WSS exposure of 12 dyne/cm2 did not affect the EC-SMC interactions, whereas WSS of 4 dyne/cm2 elevated the amount of Cx43 and reduced the amount of Cx37, with a different magnitude between the models. The reduced endothelium connectivity suggests that the presence of SMC reduces the sealing properties of the endothelium, showing a more inflammatory phenotype while the distance between the two cell types reduced their interactions. These results demonstrate that EC-SMC interactions affect EC phenotype and change the EC response to WSS. Furthermore, the interactions formed between the EC and SMC demonstrate that the 1-side model can simulate better the arterioles, while the 2-side model provides better simulation of larger arteries.Vascular functions are affected by wall shear stresses (WSS) applied on the endothelial cells (EC), as well as by the interactions of the EC with the adjacent smooth muscle cells (SMC). The present study was designed to investigate the effects of WSS on the endothelial interactions with its surroundings. For this purpose we developed and constructed two co-culture models of EC and SMC, and compared their response to that of a single monolayer of cultured EC. In one co-culture model the EC were cultured on the SMC, whereas in the other model the EC and SMC were cultured on the opposite sides of a membrane. We studied EC-matrix interactions through focal adhesion kinase morphology, EC-EC interactions through VE-Cadherin expression and morphology, and EC-SMC interactions through the expression of Cx43 and Cx37. In the absence of WSS the SMC presence reduced EC-EC connectivity but produced EC-SMC connections using both connexins. The exposure to WSS produced discontinuity in the EC-EC connections, with a weaker effect in the co-culture models. In the EC monolayer, WSS exposure (12 and 4 dyne/cm Extensive research on the effects of physiological hemodynamic forces has found that WSS causes functional switching of the endothelial phenotype from a quiescent atheroprotective phenotype under physiological and elevated levels of WSS  in vivo, VE-Cadherin expression was weak and discontinuous in areas of disturbed flow, but significantly higher and continuous where flow was laminar in vivo findings The effect of WSS on various EC-EC interactions has mainly been studied on EC monolayers . A shortThe effect of WSS on the interactions between EC and SMC has been shown to reduce the inflammatory response induced by the proliferating SMC on the EC. This was evidenced by the reduced expression levels of adhesion molecules such as P-selectin and the vascular adhesion molecule (VCAM) The direct communication of EC-SMC through myoendothelial gap junctions (MEGJ) has mainly been studied without exposure to WSS . Isakson2, a physiological WSS of 12 dyne/cm2 and a control level (0 dyne/cm2). We analyzed the morphology of the models and the expression levels of proteins responsible for the cell interactions with their neighboring cells.Most studies on WSS effects on the EC-SMC interaction have focused on the inflammatory response of both EC and SMC. However, those studies have not considered that in vivo, the EC-SMC interactions may vary between different types of blood vessels, affecting the response of EC to WSS. Based on the understanding of these interactions in vivo, we hypothesized that for the studies of the effects of mechanical forces on the endothelium the 1-side model can simulate better the arterioles, while the 2-side model provides better simulation of larger arteries. Thus, the main goal of this work was to study the ways in which the interactions between EC and their neighboring cells (i.e. EC-SMC interactions) affect the response of EC-EC interactions (i.e. VE-Cadherin) and EC-SMC (i.e. MEGJ) to WSS stimulation. To support our hypothesis we compared the response of EC to WSS using 3 different models: (1) EC monolayer, (2) 2-side model, and (3) 1-side model. The models were exposed to a low WSS of 4 dyne/cmin vitro models of the vascular wall in this work were grown in modified wells that were previously developed in our laboratory The cell cultures for the Human umbilical vein endothelial cells (HUVEC) (PromoCell) were cultured in endothelial cell media (PromoCell) supplemented with 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 12.5 U/ml Nystatin .2 in a humidifier incubator. In both cell types, only passages 4–8 were used for this study.Human umbilical aortic smooth muscle cells (SMC) (Lonza) were cultured in MCDB-131 Medium supplemented with 5% Fetal Bovine Serum, 2 mM L-Glutamine, 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 12.5 U/ml Nystatin , 2 ng/ml Insulin (Sigma Aldrich), 0.5 ng/ml Epithelial Growth Factor (Sigma Aldrich), 3 ng/ml Basic-Fibroblasts Growth Factor (bFGF)  and 1 µg/ml Hydrocortisone (Sigma Aldrich). Both cell types as well as the co-culture were cultured at 37°C and 5% CO4 SMCs were seeded on the PTFE membrane on the lower side of the well. After 24 hours, the membrane was washed to remove excess non-adherent SMC and the well was re-assembled so that the SMC cells would be facing downwards and completely submerged in the HUVEC growth medium . After 24 hours both layers of EC and SMC became confluent and ready for WSS experiments.The PTFE membrane was installed in the custom-designed well with the upper face upwards and was placed into a plastic tissue culture dish containing the SMC growth medium. About 7.5×10per side  right. A5 HUVEC were seeded on the PTFE membrane (upper side). At this step we ensured that the medium completely covered the underside of the membrane with the cell culture and without any gas bubbles. After 24 hours the HUVEC layer became confluent and ready for WSS experiments.The PTFE membrane was installed in the custom-designed well with the upper face upwards and was placed into a plastic tissue culture dish containing the HUVEC growth medium  right. Ain vitro vascular wall model was assembled in the flow chamber, so that the HUVEC side would be exposed to physiological flows while the other side (with no cells or with SMC) would stay in static conditions. Prior to conducting the experiments, all models were inspected under phase contrast microscopy to verify that the cells had reached at least 90% confluency.Once confluent layers were obtained (approximately 16 hours after the EC were seeded), the custom-designed wells were disassembled and the well bottom with the h = 1 mm) of the flow chamber is much smaller than either its length (l = 100 mm) or its width (b = 35 mm) the fluid mechanics of the channel can be described as a parallel plate flow chamber. Thus, the WSS (τω) is given by:2 is the fluid viscosity for the medium at 37°C.A custom designed parallel plate flow chamber with a rectangular cross section was built to apply uniform laminar WSS on 3 samples of the tissue-engineered vascular wall models. Since the height  model was developed to validate the uniform distribution of the WSS in the flow chamber, using the computational fluid dynamics package of FLUENT . The flow profiles at different locations were computed and compared for a uniform inlet velocity of 2 in a humidifier incubator during exposure to WSS of the endothelial side of the models. The flow chamber allows for up to 3 wells in each flow experiment. All experiments were performed using a flow rate of 600 ml/min resulting in a WSS of 12 dyne/cm2 for physiological WSS, a flow rate of 200 ml/min resulting in a WSS of 4 dyne/cm2 for low WSS and, static conditions (i.e. 0 dyne/cm2) as the control. The WSS experiments were conducted for 30 min in the case of the connexins protein quantification, and for 30 and 60 min for visualization of cell morphology, VE-Cadherin and FAK. All experiments were performed using all three models of the vascular wall as described above and all the experiments were performed at least three independent times with three wells in each test.For the WSS experiments, the well bottoms were inserted into the bottom part of the flow chamber so that the PTFE membrane was in the same plane as the chamber bottom, allowing for laminar flow on the HUVEC side of the vascular wall models. The flow chamber was then connected with tubes to a peristaltic pump and a medium reservoir. The flow chamber and the medium reservoir were maintained at 37°C and 5% CO2 and then fixed in a 4% paraformaldehyde solution for 10 min. This was followed by blocking, using a phosphate buffered solution (PBS) containing 10% normal goat serum and 0.1% Triton X-100 solution for 30 min followed by incubation overnight at 4°C with the following primary antibodies: a mouse anti VE-Cadherin (Santa Cruz Biotechnology) and a rabbit anti FAK (Abcam). This was followed by an incubation of 30 min with a secondary antibody: alexa fluor 647 conjugated goat anti rabbit, alexa fluor 488 conjugated goat anti mouse (Jackson ImmunoResearch) and TRITC conjugated phalloidine (Sigma Aldrich). Between each step the samples were rinsed 3 times with PBS. The samples were then mounted between 2 large microscope glass slides using a DAPI mounting gel (Santa Cruz Biotechnology) in order to visualize the cell nucleus. The slides were stored in a covered box at 4°C until microscopy examination. The cells were examined under a Zeiss LSM510 confocal microscope. Analysis and quantification of the images was done using the LSM image browser software (Zeiss) and the provided measuring tools.The vascular wall models were exposed for 30 and 60 min to 0, 4 and 12 dyne/cm2 and then the cells were trypsinized and centrifuged for 5 min at 7 g to separate the cells from the trypsin solution. The cell suspensions of both HUVEC and SMC were then fixed and permeabilized using ice cold methanol for 10 min at −20°C. The samples were incubated first with the appropriate primary antibody, followed by incubation with the appropriate secondary antibodies for 30 min each at room temperature. Following each step, 1 ml of PBS was added to the cell suspension and the resulting solution was then centrifuged for 5 min at 7 g to separate the cells from the solution. The solution was gently poured out of the test tube, leaving the cell pellet at the bottom of the test tube. The cells were mixed to form a homogenous cell suspension using a vortex after the next solution was added. In order to differentiate the different cells, each sample was labeled for VE-Cadherin (which is positive for HUVEC and negative for SMC) using a mouse anti VE-Cadherin antibody (Santa Cruz) and for one of the following primary antibodies: rabbit anti Cx37 alexa fluor 647 conjugated (BIOS), rabbit anti Cx43 alexa fluor 488 conjugated (BIOS) followed by the secondary antibodies: alexa fluor 647 conjugated goat anti rabbit and alexa fluor 488 or alexa fluor 647 conjugated goat anti mouse. Positive control samples were labeled with primary and secondary antibodies and negative control samples were labeled with secondary antibody alone. Samples were analyzed using a BD LSR