients with this altered breathing pattern suggests an elevation of the head and a greater extension of the head compared with the cervical spine. So, to correct the breathing pattern early, either during childhood or during adolescence, can lead to a progressive normalization of craniofacial morphology and head posture.

GPR55 and its interaction with membrane lipids: comparison with other endocannabinoid-binding receptors.

A number of integral membrane G protein-coupled receptors (GPCRs) share common structural features (including palmytoilated aminoacid residues and consensus sequences specific for interaction with cholesterol) that allow them to interact with lipid rafts, membrane cholesterol-rich microdomains able to regulate GPCR signalling and functions. Among GPCRs, type-1 and type-2 cannabinoid receptors, the molecular targets of endocannabinoids (eCBs), control many physiological and pathological processes through the activation of several signal transduction pathways. Recently, the orphan GPR55 receptor has been proved to be activated by many eCBs, thus leading to the hypothesis that it might be the "type-3" cannabinoid receptor. While the biological activity of eCBs and the influence of membrane lipids on their functions are rather well established, information regarding GPR55 is still scarce and often controversial. Based on this background, here we shall review current data about GPR55 pharmacology and signalling, highlighting its involvement in several pathophysiological conditions. We shall also outline the structural features that allow GPR55 to interact with cholesterol and to associate with lipid rafts; how the latter lipid microdomains impact the biological activity of GPR55 is also addressed, as well as their potential for the discovery of new therapeutics useful for the treatment of those human diseases that might be associated with alterations of GPR55 activity.

5-Lipoxygenase and cyclooxygenase-2 in the lungs of pigs naturally affected by enzootic pneumonia and porcine pleuropneumonia.

Enzootic pneumonia by Mycoplasma hyopneumoniae and pleuropneumonia by Actinobacillus pleuropneumoniae are among the most common and economically relevant pulmonary diseases in swine herds. We herein investigated the activity and expression of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in healthy and diseased porcine lungs, by means of immunohistochemical, immunochemical and biochemical assays. Diseased lungs showed a significantly higher activity and expression of 5-LOX and COX-2 in a wide range of cell types, thus suggesting the likely involvement of both enzymes in the pathogenesis of bacterial porcine pneumonia. Consistently, increased enzyme activities were paralleled by increased leukotriene B(4) (LTB(4)), a 5-LOX product and prostaglandin E(2) (PGE(2)), a COX-2 product, content in diseased versus healthy lungs.

Effect of the Enveloppe Linguale Nocturne on atypical swallowing: surface electromyography and computerised postural test evaluation.

AIM: Swallowing is a neuromuscular mechanism regulated by many nervous reflex arcs. Persistence of child swallowing at the end of dental eruption is called atypical swallowing (AS). This condition is related to a dysfunction of vertical maxillary growth called open bite. The authors treated this malocclusion with the Enveloppe Linguale Nocturne (ELN), or tongue positioner, created by Dr. Bonnet. The aim of this work is to evaluate the effect of ELN on swallowing and the postural variation obtained by its use. MATERIALS AND METHODS: Seven patients affected by AS were evaluated. Surface Electromyography (sEMG) testing was performed on each patient with different tongue positions, and swallowing was evaluated with and without the ELN. A surface Electromyograph (Biopack) with 8 channels was used (4 channels for the right muscles and 4 for the left) on 4 groups of muscles: temporals, masseters (MM), submental (SUB) and sternocleidmastoids. On each patient a postural test using a computerised Postural test (Lizard) was also performed. Statistical analysis was done using the Graph pad Instat 3 both for sEMG activity and for computerised postural analysis. RESULTS: All seven subjects had different results in the sEMG and footrest tests. The sEMG test results indicated that muscle activation and swallowing duration varied greatly with the use of ELN, with a reduction of time of swallow act (p = 0.002) and variation in contraction of muscles. Mean MM activation was higher without ELN than in tests performed with the appliance (p = 0.002). Mean SUB activation was higher with than without ELN (p = 0.0033). ELN has a therapeutic effect on posture too. Computerised postural test without device showed in all patients an alteration of barycentre as well as an elevated oscillatory record (A mmq; V mms). With ELN footrest kilogram difference (p = 0.0110), Oscillatory Area (P = 0.0102) and velocity of oscillation (P = 0.0102) presented a great reduction in respect to patients record without ELN. CONCLUSION: With ELN the tongue reaches the physiologic position during the swallowing and it is possible to have a low dental contact without tongue interference. ELN has no dental retention or contact. For this reason sEMG swallowing test shows that ELN induces a Mm activation reduction compared to swallowing test without ELN (P = 0.002) and an increase of SUB activation (P = 0.0033). In the same way with ELN there is a significant reduction of time of swallowing (c.f. oral phase) (P = 0.002). Patients with ELN changed their posture with a complete modification of barycentre (Footrest unbalancing kg P = 0.0110), oscillatory movement area (Footrest Area difference P = 0.0102), and oscillatory Velocity (Footrest Velocity oscillation difference P = 0.0102). These data suggest that this appliance has a function in the rehabilitation of atypical swallowing. ELN produces a physiologic neuromuscular mechanism that induces the correction of tongue position.

