This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine  by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications. However, all three nanosensors have prominent medical implications, since it is well known that there is a strong link between environment and health and that, according to the WHO , at least 25% of diseases are due to environmental risk factors.2[2 emissions can remain in the atmosphere for periods of up to tens of thousands of years. The instruments generally employed in these determinations basically consist of infrared (IR) detectors performing a continuous plotting in the site with a good degree of accuracy, but they cannot be used for extensive mapping work because of the large number of expensive devices and specialized people that would have to be involved. Long-term sampling devices such as diffusive sampling techniques are the cheapest and easiest way.Sensors for detection of CO2 accumulation takes advantages of the properties of CaO to be carbonated by this gas as shown in reference [An alternative long-term sampling method for the determination of environmental CO2.For this purpose, we studied here the variation of mass connected to the carbonation process in order to assess the quantity of gas absorbed by a fixed amount of composite in relation to the concentration of environmental COin situ directly from printed cDNAs (complementary DNAs) just before the assay [f, when a mass, Δm, was adsorbed to or desorbed from their surface according to the Sauerbrey equation:0f is the fundamental frequency, A is the gold area and ρ and l are the quartz density and thickness, respectively [in vitro NAPPA- translated protein, Ramachandran et al. substituted the use of purified proteins with the use of cDNAs encoding the target proteins at each feature of the microarray [in vitro transcription-translation (IVTT) system. Mammalian proteins can be expressed in a mammalian milieu, providing access to vast collections of cloned cDNAs. The addition of a C-terminal glutathione S-transferase (GST) tag to each protein enabled its capture on the array through an antibody to GST printed simultaneously with the expression plasmid [For what concerns the two health prototypes ,5, one bhe assay ,12. Stanectively –9. Quartectively ,12) offeectively ,9. Buildcroarray . The pro plasmid ,11. In t plasmid ,12, optiN-terminal transcription-promoting domain, thus having an oncogenic activity. The MDM2-p53 plays a major role in cancer, being also a molecular therapeutic target and its monitoring is of crucial importance in cancer diagnosis and treatment [The proteins monitored by the NAPPA-based nanogravimetric biosensor, namely p53 and MDM2, are of fundamental importance in the molecular mechanisms leading to malignant cell transformation and cancer; p53 is in fact a 53-kiloDalton phosphoprotein oncosuppressor, encoded by a 20-kilobases gene situated on the short arm of human chromosome 17 and termed as the “guardian of the genome” and the “policeman of oncogenes” ,14. Mutareatment .o-, p-diphenols, aminophenols, polyphenols, polyamines, lignin, some inorganic ions, aryldiamines, benzenthiols, and phenothiazines.In this report, our goal was finally to build the prototype of the enzyme-based biosensor for medical purposes, in which the immobilization procedure was carried out via Langmuir-Blodgett films. The enzyme implemented in our device  was lacc2.Taking into account all the considerations so far discussed and the three sensors recently introduced in the literature ,3,5, in 2.1.2 accumulation takes advantages of the properties of CaO to be carbonated by this gas. We carried out preliminary tests to assess the best concentration of CaO in the composite, individuated in the CaO/PEG weight ratio of 1/4, by studying the variation of mass connected to the carbonation process, in order to assess the quantity of gas absorbed in relation to the concentration of environmental CO2 by a fixed amount of composite. We tested the sensing properties of the composite materials via a nanogravimetric method by using a home-made glass chamber of 340 mL in volume [2 via a nanogravimetric method by performing the experiments inside an atmosphere-controlled chamber filled with CO2[2 as a function of the gas concentration inside the atmosphere-controlled chamber, thus paving the way for the possible use of these matrices for applications in the field of sensor devices for long-term evaluation of accumulated environmental CO2. In reference [The alternative long-term sampling method for the determination of environmental COad hoc designed and built Plexiglas measuring chamber, divided into two parts: the upper one, with a funnel opening, allowed the exchange of the sensing matrix with the environment and the lower one that allowed the housing of the transducer , obtained as previously reported [A LB thin film  of recomreported , was preThe surface morphology and topology of the LB thin film of laccase was investigated via Atomic Force Microscopy (AFM) . The rou3.3.1.vs. time in order to assess the variation of mass at regular intervals of time after placing the small plastic cell in a room. We also measured the mass of the deposited sample at time zero, in order to have the value of frequency related to the composite before reacting with environmental CO2. We consequently carried out acquisitions of frequency vs. time in order to sample the indoor environmental CO2 in the room, illustrated in We performed nanogravimetric acquisitions of frequency per week and consequently the average quantity of CO2 in the environment. Experimental data, summarized in 2 absorbed by the composite issued a linear absorption, coherently with the constant human activity of the sampling period. Specifically, we found the variation of mass in relation to the quantity of CO2 present in the environment (indoors), indicated the concentration of indoor CO2 during the sampling period was 10 times less than the average concentration in the atmosphere, thus indicating the good quality of the air in our laboratory.Basing on our previous experiments , we wereSince the very good results were obtained from the usage of this long-term sampling device, we are presently programming new samplings both in indoor spaces affected by strong human activities and outdoors.3.2.i.e., before reticulocyte lysate addition;At the beginning of the expression process, i.e., after 90 min at 30 °C for gene expression and protein synthesis plus 30 min at 15 °C for protein immobilization) and after washing process, at 22 °C;120 min after reticulocyte lysate addition . The ratio named normalized D factor, DN = 2Γ/I, gives information on the shape of impedance curves and on sample viscosity, while the decrease in frequency f (Hz) is related to the amount of p53 molecules being immobilized.In The resulting Michaelis-Menten constants of the p53-MDM2  suggest that the NAPPA based biosensor functioned to monitor with high selectivity the single protein being expressed even in a mixture of different genes .4.2, we have realized a new device based on the use of a sensor technology (such as the nanogravimetric one) and an array of capture, highly specific for the gas of interest. Calcium oxide may be considered a valid solution to solve the problem of the detection of carbon dioxide due to its ability to selectively absorb this gas through a chemical carbonation reaction. The carbonation reaction leads to a substantial variation of the molecular weight and was therefore taken into account in the manufacture of gravimetric detection devices. The characteristics of the reaction allowed the construction of a dosimeter for the long-term analysis of the carbon dioxide, competitive with respect to the devices already available on the market based on infrared measurements  or on measurements of the variations of a voltage across a solid electrolyte depending on the concentration of CO2.In summary, our resulting prototypes appear to yield satisfactory proof of principles in the shown specific health and environmental applications. Indeed, to measure COFor what concerns the NAPPA QCM_D conductance device the results presented demonstrated a valid response for the protein-protein interaction analysis, exploiting the great advantage of this technique that allowed the real-time, label-free characterization of molecular binding kinetics to an immobilized receptor. A proof of principle was realized immobilizing p53 plasmid, resulting in a biosensor for MDM2. The most challenging prospective of the innovative biosensors emerging from this technology is the potential capability to develop a large number of sensors for molecules of biological and medical interest, by simply changing the cDNA immobilized on the sensor, without changing the detection technology. Among the avenues being presently explored NAPPA-based vaccines identification appears to represent an additional promising future perspective in the frame of the new OMICS-based Public Health. Vaccinology has emerged as a complex interdisciplinary science, especially because of the contributions of the new OMICS disciplines . In addiFinally, with the designed and realized amperometric sensor , in ordeet al. [As far as we know, this is the first comprehensive characterization of clomipramine concentration in different biological fluids of medical interest, since drug monitoring in different biological samples is very important. Biological fluids were taken from healthy donor volunteers, who gave their informed consent, and analyzed immediately after being collected. The motivation of studying electrochemical behavior of clomipramine in different biological fluid of clinical interest was to provide a comprehensive pharmacokinetic profile. Schimmell et al. ,30 studiet al. . CSF fluet al. ,33. As f