digital flow cytometer. The Flowing software  was used for data analysis.The vascular wall models were exposed for 30 min to 0, 4 and 12 dyne/cmP value of less than 0.05 was considered statistically significant. Post-hoc Tukey-Kramer tests supplied by the SPSS software were used to perform pair wise multiple comparisons between the data obtained from the different WSS applied on the different models.The results were evaluated using a 2-way analysis of variance . A We investigated the attachment of EC to the matrix as well as the interactions of EC-EC and EC-SMC in response to WSS stimulation. For this purpose we compared the morphology and amount of proteins immediately following short term exposure to different WSS in three different models of the vascular wall made up of EC monolayer and two co-cultures of EC and SMC in which EC were cultured directly over a confluent layer of SMC or on the opposite side of a membrane with a culture of SMC.2 for 0 and 60 min are shown in 2, the HUVEC became a little more elongated and the stress fibers aligned with the flow direction in all three models , which is an important protein in the focal adhesion complex that attaches cells to the matrix, was used to evaluate the EC-matrix interaction. Stains of HUVEC actin stress fibers and FAK, following the exposure to a WSS of 12 dyne/cme models . The stre models  A to C. e models  - B.2 led to an increase in the amount of FAK in a time dependent manner. In static conditions and following exposure to 30 min of 4 dyne/cm2 (LSS) the amount of FAK in the 1-side model was very low (significantly different from the other models). Following an exposure of 30 min to 12 dyne/cm2 (PSS30) the amount of FAK in this model increased (significantly); however it was still lower than in the other two models. Following an exposure of 60 min to 12 dyne/cm2 (PSS60), however, the amount of FAK was similar to that of the EC monolayer model. The amount of FAK in the 2-side model was a little higher in all modes of WSS exposure . Following exposure to a WSS of 12 dyne/cm2 a small reduction in the expression of VE-Cadherin was found in the EC monolayer model with a non-significant reduction in the case of 4 dyne/cm2  emerged following exposure to a WSS of 12 dyne/cmdyne/cm2 .2 and 4 dyne/cm2  sample. The normalized results of the three repeats of the same test (non dependant repeats) were than averaged and presented  and 7.In static conditions, the amount of Cx43 showed a slight increase in the 2-side model compared to the EC monolayer and a significant increase in the 1-side model , and the2 the amount of both Cx43 and Cx37 did not change significantly in either the 1-side and 2-side models (2 the amount of Cx43 increased (significantly only in the 2-side model) (Following exposure to a WSS of 12 dyne/cme models  and 7. He model)  and decre model) .2 the FAK became longer and increased in its amount in a time dependent manner. In the 1-side model, this exposure caused the appearance of the FAK until it resembled the EC monolayer model. In addition, the EC-EC interactions were reduced by the presence of SMC, as demonstrated by the amount of VE-Cadherin and stain. However, when exposed to WSS, the interactions with the SMC reduced the discontinuity in EC-EC interactions seen in the VE-Cadherin stain. In the presence of SMC the EC formed direct EC-SMC interactions using both Cx37 and Cx43 to form MEGJ. When exposed to a WSS of 12 dyne/cm2 both Cx43 and Cx37 expression were elevated in the EC monolayer, suggesting higher connectivity of EEGJ, however without a significant effect in the co-culture models where it formed EC-SMC interactions. When exposed to a WSS of 4 dyne/cm2 the amount of Cx43 was elevated in all 3 models, with the highest in the EC monolayer and lowest in the 1-side model, suggesting that the interactions with SMC reduce the effect of WSS. This effect was more pronounced when there was no distance between the two cell types (caused by the membrane between the cells in the 2-side model). In the case of Cx37, a WSS of 4 dyne/cm2 increased Cx37 expression compared to the control in the EC monolayer (demonstrating higher EEGJ interactions); however, in the co-culture models (where it formed EC-SMC interactions), the expression of Cx37 was reduced, with a greater difference in the 1-side model, showing that again, a distance between the 2 type of cells due to the presence of the membrane, limits the effect of WSS on the EC-SMC interactions, and the effect is more similar to the that of the EC-EC interactions. The limited EC-SMC interactions found in the 2-side model resemble the limited interactions found in vivo in larger arteries, compared to the higher amount of MEGJ found in the 1-side model, which resembles the in vivo interactions in smaller arteries.In this study we have studied how WSS affect the interactions of the endothelium with its surrounding cells, which constructs the vascular wall. This was done using two different co-culture models of the vascular wall as well as a control model, which lacks the interaction with SMC. The results show that EC attaches to the SMC using a different FA complex, and not using FAK. Following exposure to a WSS of 12 dyne/cmThorough Inspection of the Z slices of the confocal images confirmed that the SMC and the endothelial layers remained as two separate monolayers on the opposites sides of the membrane in the 2-side model without migration of cells into the membrane or as 2 layers one on top of the other in the 1-side model. The distance between the two cell types in the 2-side model was measured to be roughly 20 µm. Although this is a large distance between EC and SMC, gap junctions have been reported to occur between the two cell types through pores present in the thin membranes In order to analyze the EC-Matrix interactions, we examined the FAK morphology of EC. In the 1-side model, the EC attached to the SMC directly and to the ECM produced by the SMC. This ECM differed from the fibronectin coated membrane that served as the attachment matrix to the EC in the EC monolayer and 2-side models. Thus the limited FAK stain -B and reOn the other hand, in a 2-side model as compared to the EC monolayer, an increase in the total amount of FAK was found in all WSS exposure modes . This isFollowing exposure to physiological WSS, the FAK stain became stronger and is easier to detect, showing an increase in the EC-matrix interaction. It is important to notice that this increase is probably not due to new formation of FAK but rather the recruitment and growth of existing FAK to the growing focal adhesion complexes. The antibody used for the FAK staining was a phosphorylated Y397 FAK (Abcam), suggesting that the increased stain was induced by increased phosphorylation of the FAK. Such phosphorylation was found in previous studies shortly after exposure to physiological WSS, and was accompanied by an increased amount of FAK at the focal adhesion sites Our finding of an increase in the amount of FAK runs counter findings reported by Davies et al, where no differences in the total adhesion area of EC to the matrix were found following the application of WSS 2, the amount of FAK plaques increased as well as the size of each plaque, resulting with an increased amount of FAK  between the endothelial cells. This could indicate a higher permeability level of the monolayer, suggesting breaks in the tight junctions or even a leaky endothelium in vitro previously, following short exposure to disturbed pulsatile flow or following long exposure to reciprocating flow and physiological WSS Following the application of physiological WSS the VE-Cadherin stain ceased to be continuously distributed at the periphery of the cells and appeared in clusters at the cell borders. This was consistent in all 3 models; however, in the 1-side model the effect seemed weaker  and the 2 the discontinuity was not as strong as after exposure to 12 dyne/cm2 in all 3 models; however, in both co-culture models this change was not significant compared to the same model in static conditions. This suggests that the EC-SMC interactions might change the EC response to WSS and the alignment sequence of the cells. In addition, the change in the focal adhesion composition could possibly be attributed to the reduced amount of fenestration in the stains of the VE-Cadherin following exposure to 12 dyne/cm2 in the 1-side model . Since the exposure time to WSS was only 30 min, this change is likely to correspond to the transient change found following the onset of the flow 2 did not induce any change in the amount of connexins. A possible explanation is that the higher amount of connexins from the MEGJ needs a longer exposure to WSS prior for a change to occur or that following exposure of the WSS the change in MEGJ requires longer exposure to WSS compared to EEGJ. A possible reason is that the connexins may move from MEGJ to EEGJ as an immediate response, before any change in the total amount of the protein takes place. Moreover, the amount of Cx37 in the EC monolayer following exposure to WSS of 12 dyne/cm2 appeared to have been equivalent to that found in the co-culture models without the WSS stimulation. It is important to note that in the EC monolayer model, Cx37 and Cx43 form gap junctions between the EC; thus differences in amount are indicative of the connectivity and communication of EC-EC (i.e. EEGJ).The effect of exposure to WSS on the EC monolayer model was a 1.5 fold elevation in the amount of connexins in all WSS modes (partly for Cx37 in the case of a WSS of 12 dyne/cm2 the response of both co-culture models was consistent with what has been reported in previous works. The elevation of Cx43 expression was higher in the 2-side model than in the 1-side model; however it was the highest in the EC monolayer model. On the other hand, although Cx37 expression was down-regulated in the co-culture models, and was the lowest in the 1-side models, it was elevated compared to the control in the EC monolayer model. These changes in the magnitudes of the response show that the response of the 2-side model had more similarities with the EC monolayer response. This suggests that the EC-SMC interactions reduce the response to WSS; however, this was more prominent when there was no distance between the two cell types caused by the membrane between the cells in the 2-side model.Subsequent to a WSS of 4 dyne/cmIn our system we found that the presence of SMC changes the response of the cells to flow. However, the presence of a membrane between the cells apparently reduces the effect of the presence of SMC in both the connexins and the VE-Cadherin response. This change could not be ascribed to the changes in the amount of contact between the cells, since the change in the amount of Cx43 between the models was not notably high. On the other hand, the membrane found between the two cell types in the 2-side model increases the distance between the cells, so that the diffusion time