Clinical and computerized evaluation in study of temporo-mandibular joint intracapsular disease.

AIM: In this work authors show a diagnostic criteria in study of TMID: neuro occlusal clinical evaluation (NOE), T-Scan 2 system and surface electromyography (sEMG). METHODS: Nine patients 25-30 years old with TMID problem and 9 healthy group control have been selected and examined. On each patients it has been performed NOE, T-Scan and sEMG test. NOE has been calculated on each patient photos lateral mandibular excursion angle called: masticatory functional angle (AFM). T-Scan System is a computerized occlusal analyzer that provide in-depth understanding of the overall balance of the occlusion. At the same time of T-Scan record sEMG tests, in resting position and in maximum clench, have been performed. RESULTS: In healthy control there were no AFM difference. In no healthy group there were difference between the two AFM greater than 6 degrees. T-Scan COF showed how in healthy group control there was never a difference of COF greater than 5%. In no healthy group the difference were greater than 5% P<0.05. T-scan showed difference of time force in maximum intercuspidation (MIFT) in healthy respect TMID patients. In healthy patients MIFT was higher than TMID patients P<0.05. sEMG test showed in non healthy group a great asymmetrical activation of masseter (MM). MM activation were greater on side affected by joint sound than the balance side P<0.001. sEMG show how in TMID patients maximum masseter activation is always lower than maximum masseter activation of healthy subjects P<0.001. CONCLUSION: Neuro occlusal clinical evaluation (NOE) in TMID patients is supported by instrumental evaluation.

[Primary and secondary prevention procedures of temporo-mandibular joint disease in the evolutive age]. / Procedure di prevenzione primaria e secondaria delle disfunzioni dell'articolazione temporo mandibolare in età evolutiva.

In the last years prevention of temporomandiboular joint (TMJ) disease had acquired great importance. According to the neuro-occlusal rehabilitation (RNO) it is possible to say that TMJ disease starts since first years of life. So it is important both for dentist and for pediatric know what are the conditions and the atypical functions which predispose to this pathology. The aim of this work was to show how it is possible to intercept since primary teeth and the correct norms of primary and secondary prevention.

[Fertility changes in women working in greenhouses]. / Alterazione della fertilità nelle lavoratrici in serra.

BACKGROUND: Greenhouse workers (GW) are exposed to environmental contaminants, including pesticides, that may not only cause known immediate effects such as acute poisoning, but also long-term effects related to chronic exposure to low dosages, a problem that has not been extensively studied This study investigated the relationship between fertility changes and exposure to pesticides in a group of women working in greenhouses. STUDY GROUP AND METHODS: The analysis is based on a retrospective cohort of 145 women working in greenhouses located in the province of Latina, Lazio Region, Italy, who were exposed to pesticides at the time of their first pregnancy. Information on health status, lifestyle, work activity, reproductive history and some confounding factors (age, smoking, alcohol abuse, drug consumption) were collected using a questionnaire. Exposure to pesticides was classified in two levels (high or low) according to the work task and the length of exposure. Changes in fertility were measured in terms of time to pregnancy (TTP), that is the number of non-contraceptive cycles that it takes a couple to conceive. A control group was selected among public administration employees in the same province. The difference in average TTP between exposed and non-exposed groups was analysed by using Student t-test. A Cox proportional hazard model was used to compare TTP between the two groups after correction for confounding factors. RESULTS: In the high-level exposure group average TTP was 10.8 months (+/- 2.0), among the non-exposed average TTP was 6.2 months (+/- 1.0). The difference between exposed and non-exposed was thus 4.6 months (p<0.05). Comparison of the distribution of TTP between the high-level exposure group and nonexposed resulted in a hazard ratio of 1.27 (I.C. 95%: 1.03-1.79); the same analysis using the low-level exposure group and non-exposed group yielded a hazard ratio of 1.12 (I.C. 95%: 0.67-1.87). DISCUSSION: The study showed reduced fertility, in terms of TTP, in the population exposed to pesticides. Among GW, TTP was as much as 50% higher than for the control group. Controlling for confounding factors, the study confirmed an increase in risk for the exposed group. The issue under investigation, however, is complex as health status is not the only factor that needs to be taken into account in studies of reproductive health; emotional status as well as congenital and acquired factors may also have a notable impact on women's fertility. Occupational exposure, therefore, can be said to be a condition requiring careful analysis while bearing in mind that other factors may influence the outcome.