Objective: A study is made of the clinical course of patients with episodic cluster headache following the injection of corticosteroids in the proximity of the sphenopalatine ganglion of the affected side. Study Design: A retrospective observation study was made corresponding to the period between 2006 and 2010. Patients with episodic cluster headache received corticosteroid infiltrations in the vicinity of the sphenopalatine ganglion. Data were collected to assess the clinical course, quantifying pain intensity and quality of life. A total of 23 patients (11 women and 12 men) with a mean age of 50.4 years (range 25-65) were included. Forty percent of the patients had undergone dental extractions in the quadrant affected by the pain, before the development of episodic cluster headache, and 37.8% underwent extractions in the same quadrant after appearance of the headache.Results: Most of the patients suffered 1-3 attacks a day, with a duration of pain of between 31-90 minutes. The mean pain intensity score during the attacks at the time of the first visit was 8.8 (range 6-10), versus 5.4 (range 3-9) one week after the first corticosteroid injection. On the first visit, 86.9% of the patients reported unbearable pain, versus 21.7% after one week, and a single patient after one month.Conclusions: The evolution of episodic cluster headache is unpredictable and variable, though corticosteroid administration clearly reduces the attacks and their duration. Key words:Episodic cluster headache, vascular pain, sphenopalatine ganglion, corticosteroid infiltration. Episodic cluster headache (ECH) is characterized by unilateral attacks of pain in the upper premolar, periorbital or temporal region, or in a combination of these locations, accompanied by ipsilateral conjunctiva injection, tearing, nasal congestion, perspiration of the affected side of the face, mitosis, and homolateral palpebral ptosis and edema. The attacks typically last 15 to 180 minutes, and patients experience one or several attacks a day in the course of an outbreak of variable duration (weeks or months). The episodic presentation of the disorder is characterized by periods of complete remission .The etiopathogenesis of cluster headache is unclear, but appears to involve the activation of parasympathetic nerve structures in the sphenopalatine ganglion, which would explain many of the associated symptoms. In addition, activation of the ipsilateral hypothalamic gray matter could explain the typical circadian and circumannual periodicity of the disorder . The sphTreatment efficacy in cluster headache is difficult to evaluate, due to the great variations in the duration of each outbreak. Until improved knowledge is gained of the etiopathogenic mechanisms of cluster headache, the most useful treatment option is to counter one of the phenomena giving rise to the pain: arterial vasodilatation. The management of ECH includes three treatment modalities: non-pharmacological , pharmacological (abortive drugs and preventive treatment) and surgical.Abortive therapy aims to arrest individual attacks, and comprises the use of subcutaneous sumatriptan, ergotamine and oxygen inhalation ,7. The aThe present study evaluates the clinical course of patients with episodic cluster headache following the injection of corticosteroids in the proximity of the sphenopalatine ganglion of the affected side.Patient selectionA retrospective, descriptive and follow-up clinical case study was carried out between the years 2006 and 2010 in patients with episodic cluster headache seen in a University oral surgery clinic. Episodic cluster headache (ECH) was diagnosed according to the criteria of the International Headache Society (HIS) (The following patient inclusion criteria were used: 1) attacks of cluster headache; 2) treatment with corticosteroid injections close to the sphenopalatine ganglion; and 3) a minimum follow-up of 12 months. The criteria used for corticosteroid injection were: 1) patients with active ECH even after the start of some other treatment; and 2) the absence of general contraindications to corticosteroid injection . Episodic cluster headache was finally diagnosed in 26 patients; three patients were excluded due to a clack of the required minimum 12 months of follow-up. We thus included 23 patients with ECH, with a mean age of 50.4 years (range 25-65) (11 women and 12 men). Forty percent of the patients had undergone dental extractions in the quadrant affected by the pain, before the development of episodic cluster headache, and 37.8% underwent extractions in the same quadrant after appearance of the headache.Data collection and follow-up of painThe following data were collected: patient age and gender, dental surgical treatment in the affected quadrant before and after appearance of the pain, and characteristics of the pain: location, frequency (number of attacks/day), duration of the attacks (minutes), and intensity , where 10 corresponds to maximum pain intensity).Patient disability and quality of life were evaluated by means of a clinical scale scored from 0-6, where 1 = no pain, 2 = mild pain , 3 = bothersome pain , 4 = moderate pain , 5 = important pain  and 6 = terrible pain . We documented the days with pain corresponding to the period from the last outbreak to the time of the first visit , and the autonomic manifestations associated with the pain.The patients received corticosteroid injections in the proximity of the sphenopalatine ganglion of the affected side. In the case of more than one attack a day, we prescribed prophylactic treatment in the form of 2 mg of ergotamine via the oral route. In patients with a single daily attack of pain we only administered corticosteroids in the vicinity of the sphenopalatine ganglion. One infiltration a week was carried out during four weeks, and in the absence of pain throughout the week, no further infiltrations were made. The possible side effects of the medication were recorded.Injection techniqueTruncal superior posterior dental nerve block was carried out using an auto-aspirating metal syringe and long needle (25-30 mm) with 4% articaine and adrenalin 1:100,000 . The injection site was on the posterolateral surface of the upper maxilla, at 1 cm above and behind the last molar. The needle penetrated mesial to the upper first molar and was advanced towards the zygomatic process until reaching the vestibular depth of the upper second molar, directing inwards, upwards and backwards to form an angle of approximately 45 degrees with respect to the upper occlusal plane. The anesthetic solution was slowly injected after negative aspiration.While waiting a few minutes for the anesthetic solution to exert its effect, the empty anesthetic carpule was filled with 40 mg of triamcinolone acetonide , and the full contents of the carpule were injected into the vicinity of the sphenopalatine ganglion (in the same way as the anesthetic block).Clinical course The patient clinical course and improvement were assessed according to the pain intensity  and the clinical disability scale . The duration of the outbreak was classified as follows: less than 10 days, 11-20 days, 21-40 days, 41-60 days and over 60 days.In 47.8% of the patients the pain was located in the premolar region, irradiating towards the orbit and temporal region; in 39.1% of the cases the pain was located in the periorbital region, irradiating towards the temple; and in 13% of the subjects the pain was diffusely located in the upper maxillary, temporal and mandibular region. The pain affected the right side in 56.5% of the patients, and the left side in 43.5%. Most of the subjects experienced between 1-3 attacks/day, and in most cases the attacks lasted 31-60 minutes .The pain had been present for under 10 days in 26% of the patients, between 11-20 days in 17.3%, between 21-40 days in 13%, between 41-60 days in 17.3%, and for over 60 days in 26% of the patients. The mean intensity of pain on the first visit was 8.8 (range 6-10). The clinical scale on the first visit showed patient disability to be moderate in 13% of the case, important in 56.6%, and terrible in 30.4%. The pain was accompanied by vegetative manifestations in 73% of the cases – particularly nasal secretion, tearing and reddening in the affected zone.Corticosteroid treatment involved a single infiltration in 26% of the cases, two injections in 52.1%, and three injections in 21.7%. A total of 73.9% of the patients received additional drug treatment.The data relating to the number and duration of the attacks after the treatment received is reported in . One weeFig. . After 1Episodic cluster headache usually manifests in the second or third decade of life, though it can also appear in older age groups. In effect, the literature describes cases of first attacks of ECH manifesting within the 8-62 years age range . In our Many patients with cluster headache are wrongly diagnosed with symptomatic headache due to dental disease – this often leading to unnecessary and ineffective dental treatments . Bahra aThe pain is located in the periorbital and temporal area, extending to the ear and even to the occipital-cervical region . In the Most patients experience 1-3 attacks a day . AccordiA variety of treatments have been investigated for prevention or symptoms relief in these patients, including triptans ,23, ergoMir et al.  administAccording to Torelli and Manzoni , 85.7% oThe evolution of the patients with episodic cluster headache is unpredictable and variable, though the administration of corticosteroids clearly reduced the duration and intensity of the pain attacks, and the duration of the outbreaks.


Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.2/cm3.The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 mThe high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured. The number based size distribution of a material and the features of its surface are predominant criteria to classify it as a NM ,2. TEM r2/cm3, excluding materials consisting of particles with a size lower than 1 nm. The VSSA of a material is generally calculated from its bulk density and its mass specific surface area. The latter is usually determined by gas absorption methodology called the BET-method [To include also aggregates and agglomerates of primary particles and complex multi-component particles with external dimensions larger than the arbitrarily specified upper size limit of 100 nm, the VSSA is proposed as a complementary qualifier to distinguish a nanostructured material from a non-nanostructured material  proposesT-method  that allT-method ,5,7,8. MSuspensions of spherical and branched gold NP were obtained from IMEC . Aggregated silica nanomaterials NM-200 and NM-203 are supplied by the European Commission-JRC  as representative reference NM. They are used as well at the OECD Working Party for Manufactured Nanomaterials programme as principal materials and international harmonization standards. The NM were brought on pioloform- and carbon-coated 400 mesh copper grids  that were pre-treated with 1% Alcian blue  to increase hydrophilicity, as described by Mast and Demeestere . Gold NPTo obtain a maximal field of view, grids were mounted in a tomography holder  such that the squares were oriented diagonally with respect to the axis of the holder. Only objects in the centre of a grid square were analyzed using a Tecnai Spirit TEM (FEI) with a BioTWIN lens configuration and a LaB6-filament operating at an acceleration voltage of 120 kV.Series of micrographs (tilt-series) were recorded semi-automatically assisted by the Xplore 3D tomography-module of the microscope control software (FEI) over a tilt range of at least 65°, or highest angle possible, at intervals of 1 degree. Shift and focus changes were corrected at every interval. Electron micrographs were acquired with a 4*4 K Eagle CCD-camera (FEI) at magnifications of 26,500 to 49,000 times and corresponding pixel sizes of 0.49 to 0.22 nm. The tilt series were aligned using the Inspect 3D software, version 2.5 (FEI) by iterative rounds of cross correlation until the alignment shifts were approaching to zero. Because of their higher signal to noise ratio, reconstructions using 10 to 20 iterations of the Simultaneous Iterative Reconstruction Technique (SIRT) algorithm were superior over reconstructions based on weighted back projection (WBP) and on the Algebraic Reconstruction Technique (ART) algorithm (not shown).For visualization in 3D, the Amira software, version 4.1.2  was used. Isosurface rendering was used to compute a triangular approximation of the interfaces between the segmented sections. The segmentation was obtained based on a single threshold. This was chosen such that the obtained surface optimally matches the boundaries of the reconstructed orthogonal digital slices (orthoslices) of the NM in the xy-plane, where resolution is highest. The resulting surface was visualized using pseudo-coloring. To reduce missing wedge artifacts, so-called streaks, the surface was smoothed using a 2 × 2 × 2 averaging of voxels (downsampling). Using the 'Create Surface' function of Amira, a surface was derived from the isosurface, which allowed measurement of the surface area of the reconstructed 3D objects and of their enclosed volume.2) and the volume (4/3πr3) of a perfect sphere, where r is replaced by the measured ECD of the projected particle divided by two. The sphericity, describing the 'roundness' of a particle by using central moments, was used to assess the hypothesis that the particle is a sphere in reality.Two-dimensional parameters of the reconstructed NP were measured from the TEM micrographs taken at 0° using the AnalySIS Solution of the iTEM software . Briefly, contrast and brightness of the micrographs were optimized, the involved particles were enclosed in a frame (region of interest) and thresholds were set to separate particles from the background based on their electron density and size. The surface area and volume of individual spherical particles were approximated by the formulas to calculate the surface area  was used.To measure the strength of correlation between the calculated VSSA and the measured VSSA, the nonparametric Spearman rank order correlation test was calculated using the SigmaPlot software, version 11.0 . To test the hypothesis that the mean VSSA obtained from ET reconstructions equals the threshold of 60 mElectron tomographic reconstruction allowed visualizing the spherical gold NP in three dimensions Figure . The par2 and 38,763 nm3, respectively. This corresponds with a VSSA of 332 m2/cm3. The mean VSSA ± SEM, determined from 10 ET reconstructions  from 60 m2/cm3.From the isosurface based volume rendering of the ET reconstructions, the total surface area and volume of their composing gold particles could be measured. For example, the total surface area and the volume of the NP shown in Figure The reconstructed gold particles showed no obvious elongation along their z-axis and image analysis of the transmission electron micrographs of the individual particles taken at a tilt angle of 0° resulted in a mean sphericity of 0.86. Hence, it was concluded that these gold particles are almost spherical and that their surface area and volume can be closely approximated by the formulas to calculate the surface area and the volume of a perfect sphere. Figure 2/cm3.Branched gold NP measure approximately 50 nm in diameter and show a highly irregular rather than a spherical morphology: they are characterized by their surface extensions or peaks. These features can be deduced from 2D images, like the original micrograph Figure  and the 2/cm3.It is not evident to envisage the structure of the silica reference materials NM-200 and NM-203 appropriately by conventional bright field TEM Figure  and 3C. By electron tomographic reconstruction based on conventional BF TEM, the general morphology of gold and silica NM was visualized in 3D. In orthoslices of the examined NM in the xy-plane, as presented in Figure Segmentation by isosurface rendering allows accessing the 3D information of an ET reconstruction in greater detail than digital slicing. Such 3D visualization and measurement of the surface features of NM can contribute to bring the second condition of the definition of a nanomaterial proposed by the European Commission  in pract2/cm3 such that these materials can be classified as NP according to the third condition of this definition. As opposed to the BET-method [From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the VSSA was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 mT-method , ET is nTo optimally characterize the morphology of a NM by ET reconstruction, it is required that (i) the projection requirement is met ; (ii) miOur results indicate that, in principle, the characterization and definition of NM can benefit from application of conventional BF ET. In the scope of putting this technique in practice for the characterization and definition of gold and silica NM, following approach is suggested to reconcile the limitations of conventional BF ET with the above-described conditions.(i) The projection requirement states that for an image intensity to be usable for ET reconstruction, it has to be a monotonic function of a projected physical quantity . The exa(ii) An important physical limitation of electron tomography arises from the fact that finite specimen thickness and tilt geometry within an electron microscope column prevent the collection of projection images spanning a complete angular range (± 90° tilt series). This results in a "missing wedge" of information in reciprocal space and results in anisotropic resolution in the resulting reconstruction . Only fo(iii) In the examined NM, the value of the threshold was selected such that the obtained surface optimally matches the boundaries of the reconstructed orthogonal digital slices (orthoslices) of the NM in the xy-plane, where resolution is highest. This threshold value was in general close to the minimal value between both peaks of the bimodal curve Figure  of the h2/cm3) is higher than the value obtained by SAXS , but lower than the values obtained by BET . For NM-203, the VSSA estimated by ET (219 ± 23 m2/cm3) is lower than both the VSSA obtained by SAXS  and BET .To evaluate the influence of missing wedge artifacts on the reconstruction and, in particular, on the validity of the quantitative results obtained from the reconstructions, the ET based analyses of colloidal gold nanoparticles with a known spherical morphology were evaluated as a control. The high correlation (r = 0.98) between the measured values of area and volume and the areas and volumes calculated from the ECD of projected NP indicates that the values measured from electron tomographic reconstructions are valid for these gold particles. On this basis, it is assumed that the surface and volume measures of the branched gold and silica NM, which lack symmetry, can be relied on also. However, it has to be stressed that the absolute numbers presented in Table The EU definition expects a resolution of at least 1 nm . It is dAs a proof of principle, it was shown that application of conventional BF ET allows 3D visualization of the examined gold and silica NM and allows measuring their surface features and VSSA. This approach can hence contribute to bring the second and third condition of the definition of a nanomaterial proposed by the European Commission in practice . Recent The authors declare that they have no competing interests.EVD and JM contributed equally to this manuscript. EVD produced most of the electron tomograms and the illustrations. JM developed the basic concept and took care of the redaction of the manuscript. PJD assisted in sample preparation and MAF assisted in the alignment and reconstruction and visualization of electron tomograms. All authors have read and approved the final manuscript.Electron tomographic reconstruction of branched gold NP. The video shows a surface rendered view of the branched gold NP shown in Figure Click here for fileElectron tomographic reconstruction of nanostructured silica nanomaterial NM-200. The video shows a surface rendered view of the nanostructured silica NP shown in Figure Click here for fileElectron tomographic reconstruction of nanostructured silica nanomaterial NM-203. The video shows a surface rendered view of the nanostructured silica NP shown in Figure Click here for file