of the secondary messengers’ increases, hence limiting or delaying the response of the cells. In addition, this change could be due to the different focal adhesion complex formed by the EC on the SMC layer as compared to the FA complex on the membrane.In vivo, the presence of MEGJ has been demonstrated in different animal models and different vascular sites; however, the frequency of MEGJ increases with decreasing vessel size in vivo interaction found in different types of blood vessels; i.e. small arterioles would be better demonstrated using the 1-side model, whereas larger arteries would be better demonstrated using the 2-side model.in vitro in the presence of SMC, in both static conditions and following exposure to WSS, as demonstrated by the amount of VE-Cadherin. This was accompanied by increased distance between the endothelial cells, which suggest a less connected and more permeable EC monolayer that resembles the inflammatory endothelium in vivo. In addition, the amount of Cx37 and Cx43 increased in the presence of SMC, indicating the formation of direct EC-SMC interactions through the formation of MEGJ. Following exposure to both physiological and low WSS, EC-EC communication through EEGJ increased in both the Cx37 and the Cx43 in the EC monolayer model. The EC-SMC interactions, however, did not vary following exposure to physiological WSS. Low WSS, on the other hand, increased EC-SMC interactions through Cx43 expression, whereas it reduced EC-SMC interactions through Cx37. The magnitude of this response varied between the different co-culture models, suggesting that the distance introduced by the membrane in the 2-side model reduces the effect of WSS on the EC-SMC interactions and thus the response in the 2-side model was more similar to the EC-EC connexin interactions. These results show that EC-SMC interactions affect EC phenotype as well as the response of the cells to WSS and that the interactions formed between the cells depends on the distance between them. Altogether, this highlights the importance of cellular interactions to vascular wall functionality, and the importance of the interactions formed when simulating different types of blood vessels.The data presented in this study demonstrate the effect of WSS on the EC-matrix, EC-EC and EC-SMC interactions. This was studied by measuring the changes in FAK, endothelial MEGJ and adherens junction proteins in response to WSS in two co-culture models and a control model of the EC monolayer. This is the first time that a reduction in the EC-EC interactions has been found


We hypothesized that post-movement beta synchronization (PMBS) and cortico-muscular coherence (CMC) during movement termination relate to each other and have similar role in sensorimotor integration. We calculated the parameters and estimated the sources of these phenomena.We measured 64-channel EEG simultaneously with surface EMG of the right first dorsal interosseus muscle in 11 healthy volunteers. In Task1, subjects kept a medium-strength contraction continuously; in Task2, superimposed on this movement, they performed repetitive self-paced short contractions. In Task3 short contractions were executed alone. Time-frequency analysis of the EEG and CMC was performed with respect to the offset of brisk movements and averaged in each subject. Sources of PMBS and CMC were also calculated.High beta power in Task1, PMBS in Task2-3, and CMC in Task1-2 could be observed in the same individual frequency bands. While beta synchronization in Task1 and PMBS in Task2-3 appeared bilateral with contralateral predominance, CMC in Task1-2 was strictly a unilateral phenomenon; their main sources did not differ contralateral to the movement in the primary sensorimotor cortex in 7 of 11 subjects in Task1, and in 6 of 9 subjects in Task2. In Task2, CMC and PMBS had the same latency but their amplitudes did not correlate with each other. In Task2, weaker PMBS source was found bilaterally within the secondary sensory cortex, while the second source of CMC was detected in the premotor cortex, contralateral to the movement. In Task3, weaker sources of PMBS could be estimated in bilateral supplementary motor cortex and in the thalamus.PMBS and CMC appear simultaneously at the end of a phasic movement possibly suggesting similar antikinetic effects, but they may be separate processes with different active functions. Whereas PMBS seems to reset the supraspinal sensorimotor network, cortico-muscular coherence may represent the recalibration of cortico-motoneuronal and spinal systems. Initiation, execution, and termination of a movement have a certain temporal spectral evolution Beta rhythm seems to play a special role in information processing of the sensorimotor system. Termination of a sensorimotor task is followed by a central, beta event-related response in the EEG and MEG recordings, called post movement beta synchronization (PMBS) PMBS is a transient, short increase of power 500 to 2500 ms after termination of a movement A similar PMBS can also be measured in the nucleus subthalamicus (STN) in Parkinson's disease In a previous study, elevated beta synchronization in the sensorimotor cortex was associated with slowing of voluntary finger movement in healthy subjects. It was concluded that high beta activity is associated with maintenance of tonic contractions and impairs the speed of the new movement Coherence is a measure of the correlation of two signals in the frequency domain. Coherence between EEG/MEG and EMG during weak-to-moderate isometric contractions is also found in the beta frequency band, typically at around 20 Hz The aim of the present study was to investigate whether PMBS and cortico-muscular coherence have a similar functional role in the physiological oscillatory drive of sensorimotor network after movement offset and to define how they are related to each other. We analyzed the parameters and sources of PMBS and cortico-muscular coherence at the same time in a common paradigm.In this study, 11 normal subjects were recruited. All gave written informed consent. The study was approved by the Ethics Committee, Medical Faculty, University of Kiel. Subjects were seated comfortably in an arm chair with their forearms supported on and their hands hanging freely from the armrests. In the first task (Task1), subjects kept a constant medium-strength contraction of the FDI-muscle (constant contraction). In the second task (Task2), superimposed on this contraction they performed a repetitive voluntary self-paced brisk squeeze of the object  approximately every 10 seconds. During the third task (Task3), the hands were also supported and the subjects executed only the brisk contraction with complete rest in between. For detection of muscle activity and marking the beginning and the end of brisk voluntary movement, a bipolar surface EMG electrode was placed above the first dorsal interosseus (FDI) muscle. EEG was recorded in parallel with a standard 64-channel EEG recording system  using a linked mastoid reference. A standard EEG cap was used with electrodes positioned according to the extended 10–20 system. EEG and EMG were band-pass filtered  and sampled at 1000 Hz. Data were stored in a computer and analyzed off-line. The EMG was full-wave rectified. The combination of band-pass filtering and rectification is the common demodulation procedure for EMG The electrode locations covering the primary sensorimotor area, with the highest PMBS power and coherence, were chosen individually contralateral and ipsilateral to the movement (C1–C6) and in the midline (Cz). Rectified EMG signal was used to identify the beginning of the movements, and we signed the EMG and EEG signal at the beginning of the movements with “on” and at the end of the movements with “off” markers. The time delay between the on and off markers was defined as movement duration. We created 8-s-long EEG segments, 4s before and 4s after the off marker position as the time 0. Ocular artifacts were controlled visually at the F1, Fz, and F2 EEG channels. Segments with visible artifacts were manually rejected. Trials were only selected when the beginning and the end of the movement could be clearly defined.The multitaper method The coherence between the two signals from the healthy subjects, in our case the EEG signal In this study, we used windows of length 1000 ms and analyzed the spectral power and coherence within a frequency band of 2–30 Hz. The signals were sampled at 1000 Hz. The time step was 50 ms with overlapping windows and the frequency resolution of 1 Hz. This method has been used earlier in PMBS analysis of patients of Wilson's disease and control subjects After calculating the absolute power spectra, in each of the three tasks, 35±5 segments were averaged and then baseline corrected with respect to a reference interval from −4 to −3 seconds.PMBS was measured in the most reactive 4-Hz-wide frequency band, which was determined for the strongest contralateral channel for each subject from the time-frequency-relative power plots . After cThe average duration of the brisk movements in all subjects was compared in the second and third tasks with Wilcoxon matched-pairs test.The average EMG activity of isometric contraction in Tasks 1 and 2 were compared, as well as the average EMG activity of brisk movements and average EMG activity of isometric contraction measured in Task2, with Wilcoxon matched-pairs test.To find out if parameters of PMBS depends on movement complexity in different electrode localizations, PMBS power and latency of EEG electrodes contralateral and ipsilateral to the movement and in the midline were compared in Tasks 2 and 3 with repeated measures ANOVA . This test was also applied separately to compare the average absolute power of the reference interval in these electrode localizations in Tasks 2 and 3 and the average beta power measured during Task1. We used Newman-Keuls post hoc test.To test if PMBS and CMC appear at the same time and in the same frequency, Wilcoxon matched-pairs test was used to compare their latencies and median frequency values. In the second task, the average power of PMBS, and the amplitude of EMG-EEG coherence of each subject were collected and analyzed with Spearman rank correlation. The level of significance was set to p<0.05.The source analysis used in this study was the dynamic imaging of coherent sources (DICS) The inverse problem is the quantitative estimation of the properties of the neural current sources underlying the neural activity. The neural activity is modeled as a current dipole or sum of current dipoles. The power and coherence at any given location in the brain can be computed using a linear transformation, which in this case is the spatial filter Using the time frequency analysis, we identified the reactive PMBS frequency band for each subject as given in The average duration of the brisk contraction was longer in Task2 (0.43±0.06s) than in Task3 .EMG activity of the constant isometric