A rare case of synovial otorrhea of iatrogenic nature in dentistry.

The authors describe a unique case of left synovial otorrhea in a masticatory phase, occurring in a 64-year-old patient after a long dental sitting. The otoscopic examination and the transverse CT of the ear revealed the fracture of the anterior wall of left external auditory canal and the presence of a small bone fragment on the external side of the glenoid fossa. This situation, at present, has been judged inoperable by otologists due to the close relationship between this area and the head of the left condyle, probably responsible for the fracture. The therapy used was based on the use of a masticatory bite plate, with the aim of increasing the vertical dimension of the jaws during mastication and, so, to keep the condyle in a forward position. The result obtained was the decrease and then the halting of the liquid exit in the auricle, through an increase of the bite plate height determined by the ex iuvantibus method. This temporary therapy could bring about a spontaneous healing of the fracture, thanks to the masticatory increase of the bite plate; but if this is not possible, a fixed prosthetic solution should then be provided for the patient. Research into the literature revealed no similar observations.

Comparative analysis of deformation of two implant/abutment connection systems during implant insertion. An in vitro study.

AIM: The implant/abutment interface represents a crucial element to maintain the structural stability of the implant restoration and to prevent mechanical problems that can compromise the long-term success of the implant treatment. METHODS: In this study deformation of the external hexagonal and internal octagonal extension of 2 implants after applying different values of insertion torque has been tested. The implants were secured in a customized resin baseplate, with the implant neck exposed, then different values of insertion torque were applied by an electronic torque controller. RESULTS: Five internal octagonal implants were tested for 60 seconds to different torque magnitudes (12 Ncm, 21 Ncm, 27 Ncm, 36 Ncm, 42 Ncm). Two external hexagonal implants were tested for 60 seconds to a torque value of 27 Ncm and of 36 Ncm. CONCLUSIONS: After torquing tests, each implant was examined by a scanning electron microscope (SEM) medium and high magnification to evaluate the deformation of the 2 different antirotational systems. The SEM analysis of the internal octagonal and external hexagonal antirotational systems of the TBR implant showed a deformation proportional to the magnitude of the applied torque during implant insertion. The geometric deformation of the antirotational extension is capable of compromising its function after a torque magnitude of 36 Ncm or superior to this value. Moreover it was demonstrated a more evident alteration of the external hexagonal extension in comparison with the octagonal system after applying a torque value of 27 Ncm.

Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins.

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins. The first shell analysis of the EXAFS modulation is consistent with the presence of n=3Nepsilon(2) imidazole nitrogens at an average distance of 1.92 +/- 0.03 A from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n=5 with average distances of 1.93 A. Alternatively, by separating the contribution of Nepsilon(2) imidazole nitrogens and of peroxide O-atoms, n=3 ligands at 1.98 +/- 0.03 A and n=2 ligands at 1.87 +/- 0.03 A are found.

Saccharose solid matrix embedded proteins: a new method for sample preparation for X-ray absorption spectroscopy.

In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy.

[Research on accidents in a tire-producing plant]. / Indagine infortunistica in una azienda produttrice di pneumatici.

In the autumn of 1987 the U.S.L. health service (prevention, hygiene and occupational safety section) began a study about the accidents in a firm manufacturing tyres, placed in its own area. The retrospective enquiry starts from the analysis of typology, diffusion and seriousness of occupational accidents. The firm's accident register has been analyzed and integrated with other necessary information provided by the firm, by I.N.A.I.L. and by the air force metereological service. The study has been carried out on data concerning the following years: 1984-1985-1986. The accidents considered, implied absence from work and were divided as follows: for absence up till 3 days (in franchise), and more than 3 days (indemnified), applying the average value calculated on one year of the three analyzed. Every accident has been analyzed per year, month, day, hour of event. According to the classes: circumstances, kind of lesion, site of lesion, period of absence from work. The indices of: frequency, seriousness, incidence, mean duration have been calculated. The average monthly values of temperature: max and min. of the area and to the average monthly amount of processed elastomer (rate of production). The statistics we obtained, justified the study and showed the operative solution. The aspect of sanitary education and the general psychological aspect regarding the accident have been considered. Moreover the general operative solutions for the firm and specific ones for every department and for every position have been shown and faced up to. In this way, according to the risks that have emerged from the enquiries on previous accidents and thanks to direct inspection. it was possible to prevent accidents.