Shape memory alloy (SMA) staples in nickel titanium with shape memory effect are effective for spinal growth control. This study was designed to evaluate the biomechanical properties of the staples and observe the stability of the fixed segments spine after the staples were implanted. According to the vertical distance of the vertebrae, SMA staples of 5, 6.5, and 8 mm were designed. The recovery stress of 24 SMA staples in three groups was measured. The pullout strength of SMA staples and stainless steel staples in each functional spinal unit was measured. Each of the six fresh specimens was divided into three conditions: normal, single staple, and double staples. Under each condition, the angle and torque of spinal movements in six directions were tested. Results show that the differences in recovery stress and maximum pullout strength between groups were statistically significant. In left and right bending, flextion, and extention, the stability of spine was decreased in conditions of single staple and double staples. Biomechanical function of SMA staples was superior to stainless steel staple. SMA staples have the function of hemiepiphyseal compression and kyphosis and scoliosis model of thoracic vertebrae in goat could be successfully created by the fusionless technique. Idiopathic scoliosis deformity is one of the common diseases in children and adolescents , 2. Its Currently, surgical or nonsurgical treatments for the near future or forward to change the natural development course of the disease of scoliosis deformity is used for the treatment of children and adolescents with idiopathic scoliosis deformity with full consideration to the premise of the disease natural history , 5. BracDuring the growth of spine, multiple organic centers are formed. It is possible to control certain organic centers in modulating spinal growth. At the same time, spinal deformity is to be corrected and partial functions are to be retained . With moHemiepiphyseal compression staples were intrasegmental fixation instrument designed by hemiepiphyseal block technique , 18. In Shape memory alloy (SMA) staples were designed according to the measurements of goat's vertebral. Maximum pullout force of the staples with different materials and design, biomechanical properties, stability of the fixed segment, and the impact on the growth of the goat's spine were evaluated in our research. It provided a reference for clinical application. Goat's thoracic has 13 segments, shaped like a human vertebral body, but the middle sag is more obviously. Six female normal goat's  thoracic spine specimens were excluded paravertebral fat, muscle, and other soft tissue retaining ligaments and joint capsule that did not damage the bone structure. Digital X-ray and CT were taken to expel tumors and congenital malformations.The vertebral body side 1/3 cross-sectional distance of each adjacent segments in T6–T11 was measured as According to the above measurement, we designed two kinds of orthopedic staples with two tines or four tines, respectively. Its length was 10 mm, the tine depth was 4 mm and thickness was 2 mm, the width was 2 mm or 4 mm, respectively. Each kind of staples was designed into 3 types which had different distances between the tines as 5 mm, 6.5 mm and 8 mm. In order to increase its antipullout and the antirotation performance, we design groove in the tine of the staples . In acco One important indicator for evaluation of shape memory effect of memory alloy is the recovery stress, which is generated by heating to a temperature above the body temperature to restore the original shape of the alloy. This maximum recovery stress test was carried out to evaluate the shape memory effect of the new designed staples. In this study two-tine SMA staples were chosen and divided into three groups: 5 mm group, 6.5 mm group, and 8 mm group. Each group had eight staples. At first, the interdental distance between the tines of orthopedic staples was stretched to 10 mm  in the ice water mixture; then the staples were placed into the corresponding fixture in thermostat water bath . At lastSpecially designed two-tine stainless steel staples, three kinds of two-tine shape memory alloy hemiepiphyseal compression staples and implantation related instruments: holding pliers, distraction pliers, and vertebral opening device, supplied by Beijing Ming Yuen Laboratory Furniture Co. Ltd. and Beijing HengShi ZiZheng Biotechnology Co. Ltd.Six fresh specimens of the vertebrae (T6–T11) in goats (age 2-3 months) were selected and excluded paravertebral fat, muscle, and other soft tissue retaining ligaments and joint capsule without any impairment on the bone structure. Each specimen of the vertebrae (T6–T11) was cut into three functional spinal units (FSU): T6-T7, T8-T9, and T10-T11, including the adjacent vertebrae and their intervertebral discs, facet joints, and ligaments connecting structure. Each FSU was randomly assigned and recombined into six vertebral body specimens, and every specimen contained the 3 FSUs of T6-T7, T8-T9, and T10-T11. Specimens were saved in −80°C and natural thawed 12 hours before testing .Stainless steel and shape memory alloy hemiepiphyseal compression staples were successively implanted into FSUs, which were divided into four groups: 5 mm group, 6.5 mm group, 8 mm group, and stainless steel staples group, and eight SMA and twenty-four stainless steel staples were tested in each group.(1) Pull-Out Test of Stainless Steel Hemiepiphyseal Compression Orthopedic Staples. Twenty-four stainless steel orthopedic staples were randomly assigned and then were implanted in the left and right side of each adjacent vertebrae of FSU from the anterior lateral across the intervertebral space, placed in front of the vertebral transverse process costal fovea. Simultaneously, the transverse process and part of the spinous process of the vertebral body were removed ( removed . The exp removed . On the (2) Pull-Out Test of SMA Staples. In order to compared the pull-out strength of stainless steel and SMA staples under the same conditions, stainless steel orthopedic staples were removed from the vertebral body specimens, and then two SMA staples of 5 mm, 6.5 mm, and 8 mm, respectively, implanted cross into each group of FSU. The center of memory alloy orthopedic staples roachback was clamped by holding pliers and then implanted in the left and right side of each adjacent vertebrae of FSU from the anterolateral across the intervertebral space, the same implantation position with stainless steel staples. And then SMA staples were treated with 55°C hot water to recover their deformation and fix firmly into the vertebral cancellous bone. The following test process was the same as above.Twelve specimens of the vertebrae (T5–T12) obtained from goats of the same age were selected and treated just as described in Specially designed 5 mm two-tine SMA staples and implantation instruments as above were used.The experiment groups were divided into four states: (1) a normal state ; (2) single staple state ; (3) double staples state ; (4) postoperative single staple state (a 5 mm SMA staple activated as described in (1) Test of Spinal Stiffness in Normal State (In Vitro). The stability of the spine under normal state without staples was tested on the MTS858 Mini biomechanical test machine, with a spine fixtures 608.30,4-of DOF sensor. Experiment dedicated fixtures were fixed at the sensor's two terminals with screws, to ensure that the vertical center axis of the upper and lower fixtures was coincided in the same straight line, and that the horizontal axes were paralleled to each other. Spine specimens embedded were placed in the hollow cylinder of up and down ends of the fixture and fixed with screws. Before each test, the direction of the sensor had to be adjusted through the computer to ensure the correct position of the fixture. The torque and angle of goat thoracic specimens was controlled at the same time, so that the spine was applied to six physiological movement including the left and right rotation, left and right lateral bending, flextion, and extention (xtention . The mov(2) Test of Spinal Stiffness in Single Staple State (In Vitro). After the test in normal state was completed, the SMA staples were implanted in the right side of each adjacent vertebrae from 1 the vertebral transverse costal fovea anterolateral across the intervertebral space, with each intervertebral space one staple. Testing process and data calculate was the same with that in normal state.(3) Test of Spinal Stiffness in Double Staple State (In Vitro). After the test in single staple state was completed, five SMA staples were implanted in the right side of each adjacent vertebrae from the anterolateral portion of staples implanted before across the intervertebral space that was two staples in each intervertebral space. Testing process and data calculate was the same with that in normal state.(4) Test of Stability of the Spine in Postoperative Single Staple State (In Vivo). Female goats of 2-3 months old, weighing 6–10 kg, were selected as specimens. Eight goats were treated with a same implantation surgery, and three goats without surgery were set as blank group. The surgical procedure was carried out as follows. The animals were anesthetized with intravenous injection of 3% pentobarbital sodium and then placed in left lateral position. The skin was straightly incised paralleled to the intercostal space between the sixth and seventh ribs and treated with electric coagulation. Then the subcutaneous tissue, the muscle tissue, and the rib periosteum under the surface layer of 7th rib were successively cut. Subperiosteal dissection was carried out to expose an 8 cm long section of rib, 1 which was cut and removed by a rib shear. After the periosteum and the parietal pleura below the rib were cut and the chest was open, the right lung showed an equal and satisfactory expansion. The thoracotomy device was installed to stretch the 6th and 8th ribs. The lungs under protection were retracted sidewards by dever retractors to reveal the thoracic vertebrae, which were arranged in neat rows, without side bend. A 5 mm shape memory alloy orthopedic staple was placed in ice water mixture and the two tines were stretched using distraction clamp to make the interdental distance 10 mm, which changed the original “C” shape of orthopedic nail into an open rectangular shape for use. The vertebral body and intervertebral space were identified. The bottom of the T6 vertebral body and the top of the T7 vertebral body in the anterior lateral portion of costotransverse joint were perforated using vertebral opening device across the T6 and T7 intervertebral space. Then the stretched shape memory alloy orthopedic staple held by distraction pliers was implanted into the channel of T6 and T7 vertebral bodies. Finally, the staple was beaten and compressed by a hammer device. Five 5 mm shape memory alloy orthopedic staples were implanted to the anterior portion of costotransverse joint and the anterior lateral of the vertebral bodies across the T6, 7, T7, 8, T8, 9, T9, 10, and T10, 11 intervertebral space successively  and pairwise comparison post hoc test. The significance level was set at P < 0.01). Therefore, the 5 mm group exhibited the maximum of recovery stresses and the 8 mm group exhibited the minimum.The maximum recovery stresses of SMA staples were determined as P < 0.05). Therefore, the 5 mm group exhibited the maximum pullout strength and the 8 mm exhibited the minimum in the SMA staple groups. All the mean values of the three SMA staple groups were higher than that of stainless steel group.Comparisons of maximum pullout strength among the SMA staples and stainless steel groups were shown in P < 0.05). And in left and right rotation, there was no significant difference between those two conditions and normal one (P > 0.05).The results of stiffness test were shown in in vivo. Through observation by X-ray, seven in eight of the goats developed scoliosis in different time, and two finally developed kyphosis. Therefore, the model-forming rates for scoliosis and for kyphosis were, respectively, 87.5% and 10.9%. Meanwhile, five in sixty SMA staples implanted into goat bodies were found loosing and shifting by continuous observation of X-ray. After the goats were sacrificed, the check of the staples showed that the shifting of staples was only developed to some extent, without dropping into the thoracic cavity from the vertebrae. The results of stiffness test of single staple state in The radiograph of one specimen was shown in In recent years, goats, calves, dogs, pigs, and other large animals were used for research and development of the intrasegmental fixation instrument by more and more scholars –24. In tThe typical characteristic of memory alloy is shape memory effect, which is generally caused in martensite inverse phase change –27. AfteThe early stability of any