contraction was higher in Task2 than in Task1 . In Task2, as expected, EMG activity increased significantly during the brisk movements as compared to the constant isometric contraction (p = 0.002).EEG beta power during continuous movement in Task1 and baseline beta power in Task2 did not differ (p = 0.99) in the frequency range of 14–26 Hz, but they were significantly higher compared with average baseline beta power in Task3 (p<0.05) in the contralateral central electrodes. In the second and third tasks post-movement beta synchronization was found in all of the subjects on both sides with contralateral preponderance, in locations C1, C3, and C5, in the frequency range of 15–27 Hz.2,20 = 18.75) owing to typical topography of PMBS in healthy subjects. The TASK factor was also significant  in all of the electrodes, PMBS power was lower in the second (1.96±1.266) than in the third task (2.24±1.395). We analyzed the average absolute power of the reference intervals (which was taken from −4 to −3 seconds relative to movement offset) in the two tasks. The ELECTRODE factor was significant as a result of higher baseline beta power in the contralateral and midline electrode locations compared with the ipsilateral ones . The significant TASK factor  revealed increased baseline beta activity during the second task (0.39±0.253 µV2) compared with the baseline activity of the third task (0.11±0.07 µV2).In comparison of PMBS power in Tasks 2 and 3, the ELECTRODE factor was significant .PMBS latency was significantly longer in the second (1.6±0.54s) than in the third task  and were the same in each subject in Task2. In the second task, latency of PMBS and the latency of maximum Coherence were not significantly different in the subjects (p = 0.65), showing that these two phenomena appear at the same time , Table 1In Task1, the source of high beta activity and the cortico-muscular coherence was in the region of the hand area of primary sensorimotor cortex. The source of PMBS was bilateral and was stronger contralateral to the movement, while the source of the coherence could be seen only on the contralateral side .In Task2, the strongest source estimated for the PMBS was identified bilaterally in the hand area of the pre- and postcentral gyrus. The second-strongest source could be seen in the secondary sensory cortex on both sides. In every subject, these sources predominated contralateral to the movement. The strongest source of cortico-muscular coherence was detected in the hand area of the primary sensorimotor cortex, a second source in a premotor area. These sources could be seen only contralateral to the movement in every subject.In Task3, we calculated again a strong source for PMBS in the hand area of primary sensorimotor cortex, a second source in the supplementary motor cortex, and a third source in the thalamus. The first two sources appeared bilateral and dominated on the contralateral side.The number of activated voxels was significantly higher for PMBS than for CMC in Task1 (p = 0.000001) and Task2  according to paired-comparisons. The first source voxel coordinates  with the maximum amplitude of PMBS and CMC were the same in 7 of 11 subject in Task1 and these coordinates were the same in 6 of 9 subjects in Task2  and 4. TIn the present study, we investigated the parameters and sources of PMBS and cortico-muscular coherence at the same time with high-resolution EEG and EMG after short self-paced brisk contraction superimposed on constant isometric weak contraction (Task2). The results of this task were compared with those of isometric contraction (Task1) and brisk contractions (Task3) alone. Our aim was to analyze how these phenomena are related to each other and whether movement complexity determines the parameters and sources of PMBS and CMC.The main findings of this study are as follows:Subjects had the same individual reactive beta frequency band for PMBS and CMC, for both simple and complex movements.PMBS was accompanied by short increase of corticomuscular coherence in the same frequency band, with the same first source of their maximum values in most of the subjects but their magnitudes are not correlated with each other. The first source of PMBS involves bigger cortex area than the first source of CMC.Sources of CMC could be estimated in the primary sensorimotor and premotor cortex, whereas PMBS could be detected in different sensorimotor networks depending on the type and complexity of the task.PMBS could be detected after short movement components during a dynamic, continuous motor task, not only after termination of the whole motor program.We could find beta synchronization in an individual frequency band, in each subject after brisk contraction in Tasks 2 and 3, mainly at central contralateral electrodes, where PMBS was previously detected During simple isometric contraction (Task1), EMG and EEG activity and cortico-muscular coherence were also highest in this beta frequency band , Table 1We have found that the power of PMBS was smaller and its latency was longer in the complex movement (Task2) compared with the brisk movement alone (Task3). The difference in power may be due to the higher baseline power in the complex task. The longer latency may reflect longer processing, e.g. of sensory information, as Task2 definitely needs more precise calibration of muscle strength level; and it may have been influenced by longer movement duration in Task2.In contrast with previous data suggesting that PMBS only occurs in phases of complete rest In one previous study, EMG coherent EEG activity could be revealed in the beta band at the same time as PMBS during phasic finger movements, though in a narrower frequency band, indicating that PMBS may have a composite nature and that only one of its components may be coherent with EMG activity During isometric contraction the source of PMBS and cortico-muscular coherence was estimated in the contralateral primary sensorimotor cortex in accordance with earlier findings Although it is known that PMBS as measured by surface EEG and MEG largely originates from the sensorimotor cortex, there are a number of hints at other brain regions playing a role in its generation. It was detected predominantly in the precentral region with MEG The source analysis in Task3 of the present study confirms that not only bilateral sensorimotor cortex but also supplementary motor and even subcortical  regions take part in the post-movement beta oscillations. Thus, the postulated micronetworks seem to connect to one large-scale sensorimotor network that is reset after the movement. In the complex movement (Task2), PMBS was not found in the SMA or the thalamus, as in the Task3. Its second source was in the bilateral secondary sensory cortex (SII), with contralateral preponderance indicating that execution of this task is highly related to precise sensory information processing; and in this case, PMBS reflects resetting mainly of sensory networks. It is known that SII has an important role in sensorimotor integration Conversely, cortico-muscular coherent activity in the beta band was limited to the contralateral side of the movement and only comprised sensorimotor and a premotor areas (Task2). These are the regions where cortico-motoneuron cells have mono-/oligosynaptic connections with the lower motoneurons of the active muscles Thus, the PMBS (beta power) sources are in keeping with a function in a widespread sensorimotor network that seems to be highly task sensitive, possibly reflecting different needs for the kind of information processing. The EMG coherent sources reflect a function of the cortico-muscular coherence, mainly in the corticospinal system. In this study the size of PMBS first source was bigger than the first source of CMC in Task1 and Task2 supporting the more limited nature of the coherent beta activity representing only a part of the more widespread PMBS network.Our results show that PMBS and cortico-muscular coherence are distinct albeit time-related phenomena at the end of a movement. Whereas it has been postulated that the PMBS resets the supraspinal central components of the sensorimotor network, the cortico-muscular coherent activity may recalibrate the cortico- lower motoneuron and spinal system


Efforts to trap early intermediates of the gold-catalyzed phenol synthesis failed. Neither inter- nor intramolecularly offered vinyl groups, ketones or alcohols were able to intercept the gold carbenoid species. This indicates that the competing steps of the gold-catalyzed phenol synthesis are much faster than the steps of the interception reaction. In the latter the barrier of activation is higher. At the same time this explains the high tolerance of this very efficient and general reaction towards functional groups. While many investigations in the field focused on methodology, mechanistic research was much less widespread NTf2 NTf2 , only 10G, formed from substrate 16, would offer the option of competition of the phenol synthesis  in the presence of lithium hexamethyldisilazide as the base. Ketoester 22 was obtained in 56% yield, however, the two-fold addition of 21 could not be suppressed completely and 14% of the corresponding tertiary alcohol 30 was also obtained. Reduction of the ketone 22 with sodium borohydride and protection of the alcohol 23 with tert-butyldimethylsilylchloride delivered 24 in excellent yield. Reduction of the ester group with diisobutylaluminiumhydride gave aldehyde 25. The addition of lithiated trimethylsilylacetylene provided the propargylic alcohol 26 and reaction with 15 under Mitsunobu conditions yielded 27. Deprotection of the alkyne 27 and the silyl ether 28, followed by the oxidation of the resulting alcohol 29 finally led to 16. It was not possible to remove both silyl groups simultaneously with TBAF, longer reaction times which would be necessary for the deprotection of the hydroxy group led to decomposition of the substrate. At 0 °C and with a very short reaction time, the alkyne was deprotected selectively. Selective deprotection of the alcohol was then possible with a mixture of acetic acid/water/THF. Another route, in which the alcohol function was deprotected first, then oxidized, followed by removal of the trimethylsilyl group from the alkyne also failed. Thus treatment of 27 with acetic acid in aqueous THF gave the desired alcohol 31in quantitative yield. However, whilst Ley oxidation . Th. Th39 wa39, catalyzed by AuCl3 in CDCl3, again only produced the expected phenol 40 (45 was similarly converted to 46. PtCl2 did not lead to a change in selectivity.The conversion of henol 40 . Not uneThe complete failure of both the inter- and the intramolecular trapping experiments shows that the gold-catalyzed phenol synthesis follows a reaction pathway low in energy. These observations also nicely explain the high functional group tolerance, for example, towards olefins and alcohols.File 1Experimental details and characterization data of synthesized compounds.


The ImmunoCAP ISAC 112 is a fluoro-immunoassay that allows detection of specific IgE to 112 molecular components from 51 allergenic sources. We studied the reliability of this technique intra- and inter- assay, as well as inter-batch- and inter-laboratory-assay.Twenty samples were studied, nineteen sera from polysensitized allergic patients, and the technique calibrator provided by the manufacturer (CTR02). We measured the sIgE from CTR02 and three patients' sera ten times in the same and in different assays. Furthermore, all samples were tested in two laboratories and with two batches of ISAC kit. To evaluate the accuracy of ISAC 112, we contrasted the determinations of CTR02 calibrator with their expected values by T Student test. To analyse the precision, we calculated the coefficient of variation (CV) of the 15 allergens that generate the calibration curve, and to analyse the repeatability and the reproducibility, we calculated the intraclass coefficient correlation (ICC) to each allergen.The results obtained for CTR02 were similar to those expected in 7 of 15 allergens that generate the calibration curve, whereas in 8 allergens the results showed significant differences. The mean CV obtained in the CTR02 determinations was of 9.4%, and the variability of sera from patients was of 22.9%. The agreement in the intra- and inter-assay analysis was very good to 94 allergens and good to one. In the inter-batch analyse, we obtained a very good agreement to 82 allergens, good to 14, moderate to 5 allergens, poor to one, and bad to 1 allergen. In the inter-laboratory analyse, we obtained a very good agreement to 73 allergens, good to 22, moderate to 6 and poor to two allergens.The allergen microarray immunoassay, ISAC 112, is a repeatable and reproducible in vitro diagnostic tool for determination of sIgE beyond the own laboratory. Component-based allergological diagnosis has opened up a new era in the study of allergies. It allows the identification of specific sensitization against proteins or specific molecular components The technique of the commercially available protein microarray ImmunoCAP ISAC® specific IgE (sIgE) 112 offers the possibility of analyzing sIgE against 112 components of purified natural proteins and recombinant proteins from 51 different allergenic sources. Since its launch onto the market, this platform has generated great expectations The objective of this study was to assess the accuracy, precision, repeatability and reproducibility of this platform. To this aim we carried out assays with the technique's calibrating sample and sera from polysensitized patients under different conditions including determinations performed both in the same assay and in different assays and considering as possible sources of additional variability the use of different batches of reagents and the performance of the technique in different laboratories.A total of 20 samples were analyzed: the calibrator sample (CTR02), provided by the manufacturer and 19 sera from polysensitized patients. The research ethical committee from the Universidad de Navarra approved the study “Technical and clinical validation of the diagnostic capacity of microarrays of allergenic molecules in allergy to pollens and/or vegetable foods” in which this work has been done. Patients provided their written informed consent to participate in this study. After inclusion for this work, the data from the patients' sera were analyzed anonymously.The CTR02 calibrator, according to information provided by the manufacturer, is composed of known amounts of chimeric monoclonal antibodies humanized against 15 molecular components . The antibodies against these 15 allergenic components are found in the calibrator in a range of 4 different concentrations expressed in ISAC Standardized Units (ISU) values for sIgE . This allows a calibration curve to be plotted from the 4 points of fluorescence intensity corresponding to the different ISU .Of the 19 sera from the polysensitized patients, sera 1, 2, and 3, contained as a group detectable amounts of sIgE against the 15 allergens making up the calibration curve and against another 80 allergens (of the 112 represented in the microarray). Sixteen additional sera, numbered 4–19, showing a broad variety of sensitizations were also included in the study.Patients' sera were frozen at −20°C after the blood collection until the immunoassays were performed and, among the different assays, samples were conserved at 4°C.The microarray ISAC 112  is a solid phase fluoro immunoassay that detects IgE antibodies against the proteins fixed on ISAC surface. One slide contains 4 microarray and one kit 5 slides, 20 microarrays in total. The technique was performed following the manufacturer's instructions. Each microarray is incubated with a serum in order to label sIgE to each protein and subsequently it is incubated with a human anti IgE detection antibody conjugated with fluorescence.Finally the fluorescence intensity of each microarray is measured by the scanner  using following parameters: laser power (LP = 60) and photo-multiplier tube (PMT = 600).2>0.85 according to the information provided by ThermoFisher.The analysis of the digitalized images is performed with the software Phadia Microarray Image Analyzer (ThermoFisher). This software allows transforming the images fluorescence intensity in numerical data according to the calibration curve built with the calibrator CTR02 sample included in each assay, as previously described . An acceThe sIgE values are expressed semiquantitatively as ISU. Results equal or greater than 0.30 ISU are considered positive, according to the indications of the manufacturer.The repeatability of the ISAC 112 technique was assessed by analyzing the sIgE from the CTR02 calibrator and sera 1 to 3 obtained in 10 determinations performed in the same assay with 2 ISAC 112 kits from the same production batch. The calibration curve was obtained with the first determination of the CTR02 calibrator in the first kit in the first slide, first microarray.The reproducibility of the technique was assessed by analyzing the sIgE results from the CTR02 calibrator and sera 1 to 3 obtained in 10 assays performed on 10 different days using 2 ISAC 112 kits from the same production batch. For each assay a calibration curve was plotted with the calibration curve performed in its own assay.The reproducibility of the ISAC 112 technique was assessed by analyzing the levels of sIgE obtained from the 20 samples studied with 2 kits from the same batch. The analyses were performed in two different laboratories i.e.: Clínica Universidad de Navarra, and the Complejo Hospitalario de Navarra, both in Pamplona, Navarra, Spain. The reading of the chips was performed with the same conditions in each laboratory. The calibration curve was plotted with the determination of the CTR02 calibrator performed in each laboratory. Both scanners had been calibrated similarly by the same technician, the same day.The reproducibility of the ISAC 112 technique was also assessed by analyzing sIgE levels from the 20 samples under study obtained from two kits from different production batch.accuracy of ISAC 112, understood as the similarity between the values obtained and those expected, and its precision, understood as the dispersion of the group of results obtained.We studied the accuracy by the analysis of the determinations performed in all the assays with the sample CTR02, after comparison of the results expected and those obtained for each one of the 15 allergens that constitute the calibration curve by means of the Student's T test.We evaluated the precision of the allergens from the calibration curve was analyzed by calculating the coefficient of variation (CV) in all the assays, as the percentage of the standard deviation of the determinations divided by the mean. We calculated the CV from the results obtained from the CTR02 calibrator.Furthermore, the repeatability, and different conditions, reproducibility, of the results obtained in the different assays by calculating the intraclass coefficient correlation (ICC) for each allergen We analyzed the strength of agreement of the determinations under the same conditions, reliability of the semiquantitative character of the technique comparing the number of allergens that modified the category of the IgE according to the range established by the manufacturer , in the interassay determinations for each serum.Also, we analysed the The Student's T test and the ICC for each allergen were performed using the statistical software package SPSS  for Windows, version 15.0 . The CV was calculated using the program Excel version 12.0, Microsoft Office 2007.2 considered by the manufacturer as acceptable The average values obtained in the sIgE determinations against the allergens of the calibration curve Der p 2, Gal d 1, Ole e 1, Can f 1, Can f 5, Phl p 5 and Pru p 3 by ISAC resulted to be similar to those expected . NeverthWhen we analyzed the global variability in the results from the different determinations of the CTR02 calibrator, we obtained a mean CV of 9.42%.The individual data of the calibrating sera allergens are expressed in Repeatability and reproducibility calculated using the ICC are summarised in The analysis of the repeatability  was very good for 94 of the 112 allergens represented in the microarray and good for the Bos d lactoferrin allergen. For the remaining 17 allergens the ICC could not be calculated as all the determinations were equal to 0 ISU.The analysis of the inter-assay reproducibility was very good for 94 of the 112 allergens and good for the Pol d 5 allergen.The reproducibility between the analyses of the determinations in two laboratories was very good for 73 allergens and good for 22 of the allergens represented in the microarray. The allergens Ara h 9, Art v 1, Bla g 5, Bos d 8, Bos d lactoferrin, and Phl p 12, showed a moderate agreement whereas in Jug r 2 and Alt a 6 agreement was mediocre. The ICC values could not be calculated for 9 allergens in inter-laboratories analysis because all the determinations for them were equal to 0 ISU.Finally, in the analysis of the reproducibility between the two kits from different production batches we obtained a very good agreement for 82 allergens and good for 14 allergens. For the allergens Bos d 8, Lep d 2, Pen m 1, Ses i 1 and Ves v 5 we obtained a moderate ICC while for Bos d lactoferrin and Alt a 6 gave a mediocre and poor agreement respectively. The ICC values could not be calculated for 9 allergens in inter-batch analysis because all the determinations for them were equal to 0 ISU.When analysing the number of disagreements in the interassay analysis determinations in patients' sera, we obtained the following results. The results of sIgE obtained modified from “not detectable” level to “low” level for 7 allergens when analysing sera 1 and 2, and for 9 allergens when analysing sera 3. The specific IgE levels modified from “low” to “moderate-high” for 7 allergens when analysing sera 1 and 2, and for 13 when analysing sera 3. The specific IgE level change from “moderate-high” to “very high” happened in 16 occasions when analysing sera 1, and in 15 occasions when analysing sera 2. Finally, the IgE level change from “not detectable” to “high” was observed for 4 and 5 allergens when using sera 1 and 2 respectively. This allergens were Act d 1, Asp f 1, Ara h 8, Ber e 1, Cor a 9, Mal d 1, Phl p 5, Phl p 12 and Pla l 1. The allergens that showed a greater number of disagreements were Jug r 2, Jug r 3 and Ses i 1, which had disagreements on the IgE level in the three sera studied.The technique of protein microarrays has been accepted as a useful method for the detection of sIgE against molecular components However, few studies have been carried out into the variability and accuracy of this technique showing improvable results in previous versions of this microarray When analysing the accuracy of the technique, 8 of the 15 allergens that form the calibration curve showed statistically significant differences between the values obtained and those expected. Despite this fact, all the calibration lines were within acceptable limits of slope and R2 established by the technique's supplier. The fact that most allergens keep their category, even at low sIgE levels, supports the reliability of the results offered by the technique. Nevertheless, it should be highlighted that this is a semiquantitative technique and the IgE levels close to the cut off points between categories can fluctuate in different assays.Regarding the technique's precision, the results obtained show a low variability when we analyse the determinations of the control sample CTR02, which is under 10% in most allergens. Also, the CV is similar both in the analysis performed in the same assay and in those performed in different assays. It is worth noting that the capacity of the allergen to attach itself to the surface of the ISAC array is lower than in quantitative techniques such as ImmunoCAP, in which the allergen is fixed under excess molarity conditions. This fact could make more clear differences in the allergen ability to bind to the microarray leading into differences in variability of specific IgE binding. Differences in reliability of some allergens from the microarray  and considering that ISAC microarray is a semiquantitative technique suggest that ISAC 112 is not the best method to monitor sensitizations and patient's follow-up.However, in our opinion these analyses demonstrated an evident improvement in the new version of the ISAC microarray. This improvement can be, among others, due to the calibration curve consisting in chimeric antibodies (sIgE) in contrast to the serum with known sIgE concentration from previous versions ISAC 103 Finally, we studied the repeatability and reproducibility of the technique, analysing the ICC in the different assays. The results obtained in the intra-assay analysis show a good repeatability, obtaining good agreement strength for almost all the allergens present in microarray. Also, the reproducibility of the interassay analysis showed good concordance strength for these allergens. The reproducibility in the inter-laboratory and inter-batch analysis still has good agreement strength for most allergens. Nevertheless, this agreement strength was lower for 8 allergens in the inter-laboratory analysis and for 7 allergens in the inter-batch agreement. This slight decrease in the results' agreement strength could be due to the fact that only two determinations were performed in each analysis instead of 10 repetitions like in the interassay analysis. Also, the performance of analysis in different laboratories and with different batches means a greater source of variability.In conclusion, ISAC 112 yields good reliability results taking into account that ISAC 112 gives semi-quantitative results. However, due to the low accuracy obtained in some of the studied allergens, the application of this semi-quantitative technique for diagnosis in clinical situations where results may have a major impact on the therapy prescribed may not be advisable. Even more, this study suggests that neither laboratory specific condition neither the change from one batch to another affect substantially microarray reliability.