intrasegmental fixation instrument implanted into bodies is crucial to the success of the surgery and it is an important evaluation method to test the pullout strength , 30. Thein vitro [in vitro environment. Memory alloy orthopedic staples were implanted into right sides of T6–T11 thoracic vertebrae and the stability test of six physiological movements including the left and right rotation, left and right lateral bending, flextion, and extention was achieved. Results showed that, along the axis of rotation, whether to implant the memory alloy orthopedic staple or how many staples to be fixated would not increase or decrease the stability of the spine, whereas on the directions of lateral bending (left/right), flexion, and extension, the stability of single staple state and double staple state of the spine samples was lower than that of the normal state. This might be explained that due to the implantation of the memory alloy orthopedic staples and its corresponding recovery stress, the cancellous bones at one side of the vertebrae were compressed; an effect approximates to a slight wedge-shaped deformity on the side of the vertebrae. Upon right lateral bending and flexion of the spine samples, the rift between the outer surface of the memory alloy orthopedic staples and the cancellous bones of the vertebrae became narrower; thus the staples played a role here as a leverage, increasing the range of motion of the left and rear intervertebral disc, as a result the entire stability of the spine was decreased. Upon left lateral bending and extension, the deformity in the right and frontal intervertebral disc was in consistence with that of the normal state, whereas the rift between the outer surface of the memory alloy orthopedic staples and the cancellous bones of the vertebrae was enlarged, causing the separation movement of the staples and bones. This might also increase the range of motion of the entire spine and decrease its stiffness. Betz et al. [in vitro anterior implants of fixtures did not necessarily increase the stability of the spine. Spinal stability refers to the ability of spine to maintain its own balance position, which reflects the relationship between the load and the displacement caused by the load. Under the same load, the smaller the displacement changes, the stronger the stability becomes. From the view of biomechanics analysis, spinal instability refers to the spinal activity abnormality increases, or the decrease of the FSU stiffness. Stiffness is the spinal ability of resistance to deformation, which is one of the indicators for evaluation of the carrying capacity of spine. The stiffness test of spinal fixed segment is commonly used for biomechanical stability test of intrasegmental fixation instrument in vitro , 32. Thiz et al.  have carin vitro showed a decrease of spinal stability, the postoperative stiffness test after 4 months presented a restoration of the stability. The cause of the recovery may be as follows. (1) In the living state, the bones of vertebral body could be repaired and rebuilt by themselves, which could narrow the interspace and alleviate the movements between the metal surface and the vertebral body. (2) The appearance of the connective tissue and new bones around SMA staples remedied the adverse effects caused by lateral compression of bones. (3) The structure of ligament could be repaired and the balance could be rebuilt.Although the immediate stiffness test In vitro, there existed an instant decrease of stability with the implantation of SMA staples. However, the spine could increase its stiffness and stability after the SMA staples were implanted in vivo for certain times and partial functions were also reserved in the fixed segments. SMA staples which have the function of hemiepiphyseal compression could be also used for kyphosis and scoliosis model of thoracic vertebrae in goat.In this research, two kinds of SMA staples were successfully designed. SMA staples presented better antipullout performance, which was superior to stainless steel staple.


Objective. To determine the effects of enzyme replacement therapy (ERT) on the hearing acuity in patients with Fabry disease. Materials. The study sample comprised 34 ears of 17 affected patients who underwent pure-tone audiometry before and after ERT. Methods. The patients were studied in relation to factors such as changes in hearing, presence of accompanying symptoms, status of renal and cardiac function, age, and gender. Data of pure-tone audiometry obtained before ERT and at the final examination were compared. Results. At the end of the follow-up period, no significant worsening of hearing acuity was noted at the end of the follow-up period. SSNHL was detected in 10 ears of 6 patients. Steroid therapy successfully cured the disease in 9 of the 10 ears. Conclusions. No significant worsening of hearing acuity was noted from the beginning to the end of ERT. The rate of improvement in SSNHL of Fabry disease was excellent in the treated patients. Hearing loss is a factor that causes marked deterioration of the patients' quality of life, and it is desirable that the hearing acuity of patients be periodically evaluated and prompt treatment of SSNHL be administered, if available. This disorder was first reported in 1898 by 2 independent investigators, namely, Anderson from the UK and Fabry from Germany [Fabry disease is a genetic inborn error of metabolism in which the enzymatic activity of The disease mainly involves the kidney, heart, and brain, and accordingly, affected patients often die in their 40 s or 50 s because of renal failure, heart failure, or cerebral infarction. In addition, angiokeratoma and hypohidrosis are present, with a variety of neurologic symptoms, including severe pain in the extremities, burning sensation, headache, dizziness, hearing loss, lack of motivation, and neurosis. According to the published literature, 54.5%  to 80% [α-Galβ (Fabrazyme) and α-Galα  were approved in Europe in 2001 and in the USA in 2003. In Japan, Fabrazyme was approved in 2004 and Replagal in 2007. Early initiation of ERT has been reported to prevent or improve renal failure and heart failure [As agents for enzyme replacement therapy (ERT),  failure , 7. Stud failure . However In this study, we examined 34 ears of 17 patients (8 men and 9 women) with Fabry disease, who underwent pure-tone audiometry before and after ERT. All these patients had a normal mean hearing level of less than 20 dB before the initiation of ERT. The mean age of the patients at the first visit was 35.0 years , and the mean follow-up period was 46.6 months (8–90 months). The patients were investigated for the course of changes in hearing and the severity of accompanying symptoms , tinnitus, and vertigo); different groups of patients were compared in terms of factors such as renal and cardiac functions, age, and gender. Hearing impairment was evaluated by pure-tone audiometry by using the five-division method, (250 Hz + 500 Hz + 1000 Hz + 2000 Hz + 4000 Hz)/5, according to the diagnostic criteria for sudden hearing loss in Japanese patients. The results of pure-tone audiometry performed before starting ERT and at the final follow-up examination were compared. The severity of SSNHL and the outcome of therapy were classified according to the classification criteria set for assessing sudden hearing loss in Japanese patients (Tables χ2 test). Thirteen of the 17 patients had tinnitus, while 9  had vertigo. SSNHL was found in 10 ears of 6 patients . The severity of SSNHL was grade 1 in 9 ears and grade 3 in 1 ear. The condition was judged as cured in 9 of the 10 ears after steroid therapy, on the basis of our criteria for improvement. Repetitive SSNHL was found in 3 (1 man and 2 women) of the 6 patients with SSNHL. The incidence of SSNHL tended to increase over the follow-up period, and all patients with renal dysfunction had SSNHL.Data collected over the study period are presented in The findings of typical cases of SSNHL are presented in  The patient developed SSNHL (grade 1) on 1 side at 41 months of age and on the opposite side at 54 months of age; at both instances, the patient was cured by steroid therapy.  SSNHL (grade 1) first occurred when the patient was 48 months of age and then again reoccurred at the opposite side at 60 months of age; both ears responded to steroid therapy. This patient showed no symptoms of the Fabry disease other than hearing loss. In this case, the patient developed SSNHL first at 20 months of age with grade 1 severity and then at 67 months with grade 3 severity. The condition was successfully treated with steroid therapy at both instances.SSNHL (grade 1) occurred at 84 months of age and did not respond to steroid therapy.Since the Fabry disease is rare and shows diverse symptoms in the initial stages of manifestation, the definitive diagnosis of this disease was often delayed in the past. However, in recent years, Fabry disease has become more widely recognized, and therefore the disease is now increasingly detected at an early stage and ERT is soon initiated. In this study, all the enrolled patients had normal hearing of less than 20 dB before ERT, and the mean follow-up period was approximately 4 years.  Because the model of Fabry disease showed a densely-stained material accumulation in the inner ear stria vascularis cell  and orga Some important findings of this study were that no eventual worsening of hearing occurred while the patients received ERT and that the incidence of SSNHL was obviously higher in this population than the incidence of sudden hearing loss in the general population. None of the patients required hearing aids, with the mean hearing level of the patients being less than 30 dB even for those who showed more than 5 dB decrease in hearing acuity. The pathologic status of SSNHL in Fabry disease remains unclear; however, some type of vascular disorder is believed to occur, similar to that observed in cerebral infarction. Although the incidence of SSNHL was higher among patients who were followedup for more than 3 years, mild cases of grade 1 severity were predominant, and the cure rate was as high as 90%. These results suggest that ERT inhibits the onset of hearing loss and SSNHL and decreases the severity of the latter. However, since this study did not include a placebo control group, the results cannot be compared against patients not receiving the therapy. Therefore, the results of the therapy over a longer period of observation should be studied before reaching a conclusion as to whether ERT is truly effective. In our previous study, several patients had severe hearing loss . These w The results of this study indicate that patients with Fabry disease should be periodically evaluated for hearing acuity and administered prompt treatment of SSNHL, if available, in order to maintain their QOL. A new finding of this study is that repeated hearing loss was observed as the only symptom of Fabry disease, albeit in 1 case (Case 14). This is suggestive of the diversity of the clinical presentation of Fabry disease and possibly points towards the existence of the auditory subtype of this disease, in addition to the renal subtype and the cardiac subtype. Further investigations of the pathologic condition of this disease in the light of these findings are desirable.No significant worsening of hearing acuity was noted until the end of ERT. The incidence of SSNHL tended to be higher in patients who were followed up for a longer duration and who had renal dysfunction. SSNHL that occurred in patients on ERT for Fabry disease was relatively mild, and the cure rate for steroid therapy was 90%. The results suggest that ERT delays the onset of hearing loss and reduces the severity of SSNHL. Periodic evaluation of hearing acuity and prompt treatment of SSNHL, if any, are desirable to maintain patients' QOL.