Arthralgia is a frequent complaint of children, and it may be a symptom of arthritis and non-inflammatory diseases.To determine the prevalence of joint pains in children of different ages.Four towns and two rural districts of the Republic of Bashkortostan (Russia) were chosen for the questionnaire. The total number of the childhood population was 50442 children under 17 years of age. Totally 43907 children or their parents were examined with the help of the screening questionnaire. The parents were questioned by medical nurses during consulting reception hours, or in kindergartens, or at school. The children aged between 11 and 17 years answered the question list on their own. The screening questionnaire included two questions:1. Did you observe any pain in joints of your child?2. Did you observe any swelling in joints of your child?Four variants of the answer were offered:A – never happened, B – happened, but does not disturb for the last year, C – happened before and disturbs for the last year, D – happened for the last year.Complaints of arthralgia in the anamnesis or for the last year  were defined in 5490 children (12.5 %). The frequency of complaints of joint pains was higher in the girls (13.1 %), than in the boys . More frequently  the joint pains were marked earlier, but for the last year they did not disturb (answer B). In 1923 children (14.4 %) the pains happened in the past and happen for the last year (answer C), in 1005 children (2.3 %) the pains happened for the last year (answer D). The frequency of complaints of joint pains was 5.8 % in the age between 4 and 6 years, and 13.3 % in the age between 7 and 11 years, and 16.6 % in the juvenile age (between 12 and 17 years of age).754 children and/or their parents answered the question positively about the joint swelling (1.7 %). 368 children and/or their parents indicated that the joint swelling had appeared or remained for the last year (0.8 %).The frequency of complaints of joint pains was defined in 12.5 % of the children. The prevalence of arthralgia was higher in the boys, than in the girls, and it increases with the age. Most children with joint pains did not suffer from the inflammatory joint disease.


Compare two groups of patients, ≥70 years old and <70 years old, diagnosed with III-IV grade haemorrhoids that underwent to stapled haemorrhoidopexy .Between May 2001 and August 2010, 320 patients underwent stapled haemorrhoidopexy (PPH Ethicon-EndoSurgery®). The database has been organised into two groups: the first composed of 30 patients (9.3%) aged≥70, while the second composed of 290 patients aged<70. The preferred type of anaesthesia was spinal with sedation (92.8%), combined with elastomeric pump of NSAIDs during the first 24 hours. The two groups were compared in order to verify their homogeneity: no significant differences were found either in the distribution of the grade of the disease, or in the spectrum of symptoms (P>>0.05). Because of the comorbidity in the elderly, the stratification of the ASA risk was different (P<0.0001).The procedure was performed in day surgery, with an average length of stay of 1 day, in 75% of the patients of the first group and in the 92% of the second group; the analysis of the surgical performance of this technique, regarding the timing and the use of haemostatic stitches, showed no significant differences between the two groups (P>>0.05). Early haemorragic post-operative complications were 2.3%, of which 1 occurred in the first group and 6/7 required surgical review. There were 10 late haemorragic complications, all of them occurred in the second group; the management of the late haemorrhages required surgical review in 40% of patients and blood transfusion in 30% of cases. During the first post-operative week several cases of significant anal pain occurred, tenesmus, faecal urgency and two cases of haemorrhoidal thrombosis. During the follow-up, which lasted on average for 4.1 years, we observed 3 relapses (10%) among the first group and 21 relapses (7.2%) among the second.Haemorrhoidal disease, although tending to relapse among susceptible patients, can be effectively treated with stapled haemorrhoidopexy (PPH). Thanks to a several years’ follow-up, our experience shows an assessment of the long-term results of this technique, focusing particularly on the comparison between the results in the elderly and in younger patients. The Longo technique is usually well tolerated by all the patients, even though not totally pain-free in the early post-operative follow-up (first week). This procedure can be performed safely in the elderly as well as in the younger patients with equivalent results.


The generated holograms and contribution maps revealed important molecular requirements to HIV-1 IN inhibition and several evidences for molecular modifications. The final models along with information resulting from molecular orbitals, 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selectivity within the chemical diversity of the data.Among several biological targets to treat AIDS, HIV integrase is a promising enzyme that can be employed to develop new anti-HIV agents. The aim of this work is to propose a mechanistic interpretation of HIV-1 integrase inhibition and to rationalize the molecular features related to the binding affinity of studied ligands. A set of 79 HIV-1 integrase inhibitors and its relationship with biological activity are investigated employing 2D and 3D QSAR models, docking analysis and DFT studies. Analyses of docking poses and frontier molecular orbitals revealed important features on the main ligand-receptor interactions. 2D and 3D models presenting good internal consistency, predictive power and stability were obtained in all cases. Significant correlation coefficients ( According to the latest UNAIDS (Joint United Nations Program on HIV/AIDS) global report, 34 million people were infected with HIV at the end of 2011. Although the number of newly infected people worldwide has been decreasing by 20% since 2001, in some parts of the world as Middle East and North Africa this number has increased by more than 35% Among the molecular targets of existing antiviral drugs that can be explored to design improved new drugs, HIV integrase (HIV-1 IN) is still a noteworthy target due to its pivotal role in the integration of the viral genome into host cell chromatin, which is a fundamental step in the replication cycle of HIV. HIV-1 IN catalyzes the integration of viral DNA into the host DNA in two steps: 3 -processing and strand transfer. The first step, 3-processing, involves a hydrolyzing reaction that removes a terminal dinucleotide to recess a CA-3 terminal. The CA-3 sequence is generally conserved on the third and fourth position from both 3-ends of long terminal repeats of the viral DNA. The second step, called strand transfer, is a transesterification reaction of the recessed 3-ends with the phosphodiester backbone of the host cell DNA. Both ends of the viral DNA are joined to the host DNA at the same time, and subsequently repair of the nick by host cell repair machinery completes the integration sequence Many advances in the discovery of HIV inhibitors have been achieved in the last decades. Inhibitors derived from 4-aryl-2,4-diketobutanoic acid containing a diketoacid moiety (DKA) were identified in 2000 by Merck N-alkyl-5-hydroxypyrimidinone derivatives acting as HIV-1 IN inhibitors were investigated by integrating molecular docking, quantum chemistry, 2D and 3D QSAR techniques. With these approaches, we were able to explore the binding modes of these inhibitors with the HIV-1 IN enzyme, as well as to examine the structure-activity relationships for the HIV-1 IN inhibitory activity of these compounds using CoMFA and HQSAR. The QSAR models generated may provide useful information about the design and synthesis of more potent HIV-1 IN inhibitors with predetermined affinity.Although some QSAR and docking studies have been performed on different series of HIV-1 IN inhibitors over the last few years, the union and rationalization of experimental results with theoretical studies, especially in the case of electronic properties, are still necessary in order to obtain predictive relations that may lead to the design of new inhibitors acting as anti-HIV drugs 50) for the complete set of compounds are listed in 50 values vary from 2 to 2000 nM  and used as dependent variables in the QSAR investigations. From the original data set containing 79 inhibitors, 64  of the data set were well represented in both training and test sets. The series of HIV-1 IN inhibitors employed in the development of 2D and 3D QSAR models is constituted by 79 compounds selected from the literature  2000 nM  and were50 values is shown in The distribution of pICThe inhibitor structures were built using the CONCORD module Docking simulations of the HIV-1 N inhibitors were performed to study the appropriate binding orientations and conformations and to provide the molecular alignment for the whole data set required for CoMFA studies. The protein structure, determined with a 2.1 Å resolution, in complex with the diketoacid inhibitor 5CITEP and a magnesium atom, was selected from Protein Data Bank (PDB code 1QS4) 2+ ion between D64 and E152 residues, using PyMOL software et al.The structural preparation of the selected target involved the following steps: (i) analysis of the main aminoacids in the active site; (ii) ligand removal of the active site; (iii) adding a second Mg2+ ion should be chelated by the oxygen atoms from the hydroxypyrimidinone group and by the oxygen atoms from D116, D64 and E152 residues . 30 runs of the GOLD search were carried out for each compound in the data set, allowing total bonds, dihedrals and angles flexibility. Ligand poses were ranked using GoldScore function Based on the structural analysis of intermolecular interactions responsible for the molecular recognition process, some features were set out in the docking process. A 5 Å cavity was defined involving residues around 5CITEP ligand. Regarding the constraints, each MgThe docked solutions of all ligands were used in the alignment process. The best 10 rank solutions for each ligand were analyzed. Each conformation was evaluated to satisfy the pharmacophoric hypothesis presented in Finally, the chosen conformations for raltegravir, the most and the least potent inhibitors of the data set were submitted to DFT  single point calculations, employing B3LYP functional 3D QSAR methods are based on the three-dimensional characters of ligand-receptor interactions and in the use of interaction molecular fields as descriptors. In this work, Comparative Molecular Fields Analysis (CoMFA) 3 carbon atom probe carrying a +1 net charge. The default value of 30 kcal/mol was set as the maximum steric and electrostatic energy cutoff. 3D models were constructed using Partial Least Square (PLS) technique, with leave-one-out cross validation and a test set (not used in the model building) as external validation test, and also the region-focusing method was employed in order to obtain a model with maximum predictive ability and to increase the resolution of CoMFA models.A crucial step in the generation of the CoMFA model is the 3D molecular alignment of the data set Hologram Quantitative Structure-Activity (HQSAR) methodology requires only 2D structures and the corresponding biological activity as input, allowing the investigation of a wide variety of bioactive compounds. This technique explains differences in the biological activity of a series of molecules by quantifying variations within their molecular holograms and using the PLS method The main steps involved in HQSAR analyses are: substructural fragmentation for each molecule in the training set; molecular hologram generation; statistical analysis and model generation; test set selection and external validation. To generate the molecular fragments and holograms, some parameters can be varied:Fragment distinction including information on A (atoms), B (bonds), C (connectivity), H (hydrogen atoms), Ch , DA (donors and acceptors of hydrogen bond);Hologram Lengths of 53, 69, 61, 71, 83, 97, 151, 199, 257, 307, 353 and 401.Fragment size: this parameter controls the minimum and maximum number of atoms in each fragment. Initially, it was used the standard size (4–7 atoms) for the generation of the fragments; then, the size was varied from 2 to 10 until the best model was found.The particular nature of the sub-structural fragments generated by HQSAR and, consequently, the information contained in the resultant molecular holograms is changed by adjusting these parameters. Finally, the correlation of the obtained descriptors with the biological property was modeled using the PLS method.2+ ions together with the catalytic triad DDE  involved in DNA strand viral binding in the strand transfer process catalyzed by integrase 2 and 37.From the molecular docking results, it was observed that all the compounds showed a similar interfacial binding mode in which the hydroxypyrymidinone group chelates Mgriad DDE , as discet al., 2). In 37), there are no interactions from this portion of the molecule with these residues.From 12), the most (2) and the least active ligand (37) studied and their complexes with Mg2+. As discussed before, the activity of these inhibitors is dependent on the Mg2+ ions from the catalytic site of HIV-1 IN, which acts as a bridge between the catalytic triad DDE, the viral cDNA and the host DNA. In order to understand the electronic features of these molecules involved in this interaction, the plots of HOMO and LUMO orbitals of the molecules alone, as well as for the molecules in complex with Mg2+ ions, were obtained by DFT/B3LYP/6-31G** calculations.2+ ion, with a higher electronic density on oxygen atoms. Thus, the rings at 8-hydroxy-- naphthyridine -7-carboxamide group contribute with a nucleophile character to these molecules, in contrast with the 1,3-diketoacid group, which lacks aromatic rings. Consequently, naphthyridine derivatives are more active at HIV-1 IN than diketoacids. Analyzing the molecules in complex with Mg2+, one can see that LUMO is concentrated on Mg2+ ions in the most active molecules, since this is the site for putative electronic transfer. In the case of the least active molecule, LUMO spreads over the whole molecule, which can in part accounts for the less affinity of this molecule at HIV-1 IN.From 2+ ions and the catalytic triad DDE.The 79 molecules aligned for the 3D QSAR modeling are shown in 2q = 0.401, with four PLS components). Thus, we have applied the region-focusing procedure, which weights the lattice points in a CoMFA region in order to enhance or attenuate their contribution to the PLS analysis, allowing to increase the resolution of CoMFA models. So, our grid points were weighted by standard deviation coefficient values ranging from 0.3 to 1.2, with a grid spacing varying from 0.5 to 1.5 Å. The best statistical results were obtained when region focusing was weighted by a standard deviation coefficient of 1.0 . Within this CoMFA model, the contributions of steric and electrostatic fields correspond to 49% and 51% of the total variance, respectively.As the first CoMFA model showed unsatisfactory statistical parameters, we have applied the region-focusing procedure. The best statistical results obtained are displayed in 12), the most active (2) and the least active (37) molecules are shown in From the best CoMFA model obtained, 3D contour maps were generated, i.e. graphical results can be analyzed considering steric and electrostatic fields. Favorable and unfavorable regions for substitution by bulky substituents are represented in green and yellow, respectively. Electrostatic features are characterized in such a way that red contours represent regions in which electronegative substituents may increase the biological activity, whereas blue contours indicate regions in which electropositive groups would contribute to enhance activity. The contour maps for raltegravir  according to the electrostatic contour maps, a ketone group linked to pyrimidine ring can enhance the biological activity; (2) from the steric contour maps, a compound should have bulky substituents at the pyrimidone ring, for example, tetrahydrofuran, tetrahydropyran or morpholine.The presence of green polyhedra suggests the introduction of bulky groups, and the presence of blue polyhedra, more positive groups. This information is consistent with the docking studies performed to obtain the structural alignment. The best positioning of the molecules in the enzyme active site allows bulky groups to be added in these positions. When compared to the least active inhibitors, raltegravir has a bigger fragment A with more positive groups as oxadiazole ring and carboxamide group, both with nitrogen atoms. The presence of these groups is required for the interactions with the polar residues K156, K159 and T66 through hydrogen bonds. The presence of the red polyhedra in the chelating region indicates that high electron densities may increase the ligand affinities. Electronegative substituents in this position exert a greater effect on the affinity for Mg2q = 0.643 and 2r = 0.908), which were derived using A/B/C/H/Ch/DA, respectively, and six being the optimum number of PLS components. This model was selected to investigate the influence of different fragment sizes on the main statistical parameters. The results obtained after the variation of the fragment sizes are displayed in 2q = 0.643 and 2r = 0.908). Therefore, the final HQSAR model generated with the training set has the good cross-validation correlation coefficient as an indication of model robustness.From the patterns of fragment counts, several HQSAR models were generated and investigated. The statistical results from the PLS analyses for the 64 training set compounds, using several combinations of fragment distinctions, are presented in 12), the most (2) and the least (37) potent inhibitors of the data set.The information contained in the best HQSAR model (model 5) is encoded in a contribution map, which shows every atom of a given molecule and its contribution to the biological activity. On this map, colors in the red region of the spectrum reflect unfavorable or negative contributions, and colors in the green region reflect favorable or positive contributions. Atoms with intermediate contributions are colored in white. The contribution map for raltegravir and the most potent inhibitor (2), displayed in In order to ascertain the abilities of the obtained models in making predictions for unknown compounds, a test set chosen in such a way that it represents the chemical diversity and the distribution of biological activity, was employed to perform the external validation. The low differences between experimental and predicted values indicate that both models present a good predictive ability. The external validation process shows that these models are able to predict the biological activity of other molecules within this chemical class, represented by the diversity within the chemical space explored.2+ ions. Additional interactions are observed in raltegravir drug and the most potent molecules, which show a better positioning of fragment A into one of the active site cavities, due to interactions with T66, K159 and K156 residues. The DFT results indicated that, for the most active molecules, the HOMO molecular orbitals are located mainly in the chelating domain. So, there is an important contribution of the negative charges on the oxygen atoms that are involved in coordination with the Mg2+ ions. This orbital effect produces a high nucleophilic character on these active sites. On the other hand, LUMO of the more active molecules is located in the Mg2+ region, enabling interactions with oxygen atoms of aspartate (D64 and D116) and glutamate (E152) residues in the enzyme active site. The resulting 3D QSAR model was able to provide a good correlation between the steric and electrostatic fields with the docking results. The contour maps indicated that in the chelating region, more electronegative groups are needed, and that in the fragment A, substitutions by more positive and bulkier groups contribute to interactions with polar T66, K159 and K156 residues. Additionally, information obtained from HQSAR contribution maps indicates the importance of substituents in fragment A to the design of new HIV-1 IN Inhibitors.In this study, we presented molecular docking and quantum chemical studies, along with CoMFA and HQSAR models, successfully applied to a series of HIV-1 IN inhibitors. By the analysis of molecular docking, it was possible to verify the binding modes of the inhibitors at the enzyme active site, which occur by chelation of the two MgTable S150 values for the HIV-1 N inhibitors.Chemical structures and IC(DOCX)Click here for additional data file.


Potential methods of application of self-administered Web-based pure-tone audiometry conducted at home on a PC with a sound card and ordinary headphones depend on the value of measurement error in such tests.The aim of this research was to determine the measurement error of the hearing threshold determined in the way described above and to identify and analyze factors influencing its value.The evaluation of the hearing threshold was made in three series: (1) tests on a clinical audiometer, (2) self-tests done on a specially calibrated computer under the supervision of an audiologist, and (3) self-tests conducted at home. The research was carried out on the group of 51 participants selected from patients of an audiology outpatient clinic. From the group of 51 patients examined in the first two series, the third series was self-administered at home by 37 subjects (73%).The average difference between the value of the hearing threshold determined in series 1 and in series 2 was -1.54dB with standard deviation of 7.88dB and a Pearson correlation coefficient of .90. Between the first and third series, these values were -1.35dB±10.66dB and .84, respectively. In series 3, the standard deviation was most influenced by the error connected with the procedure of hearing threshold identification (6.64dB), calibration error (6.19dB), and additionally at the frequency of 250Hz by frequency nonlinearity error (7.28dB).The obtained results confirm the possibility of applying Web-based pure-tone audiometry in screening tests. In the future, modifications of the method leading to the decrease in measurement error can broaden the scope of Web-based pure-tone audiometry application. The development of Internet technologies combined with technological progress in the construction of personal computers, especially in terms of improving performance of sound cards, offer the possibility of conducting self-administered hearing tests at home. Moreover, in a group of elderly people who often suffer from hearing loss, there is an increasing number of Internet users. The validity of such hearing tests needs to be evaluated before applying the test results in the diagnostic and therapeutic process.The remote examinations of hearing conducted with the use of PCs have been carried out for about 10 years -4 and exThe hearing screening tests done remotely with the use of a PC sound card are most often in the form of a speech-in-noise test ,7,11,12.The potential application of pure-tone audiometry conducted on a PC depends on the value of the measurement error. The PC-based test will not substitute the clinical pure-tone audiometry. However, it can be applied for conducting self-administered check-ups in cases of limited access to clinical devices, eg, at the general practitioner. Alternatively, it can be used as an initial telemedical examination combined with a survey, which will help determine the direction of future treatment. The aim of the research was to identify the measurement error connected with determining the hearing threshold conducted by means of self-administered Web-based pure tone audiometry, as well as identify and analyze factors affecting its value.The hearing threshold evaluation was done in three series: (1) audiologist-performed tests on clinical audiometer at an audiology outpatient clinic, (2) self-administered Web-based tests on a specially calibrated computer at an audiology outpatient clinic under the supervision of an audiologist, and (3) self-administered Web-based tests conducted at home.In series 1 and 2, 51 participants , aged 11-60 years (the median age was 34) underwent examination. The research participants were selected from among the patients of an audiology outpatient clinic. The qualification criterion was the willingness to participate in the research, owning a PC, basic skills to operate it, and having an email account. 