Before the deposition, optical microscopy and Raman spectroscopy were utilized to identify the number of the graphene layers. After the deposition, scanning electron microscopy was used to observe the morphologies of the particles. Systematic analysis revealed that the average sizes of the nanoparticles increased with the number of graphene layers. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. The effect of the substrate such as SiO2/Si was investigated by fabricating suspended graphene, and the size and density were similar to those of supported graphene. Based on a comparison of the results, the different morphologies of the silver nanoparticles on different graphene layers were theorized to be caused only by the variation of the diffusion barriers with the number of layers of graphene.The distributions of sizes of silver nanoparticles that were deposited on monolayer, bilayer, and trilayer graphene films were observed. Deposition was carried out by thermal evaporation and the graphene films, placed on SiO It, therefore, has various unique electrical and optical properties of interest to scientists and technologists[2 and Al2O3 with special thickness between graphene and the substrate are typically used to make graphene optically visible[A single atomic layer of graphene is the thinnest spologists-3. Graph visible-7. The e visible. Raman a visible-14. Rece visible-20. The  visible,22.In this work, layers of graphene were fabricated in a sample by micromechanical cleavage. The number of layers of graphene was determined by micro-Raman spectroscopy and optical microscopy. After silver nanoparticles were deposited on the sample using a thermal deposition system, the distribution and sizes of the particles on flakes with different numbers of layers were systematically analyzed. To analyze the effect of the substrate, suspended graphene was fabricated, and the size and density thereof were found to be similar to those of supported graphene. The different results for the mono-, bi-, and tri-layer graphene are theorized to be caused only by the variation among the diffusion barriers of the various graphene layers, which provides a method of determining the number of graphene layers and provides information that can be utilized to elucidate the interaction between a graphene flake and its substrate.2 over a Si substrate. Graphene flakes that contained different numbers of graphene layers were distributed on different areas on the same sample. The variation in the color contrast with the number of graphene layers is observed under an optical microscope, as presented in FigureA graphene flake was fabricated by micromechanical cleavage with scotch tape. It was capped with 300-nm-thick layer of SiO2/Si substrate and the monolayer, bilayer, and trilayer graphene flakes were obtained, and shown on right-hand sides of Figure2/Si substrate.Silver nanoparticles were deposited on graphene flakes at a rate of 0.5 nm/min using a thermal deposition system to a thickness of 5 nm at a fixed temperature of the deposition system of 300 K. To elucidate the surface diffusion, the sample after deposition was maintained at 373 K in a vacuum for 1 h. To study the surfaces of the graphene flakes, scanning electron microscopy (SEM) images of the distribution of nanoparticles on the SiO2/Si substrate, monolayer, bilayer, and trilayer graphene. The sizes of the nanoparticles on the monolayer graphene flake are distributed in the range of 0 to 50 nm, whereas those of the nanoparticles on the trilayer graphene flake were distributed in the range of 10 to 70 nm. Whereas the sizes of the nanoparticles on the SiO2/Si substrate were distributed in the range of 0 to 50 nm, the majority of them were in the range of 10 to 30 nm.To investigate the density and size of the nanoparticles, average size and density were determined by histogram analysis. The histograms, on the left-hand sides of Figure2/Si substrate without any graphene flake. The nanoparticles on the trilayer graphene are largest, with a mean size of approximately 36 nm; as the number of graphene layers decreased, the average size fell to 32, 25, and 18 nm. The density of nanoparticles on the trilayer graphene was also the lowest, at around 3.1 × 1014/m2. Reducing the number of graphene layers increased the density from 3.1 × 1014 to 9.1 × 1014/m2. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. Different morphologies of silver on the differently layered graphene are obtained when silver particles are deposited on the graphene surfaces because of the differences of surface free energy. The variation in surface free energy with number of layers is responsible for the variation in the morphologies of silver on the differently layered graphene. The particles are randomly deposited on the graphene surface, and surface diffusion causes randomly arranged particles to combine to form relative large islands. The surface diffusion coefficient D ∝ exp(−En/kT) indicates the interaction between particles and the substrate surface, where En and k denote the diffusion barrier and the Boltzmann constant, respectively. The density of the particles on the substrate (N) can be expressed as N ∝ (1/D)1/3 for isotropic surface diffusion. The diffusion barrier specifies the interaction between particles and the substrate surface. The difference between the diffusion barriers of the substrate and monolayer can be calculated as ΔE = E0 − E1 = 3kT ln(N0/N1) = 61.7 mev by combining the above equations. The difference between the barriers of the substrates with other numbers of layers can be similarly obtained, and they are 39.1 meV (E1-E2) between the first and second layers and 3.1 meV (E2-E3) between the second and third. The difference between the diffusion barriers of silver decreased as the number of graphene layers increased. According to one review[2/Si substrate and different graphene layers, which the most is trilayer, is in the range of 9 × 1013 to 1.25 × 1013/m2, and the difference between diffusion barriers of different layers is in the range 500 to 291 meV. The area density of the silver nanoparticles herein is approximately ten times this value, indicating that silver nanoparticles can be more easily prevented from moving along the graphene surface, revealing a stronger interaction between silver and graphene than between gold and graphene.In Figuree review, the are2/Si is responsible for the variation in morphology, suspended graphene was fabricated by mechanical exfoliation of the graphene flakes onto an oxidized silicon wafer. First, ordered squares with areas of 6 μm2 were defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of HOPG with scotch tape was then used to deposit the suspended graphene flakes over the indents. The deposition conditions and silver nanoparticles were the same for both types of graphene. Figure2 does not affect the distribution of silver nanoparticles on the surface of graphene.To determine whether the substrate effect of SiO2, suspended graphene was also fabricated. The size and density of suspended graphene were found to be similar to those of the supported graphene. According to these results, only variations in the interactions between n-layer graphene and the silver nanoparticles were responsible for the variation in their distribution.In this work, the distribution of sizes of silver nanoparticles that were deposited on graphene films with different numbers of atomic layers of graphene was investigated. A systematic analysis revealed that the average size of the nanoparticles increased, and the area density and the difference between diffusion barriers of the nanoparticles decreased as the number of graphene layers increased. To analyze the effect of a substrate such as SiOThe authors declare that they have no competing interests.C-WH carried on the experimental parts: the acquisition, analysis, and interpretation of data. C-WH was also involved in drafting the manuscript. H-YL and C-HH took the analysis and interpretation of data. They also had been involved in revising the manuscript. R-JS and W-HW  prepared the graphene samples using micromechanical method and obtained the OM images. C-YL has made substantial contributions to the conception and design of the study, and in the critical revision of the manuscript for important intellectual content. H-CC, the corresponding author, had made substantial contributions to conception and design, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.C-WH received his BS degree in Electrical Engineering from the National University of Kaohsiung, Kaohsiung, Taiwan, in 2008. He studied his MS degree in 2008 and Ph.D. degree directly in 2009. Currently, he is a Ph.D. candidate in the Department of Photonics, National Cheng Kung University, Tainan, Taiwan. He focuses on the property of graphene and surface plasmon resonance of nanoparticles. H-YL received her doctoral degree from the Institute of Biomedical Engineering, National Cheng Kung University in 2010. Her current research interest focuses on the SERS properties of graphene and possible sensing application. C-HH received his doctoral degree from the Institute of Electro-Optical Science and Engineering, National Cheng Kung University in 2009. His current research interest focuses on the Raman and SERS properties of graphene incorporating with metal nanoparticles.