102 ears were examined from which 45 (44%) were ears without a hearing loss, ie, with the hearing threshold of 25dBHL and less, 17 (16.7%) were ears with hearing impairment below 40dBHL, 31 (30.4%) with hearing impairment above 40dBHL and below 70dBHL, and 9 (8.8%) were above 70dBHL. From the group of 51 patients examined in the first two series, series 3 was self-administered at home by 37 subjects (73%). The examinations in series 1 and 2 were conducted on the same day, and examinations from series 3 were conducted up to 196 days later (median of 9 days). In the case of failure to conduct test in series 3 in a time of 2-3 weeks, the patients were reminded by a phone call and then after about 1 month, they were reminded by an email.All tests from series 1 were made in an acoustic cabin with the use of clinical audiometer Interacoustic AD229e and headphones TDH-39. The calibration of the audiometer was conducted according to ISO 389-1:1998. The hearing threshold in pure-tone audiometry was determined with the use of the ascending method, according to ISO 8253-1:2010. The level of the tone was reduced in 10dB steps until no further response occurred, and then it was increased in 5dB steps until the subject responded. The threshold was defined as the lowest level at which responses occurred in at least half of the series of ascending trials with a minimum of two responses required at that level . The exaThe examinations in series 2 and 3 were conducted on personal computers following their calibration. Both the calibration and the examination were performed at system volume set at the maximum level. The calibration consisted of determining the reference sound level by a person with good hearing. During calibration, two sounds differing in intensity were presented bilaterally in turns for 1 second at a frequency of 1kHz. The difference in sound intensity was stable and equalled 5dB. The task of the reference person was to set the volume in a way that ensured that only the louder of the two sounds presented was audible. The volume was controlled by means of a slider with the step of 1dB. The reference person could listen to the sound for unlimited time, adjusting the volume using the slider with 1dB step any number of times, in order to finally confirm the selected level with a button. The mean intensity of the two presented sounds determined the hearing level of the reference person and was marked 0dBRP-HL. The reference values at frequencies differing from the calibration frequency, ie, 1kHz, were calculated using the model based on the A-weighting filter .The procedure of determining the hearing threshold during the test, both in series 2 and 3, differed from that applied during calibration. The task of the subject was to set the intensity of the presented pure tone modulated by sinusoidal envelope with a 2-second period at such a level that the sound was on the verge of audibility. Patients could listen to the sound for unlimited time and change the intensity of the presented sound signal by themselves using a slider with 5dB step any number of times, and then confirm the chosen level with a button. The hearing threshold was determined at frequencies as in series 1, ie, 250Hz, 500Hz, 1kHz, 2kHz, 4kHz, and 8kHz. For hearing thresholds below 0dBHL-RP, the value was assumed to be 0dBHL-RP. The time spent on calibrating and examinations was not recorded.All the examinations in series 2 were conducted on a notebook Dell Vostro 1320 with operational system Microsoft Windows 7 and headphones Technics RP-F290 placed in an acoustic cabin of an audiology outpatient clinic and connected to the Internet. The calibration was repeated six times by 3 people whose hearing threshold in pure-tone audiometry at the calibration frequency was 0dBHL. Each person performed calibration twice. The final value of the calibration coefficient was determined as the mean of all values obtained from single calibrations and was the same for all examinations in series 2. Examinations were supervised by an audiologist, whose task was to train a patient and detect hisher mistakes, eg, changing the sides of headphones, omission of the frequency, or accidental marking of a threshold that was significantly different from the actual one.Examinations in series 3 were conducted by the patients themselves on their own personal computers at home. Each examination was conducted on a different computer using different headphones. The test station was calibrated by other household members with no previous hearing problems. In that series, the hearing threshold of the reference person was not controlled. The trial participants were instructed to conduct the test at home in a quiet place, preferably in the evening or at night. Moreover, they were informed that the calibration should be performed by a person up to 35 years old, with no previous hearing problems.The hearing threshold determined in series 1 was compared to hearing thresholds from series 2 and 3. If there was no response at a given frequency, measurement was not taken into account for further calculations. The mean difference between thresholds in series, its standard deviation, Pearson’s correlation coefficient, and linear estimators  were calThe mean difference between thresholds in series, its standard deviation, Pearson’s correlation coefficient, and linear estimators of Deming’s regression were determined for the hearing loss below 40dBHL as well as greater than or equal to 40dBHL . The divP=.05, and the specificity was 0.96 with confidence intervals of  at P=.05. For series 3, the respective values were sensitivity 0.89  and specificity 0.89 .On the basis of the obtained results, the sensitivity and specificity of noise-induced hearing loss detection was calculated, according to the criteria proposed in  adopted This paper attempts to identify and assess the values of the sources of error in determining the hearing threshold of Web-based examinations. The literature data on standard deviation of the hearing threshold determined in test-retest examinations carried out in clinical settings are presented in X and Y is the sum of their variance  at the level of literature data σclin, the standard deviation of the measurement error in Web-based hearing tests for series 2 σ(ii) was calculated at 6.64dB based on the value σ(i) and the value σ(i)(ii), which is the standard deviation of the hearing threshold difference between the values determined in series 1 and 2. Similarly, for series 3, we obtain the standard deviation σ(iii) equal to 9.78dB , both for series 2 and for series 3.gain≈0dB).With the increase in intensity, increment of the standard deviation of measurement error in series 3 is slightly smaller than in series 2. In series 2, the standard deviation of the gain error equals zero since all tests are done on the same set. The lack of increase in standard deviation in series 3 above the values observed in series 2 indicates a negligible effect of the gain error on the measurement values . Taking into account the above, we obtain the calibration error σcal equal to 6.19dB , the calibration error , and additionally at the frequency of 250Hz, the frequency response nonlinearity error (250Hzσnonlin=7.28dB).In series 3, the standard deviation values were most influenced by the error connected with the procedure of determining the hearing threshold, the same as the error in series 2  may be attributed to the difficulty connected with self-assessment of the hearing threshold. Standard deviations between series 1 and 2 as well as between series 1 and 3 increase together with the increase in the hearing loss  is connected with the error of hearing threshold assessment σproc_cal and the standard deviation in the hearing threshold of the reference persons σpop at the calibration frequency, ie, 1kHz. The standard deviation of the hearing threshold determined by means of the ascending method among the population of young subjects without prior hearing problems, σpop_asc, can be estimated on the basis of literature data [pop_asc=5.6dB. Assuming the standard deviation of the ascending method σclin is at the level of 4.25dB, we obtain σpop at 3.65dB and then σproc_cal at 5.00dB , despite the possibility of setting the threshold with the accuracy of 1dB step, is characterized by a slightly higher error than in the ascending method σclin=4.25dB, whose step equals 5dB. Therefore, accuracy improvement of the Web-based examination can be achieved by modifying the method. An interesting solution is offered by, eg, the Békésy’s method, as it is characterized by lower standard deviation in the test-retest examination compared to the ascending method [The method of the hearing threshold evaluation used during calibration  of the headphones used in the examination and the frequency nonlinearity of the sound card. The biggest differences between the RETSPL values occur at low frequencies [The frequency nonlinearity error is the difference between the actual values of the reference sound level and the values determined by the model. The greatest value of quencies , also inTaking into account the significant value of the calibration error and frequency nonlinearity error, it seems interesting to conduct pure-tone audiometry examination on generally available appliances with known electro-acoustic parameters, eg, smartphones. On determining frequency characteristics of the selected smartphone model with bundled headphones, it seems possible to omit the calibration stage and thus obtain more precise examination results.Web-based pure-tone audiometry can be used without previous training in its conducting. However, it requires the knowledge of basic terms such as the hearing threshold or frequency. On the other hand, an attractive, intuitive and user-friendly interface can largely replace training. Prior to the tests in series 3, patients were instructed on how to perform the test and had performed similar tests before in series 2 under the supervision of an audiologist. The knowledge of the application could lead to reduction in the value of the measurement error.The obtained results of sensitivity and specificity confirm the possibility of applying Web-based pure-tone audiometry in screening tests. Moreover, Web-based pure-tone audiometry may be used for self-monitoring of hearing, especially if tests are to be conducted under the same conditions. If the same equipment is used, the relative error between subsequent examinations will be reduced by frequency nonlinearity error, and in the case of the same calibration coefficients, relative error will be reduced by the calibration error. Self-monitoring of hearing may become applicable in hearing disorders, such as fluctuating hearing loss, tinnitus, sudden hearing loss, otosclerosis, Ménière’s disease, as well as during treatment with ototoxic drugs. In the future, modifications of the method leading to the decrease in measurement error can broaden the scope of Web-based pure-tone audiometry application.


In Algeria, improving the quality of care is a priority since 2005, and the impact of actions was felt by patients and caregivers so subjective and varies from one institution to another, because no analysis rational and no research on assessment tools do exist nationwide. CHU of Tizi-Ouzou is one of the pilot sites for the implementation of these indicators.1 - To determine the incidence of adverse events leading to adverse effects for patients; 2 - Evaluate the degree of patient satisfaction with regard to various hospital services; 3 - Find the underlying causes of iatrogenesis; 4 - Define a strategy to improve the quality of care.THERE are two surveys, one descriptive cross-sectional, followed by a prospective, analytical type, with patients hospitalized for a minimum of 24 to 48 hours over a period of two years.Preliminary results showed that of 656 patients hospitalized for an average stay of 7 days, the incidence of adverse events was 16.5%, the confirmation rate is 87.96%. The event most frequently encountered in the first passage is nosocomial infection (11.1%) in the second passage, unwanted physical harm (10.2%) in the third passage, nosocomial infection (3.7%).As the survey of patient satisfaction, weaknesses in the quality of care in our institution are by far the advice given to the patient's discharge (57.8%), the information given by the doctor (57.01% ), and the identification of the function of the staff (49.5%).Despite the constraints, this study allows us to better know the quality of care at our institution and the procedure to be adopted to establish the assessment tools, however, the implementation of a quality project requires a system efficient information in order to analyze a priori and a posteriori risk inherent in each activity or process of care measures withdrawal, reduction and / or recovery and a battery of indicators applicable to our care facilities and generalizable at regional or national level.None declared