Adenosine 5-triphosphate (ATP) and its breakdown products ADP and adenosine can act as extracellular messengers in a range of biological processes. Extracellular adenine nucleotides are metabolized by a number of enzymes including NTPDases and 5'-nucleotidase, which are considered to be the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATPase and ADPase activities in rat serum exhibit a 24-h temporal pattern, with higher enzyme activity during the dark (activity) phase. It was found that stress can cause disruptions in biological circadian rhythms and in the cardiovascular system. Therefore, the aim of the present study was to examine the influence of acute stress exposure upon temporal patterns of NTPDase and 5-nucleotidase enzyme activities in rat blood serum.Adult male Wistar rats were divided into 4 groups: ZT0, ZT6, ZT12 and ZT18. Each group was subdivided in 4 groups: control, immediately, 6 h and 24 h after one hour of restraint stress. ATP, ADP and AMP hydrolysis were assayed in the serum.All stressed groups showed significant decreases in all enzyme activities at ZT 12 and ZT 18 when compared with control.Acute stress provokes a decrease in nucleotidase activities dependent on the time that this stress occurs and this effect appears to persist for at least 24 hours. Stress can change levels of nucleotides, related to increased frequency of cardiovascular events during the activity phase. Altered levels of nucleotides in serum may be involved in cardiovascular events more frequent during the activity phase in mammals, and with their etiology linked to stress. Extracellular adenosine 5'-triphosphate (ATP) and its breakdown products, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine, can act as extracellular messengers in a range of biological processes through binding to purinergic receptors. In addition, they have been shown to have pronounced effects on a variety of biological processes such as neurotransmission, regulation of cardiac function and platelet aggregation , as wellExtracellular nucleotides can be hydrolyzed by a variety of enzymes that are located on the surface of cells, or by soluble forms in the interstitial medium or within body fluids . NucleosCircadian organization is important to enable an organism to maintain equilibrium in response to the daily changes in the external environmental and prepare accordingly . In mammSome studies have demonstrated the influence of stress in modulating the purinergic system. Mild stress, such as a mild foot shock, is enough to promote specific changes in the hydrolysis of ATP and ADP in some tissues such as cerebral cortex . MoreoveConsidering that the temporal variation of nucleotidase activities  may be oA total of 98 naive adult male Wistar rats  were used. The animals were maintained under a standard 12/12-h light/dark cycle , in a controlled environment (22 ± 2°C), with rat chow and water ad libitum. The Zeitgeber time was used as a reference to detect the rhythmicity of the variables under study. The experimental protocol was approved by an Institutional Review Board for use in animals with procedures in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals , the UK Animals Scientific Procedures Act 1986 and the European Community's Council Directive of 24 November 1986 (86/609/EEC).Rats were habituated to the vivarium for 2 weeks before the beginning of the experiment. On the day of the experiment, the animals were divided into 4 groups according to time of day  and each of these was subdivided into 4 groups according to time of death  . Comparisons between groups were analyzed by one-way ANOVA followed by Tukey's test. Differences between groups were considered significant at A summary of all the results of stress effects upon temporal patterns of ATPase, ADPase and AMPase activities are presented in Figures 15,82 = 8.205, P < 0.001, Figure 15,82 = 9.911, P < 0.001, Figure The hydrolysis of ATP and ADP were determined in blood serum at 0 h, 6 h and 24 h after the stress procedure during 24-hour at ZT 0, 6, 12, and 18. One-way ANOVA followed by Tukey's test revealed significant differences in the effect of stress on the 24-h profile of ATPase  than during the light period (ZT 0 and ZT 6) in control rats is consistent with a previous study in which we demonstrated that ATPase and ADPase activities (probably the NTPDase 1-like soluble enzyme) exhibit a 24-h temporal pattern . In the This result is consistent with previous findings in humans  and in rAdditionally, mechanisms involved in the acute stress response promote the critical secretion of glucocorticoids that is linked to the modulation of ATPase, ADPase and AMPase activities . Here, iIt was verified that soluble nucleotidases seem to be co-released with the neurotransmitter ATP via stimulation of sympathetic nerves . As is wAdditionally, stress causes release of epinephrine from the adrenal glands, and norepinephrine is released together with ATP by the terminals of the sympathetic nervous system . The eleIn conclusion, acute stress decreased the hydrolysis of nucleotides in rat blood serum during the night (activity) phase, and this effect persisted for up to 24 hours. It is tempting to suggest that altered levels of nucleotides and nucleosides in serum may be involved in the more frequent occurrence of cardiovascular events during the activity phase in mammals , which hThe authors declare that they have no competing interests.AS and BDC carried out the design of the study and performed the statistical analysis; LFM and JRR carried out the enzymatic assays and helped to shape the manuscript; WC, MPLH and AMOB participated in the design of the study; ILST coordinated the study, performed the statistical analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.


According to the National Heart, Lung, and Blood Institute of the National Institute of Health, hypotension refers to an abnormally low blood pressure (BP). In childhood, hypotension can be determined according to two different definitions: BP below the 5th percentile or below two standard deviations (SDs) of the mean for age and gender . A graphThe objective of this study was to update the graphic representation of pediatric hypotension.We used a computerized calculation method to develop high-resolution graphics containing 5,841 points each, to depict the main percentiles associated with low BP for boys from 1 to 17 years old in the 50th percentile of height. Each point represents the calculation of the polynomial equation that includes the statistical processing of the last Report on Blood Pressure . We alsoFive monotonic curves of diastolic BP for boys representing the 50th, 25th, 10th, 5th, and 2.275th (-2SD) percentiles were built (Figure The correction of diastolic BP for height is of little significance, and this updated graphic can be used to diagnose low diastolic BP for boys. Clinical studies are necessary to determine the SBP percentile that better represents clinically significant hypotension.


Knee osteoarthritis (KOA) is a degenerative joint disease that occurs mainly in the elderly population. However, there are currently no effective treatments for treating this condition. In this study, the efficacy of needle-knife therapy, a technique of traditional Chinese medicine that has been widely used to treat KOA was investigated. Patients (n=170) with KOA were randomly divided for needle-knife therapy (treatment group) and acupuncture therapy (control group). Outcome evaluation included stiffness, pain, physiological function, overall changes, total symptom score, clinical curative effects and the concentrations of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the synovial fluid. The trial was completed in 151 patients (233 knees) from a total of 170 patients (264 knees); the treatment group comprised 76 patients (117 knees) who completed the trial and 9 patients (14 knees) who were removed from the study, and the control group comprised 75 patients (116 knees) who completed the trial and 10 patients (17 knees) who were removed from the study. The symptom scores of KOA in stages I–IV were reduced significantly in the treatment group and those of stages I–III were decreased significantly in the control group. The effective rate of the KOA therapy in the patients of stages III and IV in the treatment group was significantly higher than that in the control group. After treatment, the decrements of IL-1β, IL-6 and TNF-α in the treatment group were greater than those in the control group. These results showed that the use of needle-knife therapy to treat KOA effectively improved the clinical symptoms by inhibiting the expression of inflammatory cytokines. Knee osteoarthritis (KOA) is a degenerative disorder that results from cartilage failure or an imbalance of the degradation and repair processes of chondrocytes, cartilage matrix and subchondral bone, which are induced by a complex interplay of biochemical and biomechanical factors with secondary components of inflammation ,2. StudiConventional therapies for KOA, which are designed to regulate the symptoms of stiffness and pain, include topical analgesics, glucosamine, nonsteroidal anti-inflammatory drugs, intra-articular injection of sodium hyaluronate and surgical treatment ,8. HowevKOA is a chronic, retrogressive knee disease often occurring in the elderly, with most cases belonging to the TCM ‘stasis of the channels’ type . In needThe aim of the present study was to identify a more effective therapy for reducing joint pain and disability, as well as to prevent and mitigate cartilage degradation. A randomized, controlled study was performed to evaluate the safety and efficacy of needle-knife therapy for treating KOA. The basic design of this study involved the quantification and comparison of the efficacy of needle-knife therapy and acupuncture for KOA. The effects of the two treatments on the clinical symptoms of KOA and the expression of inflammatory cytokines were investigated.A total of 170 patients with KOA from the Fuzhou General Hospital of Nanjing Military Command  from April 2010 to March 2013 were enrolled in this study. The clinical characteristics of the patients were recorded in a uniform data collection table.All patients enrolled were randomly assigned to two groups by lottery and their general characteristics are listed in The diagnosis of KOA was formulated according to the standards issued by the American College of Rheumatology ,19. PatiThe TCM syndrome of ‘stasis of the channels’ type was differentiated with reference to the standards for diagnosis in Efficacy Evaluation of TCM Diseases and Syndromes and the Guiding Principle of Clinical Research on New Drugs of Traditional Chinese Medicine . DiagnosThe patients with the following conditions were excluded: Patients aged >70 years; patients with concurrent rheumatoid arthritis, psoriasis, syphilitic neuropathy, ochronosis, metabolic osteopathy, acute trauma and other diseases affecting the joints; women in pregnancy or lactation; patients with accompanying severe cardiovascular, hepatic, renal or with mental disease; patients who had been treated with other methods that may have influenced the observation of indices in this study; and patients who discontinued treatment during the study or refused to objectively provide evaluation data in this study.The patients in the treatment group were treated with needle-knife  therapy at the dominant acupoints of Neixiyan (Ex-LE4) and Waixiyan (Ex-LE5), as well as the conjugate points Xuanzhong (GB39), Xuehai (SP10), Dubi (ST35) and Taixi (KI3)  and 2. TThe patients in the control group were treated with acupuncture at the dominant acupoints of Neixiyan and Waixiyan, as well as the conjugate points Xuanzhong, Xuehai, Dubi and Taixi. The patients were seated with the knee joints flexed. After the acupoints were disinfected, routine, disposable needles were inserted at the dominant acupoints for 1.5 Cun, with twisting up and down to induce ‘De-Qi’. The treatment was conducted daily for 5 days as one therapeutic course, and two courses were administered to each patient with a 2-day interval between the two courses.The clinical symptoms scores of the patients were evaluated for 11 items following the criteria set in reference to the Guiding Principle of Clinical Research on New Drugs of TCM : i) MornThe symptoms of items i)–vii) were ranked by severity in three grades and scored from 1 to 3; the more severe the symptom, the higher the score, and no symptom scored 0. A similar method was performed on the symptoms of items viii) and ix), but they were ranked in two grades and scored from 1 to 2; as for items x) and xi), when they occurred, the score was 1, otherwise it was 0. The severity of KOA was estimated in three grades based on the total score of symptoms (the sum of various symptoms): mild grade, the total score was <10; moderate grade, the total score was from 10 to 18; and severe grade, the total score was >18. The maximum possible symptom score was 33.With reference to the Guiding Principle of clinical Research on New Drugs of TCM , accordiFor the assessment of the IL-1β, IL-6 and TNF-α levels in the synovial fluid, the synovial fluid collected from the patients with KOA prior to and following treatment was dispensed into 1-ml aliquots. The synovial fluid was diluted with diluent’s buffer  to the appropriate detection range for evaluation by enzyme-linked immunosorbent assay (ELISA). The levels of the proteins of interest in the synovial fluid were measured using commercially available ELISA kits , following the manufacturer’s instructions.The symptoms and physical signs of adverse reactions that occurred following the two treatments (needle-knife therapy and acupuncture) were recorded, and routine blood, urine and stool tests, electrocardiogram (ECG) tests, and evaluations of liver and renal functions were performed before and after treatment.All data were analyzed using SPSS software for Windows, version 13.0 . Statistical analysis of the data was performed with Student’s t-test and one-way analysis of variance. The enumeration data was analyzed by the chi-square test. P<0.05 was considered to indicate a statistically significant difference.From the 170 patients (264 knees) enrolled in this study, 151 patients (233 knees) completed the trial and 19 patients (31 knees) were removed. In this study, 76 patients (117 knees) and 75 patients (116 knees) completed the trial in the treatment and control groups, respectively. The nineteen patients (31 knees) that were excluded due to uncompleted prescribed therapeutic courses included nine patients (14 knees) in the treatment group with two joints of stage I, three joints of stage II, five joints of stage III and four joints of stage IV; and 10 patients (17 knees) in the control group, which comprised three joints of stage I, six joints of stage II, five joints of stage III and three joints of stage IV. The exclusion rates in the two groups were not significantly different and the influence of exclusion on the intergroup proportionality of the baseline was negligible.The symptom scores of KOA at the corresponding stages were not significantly different between the two groups prior to treatment (P>0.05). Following treatment, the symptom scores of KOA in stages I–IV were reduced significantly in the treatment group (P<0.05 or P<0.01), and the scores of KOA in stages I–III were decreased significantly in the control group (P<0.05 or P<0.01), while those of stage IV were not significantly changed (P>0.05), although a slight reduction was detected .In a comparison between the two groups, with the exception of a significant reduction in the total symptom scores of KOA in stage III in the treatment group compared with that in the control group (P<0.05), no significant differences were observed in the symptom scores of KOA at other stages of the disease (P>0.05), although a few divergences were revealed .No statistically significant difference between the two groups was detected in the excellent rates of the treatments in the patients at stages I–IV and in the effective rates of the treatments in the patients of stages I and II (P>0.05). The effective rate in patients of stages III and IV in the treatment group was significantly higher than that in the control group (P<0.05) .To evaluate the effect of needle-knife therapy on the expression levels of inflammatory cytokines, the concentrations of IL-1β, IL-6 and TNF-α in the synovial fluid of the patients with KOA were investigated by ELISA. As shown in No significant changes in the X-ray features between before and after treatment were observed in either group, indicating that although treatment in the two groups effectively alleviated the symptoms of KOA, it did not improve the organic changes that had previously occurred.No adverse reaction-associated signs and symptoms or laboratory indices were identified during the whole treatment course.The results of the present support the use of needle-knife therapy as a safe and effective method for the treatment of KOA. It was shown to be more effective than routine acupuncture for the alleviation of pain and improvement of physiological function by inhibiting the expression of inflammatory cytokines. In addition, the results also indicated that acupuncture may relieve knee stiffness and pain and improve the function scores of KOA.KOA increases in prevalence with age and is a major cause of pain and locomotor disability worldwide. It belongs to the category of Gu Bi in TCM, which means either the limbs or the joints are suffering from pain and malfunction. TCM suggests that the disease is based on Gan-Shen insufficiency and weakness of tendons and bones, and is importantly linked to blood stasis . CurrentPatients with KOA require better pain control and reduced adverse events. Therefore, as comorbidities and aging increase, a more convenient approach is necessary . Needle-According to the features of KOA, two widely applied acupoints, Xuanzhong (GB39) and Taixi (KI3), were selected to dredge the meridian-collaterals and nourish Shen to strengthen bone, which may effectively improve the Gan-Shen insufficiency and weakness of tendons and bones, raise the endurance of peri-articular tissue to inflammatory stimulation, and be helpful for knee joint pain alleviation and knee joint function restoration to attain good therapeutic effectiveness. Hence, needle-knife therapy may effectively alleviate pain in the knee joints and improve the scope of motion. Results of this study showed that the clinical symptoms in patients with KOA of stages I–IV were improved significantly following treatment with needle-knife or acupuncture therapy, while needle-knife therapy showed a better efficacy than acupuncture for the patients of stages III and IV.KOA is characterized by an imbalance of matrix synthesis and matrix degradation in cartilage at the cell and tissue levels. Chondrocytes, the only cell type present in articular cartilage, are responsible for the synthesis and breakdown of the extracellular matrix (ECM) . SignalsThe preliminary findings in the present study indicate that needle-knife therapy is effective in improving knee pain, stiffness and physical function in patients with KOA. The curative and the long-term curative effects are to be determined in further follow-up visits. The major limitation in the present study was the small sample size, and a randomized controlled study with a larger sample size will be conducted in the future.